MicroBiology Class Notes

Notes 01/27/2022

-Cytoskeletal components involved in growth and cell shape --Division plane -Growth rates can vary depending on nutrient availability, temperature, O2, osmotic pressure, etc. --Types of e- donors and acceptors available --How much Biosynthesis of amino acids, vitamins, etc. is required -Prototrophs can make all amino acids, nucleotides, vitamins, etc. --Autotrophs (carbon fixers) (photosynthesizers, chemosynthesizers... growing on inorganic stuff so they have to make the carbon stuff!) --Free-living organotrophs in variable or dilute habitats -Auxotrophs require one or more "primary metabolites" --Amino acids, nucleotides, vitamins, etc. --Typically live in consortia or on hosts --"That which is unavoidable becomes essential" GROWTH OF POPULATIONS -Methods for counting cells ---Direct Microscopic Counts (Specialized microscope slide with a grid. Each box has a specific volume of medium allowing you to count the cells) (not used often for small, translucent bacteria) -Mathematics of population growth Viable Cell Counts -Samples can be diluted or concentrated as needed -Cells can be concentrated from dilute media by filtration -Requires overnight incubation ***Petri dish and counting colonies OPTICAL DENSITY OR "TURBIDITY" -Cells scatter light so it's optical density Generation Time ≡ Doubling time ≡ "g", t or τ (tau) N = N_o2^n , where N = # cells, n = # generations EXPONENTIAL GROWTH -Semi-log paper: -Straight lines indicate constant growth rates -Slopes indicate growth rate -Deviations from linearity indicate changing rates Diauxic Growth Curve (from Greek "auxein," to grow) ***Example of Catabolite Repression (Glucose prevents activation of other things) ***Lag phase is where cells are not growing, but regulating gene expression to the medium -Cells prefer glucose -Glucose is used until it is exhausted -When glucose is gone, genes for utilizing lactose expressed -Only then is lactose used -Growth on glucose is faster (steeper slope) than growth on secondary sources GROWTH IN LIQUID BATCH CULTURES -Pseudo-steady state during "log" or exponential phase -If growth rate is constant, cell physiology is constant ***Transition from Exponential to Stationary phase until the cultures run out of nutrients, then they start to die out CELLULAR GROWTH -Accumulation of biomass --Energy and Biosynthetic Metabolism -Peptidoglycan synthesis -Replication -Coordinated division --Peptidoglycan reorganization and synthesis at the Septum --Chromosome segregation GROWTH AND DIVISION Gene nomenclature: -fts for "filamentous, temperature-sensitive" phenotype --ftsZ = gene, FtsZ = protein -min for "minicell" (cells that do not divide in the right place all of the time, divide in random places making small cells) -mre for "maintenance and regulation" (of cell shape) (cell shape mutants) --FtsZ is tubulin-like --MreB is actin-like Replica Plating for Temperature-Sensitive Mutants -Mutagenize culture and plate at 30o -Replica plate and incubate replicates at 30o and 42o --Find colonies that do not grow on a plate incubated at 42o -Examine "ts" mutants for filamentous growth at 42o --"filamentous, ts" or or "fts" mutants ***Mutation in something that is functional at 30 degrees but not at 42 degrees Min Proteins and the FtsZ Ring -MinE forms a spiral band throughout cell and pushes MinC,D towards the poles -MinC,D inhibit FtsZ ring formation -Thus the FtsZ ring forms in center, where MinC,D are at their lowest concentrations Peptidoglycan Synthesis -Subunits synthesized in cytosol, transported out on bactoprenol (bactoprenol can change its orientation but it is hydrophobic and inside of the membrane) -Autolysins cut existing wall -Transglycosylases insert new subunits into cuts -Transpeptidases crosslink across strands CELL SHAPE DETERMINANTS -Advantages to be a rod or helix? --S/V ratio and nutrient uptake --Adherence --Motility -MreB is an actin-like protein. --Stimulates peptidoglycan synthesis. --Synthesis at several points elongates cell as a rod. CELL WALL SYNTHESIS IN COCCI -Advantages to be a coccus? (lower S/V, good for avoiding detection by immune system) --Cell packing avoids phagocytosis --Aids gene transfer? -Cocci have no mreB gene -PG synthesis starts at the FtsZ ring and produces "growth zones" *Strep has a chain of division planes *Staph has random division planes FACTORS THAT AFFECT GROWTH -Oxygen -Temperature -Osmolarity -pH OXYGEN AND GROWTH Agar medium with a few cells, Thioglycolate and Reazurin. Incubate... ***Thioglycolate binds oxygen and keeps conc low throughout the tube except near air at top -Reazurin turns pink in presence of oxygen -Strict Aerobe (grows best at high oxygen conc) -Strict Anaerobe (grows best at low to no oxygen conc) -Facultative anaerobe (can use oxygen or can not use it, not required oxygen, but tend to grow better in oxygen) -Microaerophile (Do not grow well where high oxygen conc, but they do require some of it) -Aerotolerant anaerobe REACTIVE OXYGEN SPECIES AND THEIR DETOXIFICATION -Each e- hungry Hydroxyl Radical (OH"dot") causes a chain reaction, destroying many biomolecules -Process ends when an antioxidant gives up an electron -Enzymes that detoxify reactive oxygen species --Catalase --Peroxidase --Superoxide Dismutase --Superoxide Dismutase/catalase in combination: --Superoxide Reductase Antioxidants Can Quench Radicals and Singlet Oxygen -Vitamin C "quenches" radicals by giving up or acquiring H"dot" -Energy is distributed across many atoms, reducing stress -Same principle operates for carotenoid antioxidants Staphylococcus aureus Coagulase and Pigment -Clot blocks phagocytes -Carotenoid pigment protects phagocytosed SA from toxic oxygen species -Virulence Factors (invasive forms lack coagulase that stops the spread through blood clotting) TEMPERATURE -"Cardinal" temperatures --Minimum at which growth can occur and a Maximum above which growth will not occur.... Also an optimum in which growth is highest, it is typically close to the maximum ---At minimum protein conformational changes stop and membrane gelling occurs (does not kill them) ---At maximum, protein denaturation occurs which kills them EXAMPLES OF TEMPERATURE PROFILES -Strains that have unusually large ranges are sometimes considered "tolerant." Thermotolerant or Psychrotolerant, eg.

Notes 02/24/2022 (Mobile Elements)

Bacterial Evolution -Random mutation produces variation --Selection causes favorable mutants to outgrow parents --As population increases, new variants become more likely -Many types of mutation are possible --Base changes --Insertions, duplications, deletions -What we observe are selected variants! Gene Gain and Loss -Chromosomes (blue) can gain and lose genes as they adapt to new environments ***Possible to gain genes useful in new environment or lose genes detrimental in new environment DNA Movement-Horizontal Gene Transfer -Three major mechanisms for moving DNA among cells: --Transformation by environmental DNA fragments ---Incoming DNA can be incorporated into genomes --Transduction by viruses (bacteriophages or "phages") ---Move DNA from one cell to another --Plasmids ---Move from cell to cell -Can rapidly create new genomes -May be common in complex communities, like those in biofilms Transformation -Many species can incorporate new DNA --Competence --Usually taken from a relative ---Requires considerable sequence similarity --Known to occur within biofilms -Recombination mechanisms --SSB, single-stranded binding protein --RecA, recombination (and repair) --"Heteroduplex" formation --Can result in allelic changes as well as insertions or deletions Gain And Loss Of Dna -The incoming fragment has extra material (blue strand), producing an insertion of B -Or, the incoming fragment may have less material (red), producing a deletion of B ***(50 percent of daughter cells get insertion of B) Bacteriophages or "Phages" -Infectious, selfish elements that exploit host cells --Replicate at expense of the host cell --Many kill host cells --Others can become incorporated into host genome -"Transduction" is phage-mediated gene transfer --Some phages can transmit host chromosomal pieces from one cell to other host cells GENERALIZED TRANSDUCTION -Phages can "transduce" DNA from donors to recipients -Phages may also facilitate transformation by releasing DNA as they lyse infected cells SPECIALIZED TRANSDUCTION (only transmit certain set of genes close to the site of insertion) -Temperate or "lysogenic" phages --Can either undergo lytic or "lysogenic" response --Lysogens can remain dormant in the host for many generations --Lysogens can be induced by DNA-damaging agents -Studied extensively for phage lambda of E. coli -Can transfer genes among host cells PLASMIDS -Circular Extrachromosomal DNAs -"Selfish" elements -- Parasites on host cells --May have genes that increase their horizontal transmission ---Genes that promote cell-cell transfer like pili --May have genes that increase vertical transmission ---Stable Poison -- Labile Antidote systems ---Bacteriocins kill cells that lack the plasmid -May have genes that confer advantage in some habitat --Bacteriocins --Catabolic genes to exploit more carbon & energy sources --Virulence factors to exploit hosts --Antibiotic resistance genes for medically important strains PLASMIDS AND CONJUGATION -F Plasmid --Could cause transmission of genes from one cell to another (F is for fertility) ---Horizontal Transfer/Transmission throughout a population... a mechanism for getting genes from cell to cell TRANSPOSABLE ELEMENTS -Insertion Sequences --Selfish elements that replicate at the host's expense ***a mutation persists due to mobility outcompeting ability to be eliminated -Composite Transposons ***Has two insertion sequences Transposition from Chromosome to Plasmid, then to New Cells -Composite Tn (Transposon) (insertion sequence) with XYZ copies to plasmid (COULD BE ADVANTAGEOUS, IF SO, SEE BELOW) -Plasmid with XYZ moves to new cells by conjugation The Original "R" Plasmid (R for RESISTANCE) -Discovered during a dysentery epidemic in Japan in the early 1960's -The F plasmid with composite transposons in it -Emerged due to antibiotic treatments ***Insertion sequences conferred antibiotic resistance, making a strain more difficult to treat. It is an F plasmid that has picked up two composite transposons. Integrons -Gene-sized pieces from elsewhere incorporated into the att site. -Left-most gene expressed and, if fitness increases, host expands. -Over time, cassettes accumulate as the host encounters new habitats. -Deletions can change the expression site cassette. (left-most no longer the expressed, red one becomes the expressed left-most gene) SUMMARY -Transposable elements. Can move genes among DNAs, like chromosome to plasmid or phage, or vice versa -Plasmids. Move from cell-to-cell taking genes with them -Phages. Can acquire and move genes -These mechanisms create bacteria that have new gene combinations -Discovered for antibiotic resistance, but other types of genes also involved. -Virulence factors in hospital strains -Catabolic genes in environmental isolates ***These gene transfers can have very far gene transfer reaches, not just within their own species so to say Gene Loss -Loss of genes can facilitate adaptation to a new lifestyle -Deletions are common mutations --Errors in replication can skip sequences --Errors during excision of mobile elements --Certain Recombination events -Examples: --Loss of coagulase gene in invasive Staphylococcus aureus (Golden Staph) ***Changes in lifestyle of organism can be advantageous or disadvantageous, mutations can give organism new resistances tetC, tetA, tetR -R is a regulator, A and C and part if the composite transposon gene ***pick up genes for resistance ***stepwise gain of multiple antibiotic resistance, moved from cell to cell, picking up composite transposons as S. TYPHI plasmid CURRENT PAN GENOME MODEL -Core genome encodes genes for central metabolism, molecular biology, membrane, cell wall, etc. -Variable genome or "mobilome" is comprised of mobile elements and varies among strains --Some mobile elements cross species, genus and even phylum lines -Together all genes in a related group is the Pan Genome --Any given strain has some subset of the pan genome Many Examples Involving Pathogens -Vibrio cholerae. Cholera (massive diarrhea) --Adherence pilus (allows to adhere to our guts, important property for organisms), potent toxin (second phage brings in a toxin that causes diarrhea), both phage-encoded -E. coli O157:H7. "Hamburger-associated" pathogen( https://www.youtube.com/watch?v=Ull53xGkRT4 ) -MRSA strains, "SCC-Mec", integron-like antibiotic resistance genes -Corynebacterium diphtheriae -- Diphtheria -Streptococcal syndromes -Yersinia pestis -- Bubonic plague Mobile Element Evolution -Phages, plasmid and transposons are genetic toolkits! -They can exchange genes to create novel elements -Phages that are plasmids or transposons, etc. -Also applies to eukaryotic systems -- retroviruses are both viruses and transposons Crisprs -Clustered Regularly Interspaced Short Palindromic Repeats -Pieces of invading phage DNA are inserted into the host chromosome -Expressed CRISPR RNA is used to guide cutting of invading phage genomes -Like other defensive systems like restriction/modification systems, they can be found on mobile elements -CAS are "CRISPR associated" proteins -Various CAS act at different points in the DNA capture, RNA processing and viral DNA cleavage pathway -2020 Nobel Prize to Emmanuelle Charpentier and Jennifer Doudna CRISPR-Based Gene-Editing Methods -Isolate stem cells from patient -Introduce CRISPR enzyme, guide RNA and "predesigned" DNA -CRISPR cuts at specific site, directed by the guide RNA -Cellular repair enzymes will stick predesigned DNA into the cut site -Not 100% efficient, so screen colonies for those that have the desired DNA insert

Notes 03/17/2022 (Virology)

Virus Particle = Virion -Genomes: RNA or DNA, single- or double-stranded -Surface proteins bind cellular receptors -- Tropism ***Naked Viruses are just protein and a nucleic acid of some sort ***Enveloped Virus is a protein with a nucleic acid that has been placed in a lipid envelope that has been obtained from a host membrane Entering and Exiting Eukaryotic Cells -Endocytosis for non-enveloped viruses --ATP synthases acidify vesicle, releasing nucleocapsid --Virus exit by cell lysis -Membrane fusion for enveloped viruses --Virus exit by reversal -- budding --Protease cleavages important for endocytosis and/or budding Helical Nucleocapsid Symmetry -Capsomers made of one or a few kinds of polypeptides -Few genes required to make capsid -Simple construction allows for ease of assembly ***Capsomeres bind the virus RNA and form a helical structure, most self-assemble, sometimes chaperones Icosahedral Symmetry -Icosohedrons have 20 triangular faces and 12 corners --Hexons and Pentons -"Quasispherical" (not exactly spherical, but close) -Hexon capsomers have 3 copies each of red and yellow polypeptides -Penton capsomers have five blue polypeptides Complex Viral Structures -Phage T4 -Influenza Virus (envelope virus with a bag of eight separate helical genes, halo around the outside are the surface proteins that bind to get into cells and help to get out of cells) PLAQUE ASSAYS for Counting Viruses -Phage plaques (cells grow and phages grow on cells, in agar plate) -Holes in lawns -Animal Virus Plaques -Crystal violet stains living cells Origins Of Viruses -Regressive: Pathogenic cells that lose materials? --Chlamydia as an intermediate? ---Very viral-like lifecycle --Mimiviruses, Pandoraviruses -- giant viruses that have some "housekeeping" genes -Progressive: Derived from mobile elements? (fewer genes but built from transposons, plasmids, etc.) --Some viruses are transposons --Some viruses are plasmids --Some transposons are plasmids, etc. -Genetic Toolkits Lytic Cycle -Early genes: --Host "shut-off" --Prepare to replicate -Replication phase: --Replication --Genome Processing -Late genes: (involved in formation of nucleocapsid) --Capsid --Envelope --Release Phage λ Lytic Vs. Lysogenic Cycle -Rapidly growing host cells express a scavenging protease -Protease degrades phage repressor ***Phage expresses a repressor protein LYSOGENIC -Slowly growing cells lack protease activity -Repressor blocks phage gene expression -Phage can induce later and undergo lytic cycle SOS Response and Lambda Induction -Single-stranded DNA bound by RecA protein -RecA-ssDNA stimulates LexA self-cleavage to activate SOS response (No longer a good match for DNA polymerase, then SOS turns on) -Some phage repressors also self-cleave, others inactivated by binding to either a host or phage "antirepressor" protein ***If SOS system is activated, then the phage genome is also activated by inhibition of the repressor!!! PHAGE MU (MUTATOR) -Replicates by transposition to many new locations -Occurs slowly over many generations -Mu c(Ts) transposes at a high rate at high temperatures Recombination Between Inverted Repeats Flips Sequences in Between -Changes Tail Fiber expression from US to U'S' -Increases range of host strains the progeny can infect Animal Virus Genome Classes -Herpes (dsDNA) -Parvo (ssDNA to dsDNA) -Rota (dsRNA) -"Picorna", Corona ( (+) ssRNA goes to (-) ssRNA) ***virus enters the cell and gets translated -Flu ( (-) ssRNA) -Retro (ssRNA-RT, DNA/RNA, dsDNA) -HepB (dsDNA-RT) ***RT=Reverse Transcription as it replicates Mutation Rates In RNA Viruses -Replicating RNA genomes have ~one error per ~104 bases synthesized -Genomes may be unstable if they contain too many errors --Means that RNA viral genomes limited to ~10^4 bases (or bp) ---Coronaviruses are an exception at 3 x 10^4 bases -Viruses evolve quickly --Produce enormous numbers of virions per infected cell, with an average of one mutation/viral genome --Evolution during epidemics and even within individual patients common ***No repair mechanisms so they can't be very large because this would include too many mutations... coronaviruses do have repair mechanisms! Weekly Flu Reports from CDC -Mild flu year due to social distancing and masking, if you are stopping coronavirus, you are also stopping the flu!!! Influenza Virus -Eight genomic segments --RNA pol (Replicase) & endonuclease -Flu group A has many H,N subgroups --Hemagglutinin (H) --Neuraminidase(N) --E.g., H1N1, H3N2 -Antigenic drift and shift --Mutational drift and Seasonal flu drift --Reassortment causes shifts and and Pandemic flu -Internal genes also important, but less known about their roles in host species specificity H and TROPISM -Several avian strains have transmitted weakly to humans, killing ⅓ to ½ of victims --H5N1, H7N9 -Sialic acid and tropism -Avian flu tropism can be modified to binding 2,6 by single mutations! REASSORTMENT AND THE 2009 PANDEMIC VIRUS -TRIG: Triple Reassortment, Internal Genes -The emergent virus was a further reassortment -Fortunately, it wasn't very deadly FLU VACCINATION STRATEGY HAS EVOLVED -Vaccinate children between 5 and 18 and death rates for a disease will go down due to much less transmission! Transmissibility Within A Population: Ro, R -Ro ≡ "Basic Reproductive Number". Number of new cases per current case in a immunological naive population -How to decrease R? -R depends on many factors including social practices that affect transmission (maks, crowding, hand washing, etc.), and the level of acquired immunity in the population -For flu, crowding in schools and vaccination are important ***greater than one means it can exponentially spread Optimal Strategy For Minimizing Deaths In The 2009-2010 Pandemic -Computer simulations to determine optimal strategy for achieving R < 1 . -Average pre-vaccination Ro = 1.4 -Solid line is current US population profile -Gray bars are numbers vaccinated in specific age groups -Can be done with only 61 million doses. We currently administer ~160M doses/yr ***NEED TO VACCINATE KIDS, YOUNG ADULTS, AND THEIR PARENTS Minimizing Various Costs -YLL = Years of Life Lost -"Cost" includes costs of vaccination and of disease -CV = Contingent Valuation (based on surveys) Vaccine Production -In April, identify 3 or 4 antigenic variant strains -Clone H and N genes and combine with plasmids for the internal genes of Production Strains -Grow virus in eggs --Approximately 1 egg per dose --Requires ~160 million eggs! --Kill virus by chemical treatment -Animal cell culture-based production decreases time --May produce vaccines that are better matches --"Flumist" -- cold-adapted virus (33o maximum) Flu "Imprinting" -Your first flu infection can affect how well you respond to future vaccines and infections -- Immune Imprinting --Those born before 1957 most likely saw an H1N1 type flu early in life and responded well in the 2009 H1N1 pandemic --Those born between between 1968 to 1985 most likely saw H3N2 as their first flu and responded more poorly in 2009 -Imprinting may also account for varying vaccine efficacies for different age groups -https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6891942/

Notes 04/12/2022 (Infectious Disease Emergence)

Zoonotic Spillovers -Cumulative number of outbreaks -Most pathogen spillovers are from rodent or bat reservoirs -And see: Spillover, David Quammen. 2012. W.W. Norton & Co. --In-depth coverage of many emerging infectious diseases Newly Discovered Emergent Zoonotic Pathogens -Most common characteristics: --an RNA virus --a taxonomically broad host range --use of phylogenetically conserved cell-receptor --transmissible between humans but at first causing only small outbreaks; and --occurring in regions undergoing ecological, demographic, or social change Stages of Emergence from Zoonoses -Diseases that went from 2 -> 5 after the onset of agriculture include smallpox, measles, flu, diphtheria, pertussis, mumps -Each spillover provides an opportunity for pathogens to adapt to human hosts -Factors that promote progression on pathway --Ease of transmission among humans --Isolation of human from animal reservoirs --Population size, etc. Bats As Reservoirs -20% of mammalian species are bats (~ 925 / ~4500) -Large populations, large supply of new hosts -Long lives -> chronic carriers --Low virulence in bat reservoir species, but not necessarily in other potential hosts -Wide geographical ranges -> spread -66 "known" viruses isolated from bats including: --Ebola, rabies, hanta, chikungunya, rift valley fever, many encephalitis viruses -Bat immune systems may make them less likely to develop severe symptoms Human Ecological Factors That Affect Emergence -Changes in: --Technology -- E. coli O157 in corn-fed cattle --Land use -- Ebola & palm plantation, Lyme disease & development --Human behavior -- HIV and prostitution, SARS and "wet markets" --Demographics -- Cholera, HIV and development of cities -Travel -- Global dispersal of emerging pathogens -Lack of public health controls -- antibiotic misuse, and lack of vaccination -High mutation rates can lead to adaptation to new hosts Programmed Frameshifting -TheCatAteTheBigRatAnd... EbiGraTanD... -In retroviruses, translation to produce gag-pol fusion protein --Without the frameshift => gag --With a frameshift => gag-pol --Proteolytic processing generates final protein products -Programmed Frameshift site is a slippery run followed by pseudoknot ***pseudoknot can stall ribosomes, called a pseudoknot because it can be pulled apart, common in retroviruses Programmed Frameshift Sites -In many viruses and transposons --Many retroviruses --Coronaviruses (SARS-CoV, SARS-CoV-2, MERS, some cold viruses) ---Without frameshift, protein 1a; with shift, protein 1b ----Together, 1a + 1b make up the replicase enzyme --Also in many transposases; the two products make the active transposase -Some cellular genes --E. coli: prfB (RF2), dnaX ---Autoregulation by RF2 ----High RF2 activity terminates, otherwise frameshifts past the UGA stop codon --Euks: "antizyme" gene frameshifts to produce antizyme ---Autoregulation of the gene ----High polyamine concentration promotes frameshifting, and antizyme blocks ornithine decarboxylase, decreasing polyamine production ***Termination regulates, lack of termination frameshifts HIV Infection -Mutation occurs during infection --"Infectious dose" contains a mixture of Env protein (gp120) variants -gp120 determines "tropism" --CCR5 on Macrophages -- M-Tropic --CXCR4 on T cells -- T-Tropic -Humans lacking CCR5 are unable to become infected -Infection and disease model: --M-tropic variants initiate infection --As they proliferate T-Tropic variants appear and infect T-cells --Ultimately, the loss of T-cells results in AIDS ***HAS TO BE AN M-TROPIC STAGE IN AN HIV INFECTION (initially does not infect T cells but infects macrophages, must pass through CCR5 stage to later infect T Cells) Strains Diverge into "Clades" Covid Clades -Greatest diversity in China (A, B, and their subclades) -Subclades moved to different locations and then diverged further HIV Origins -HIV has been in humans for about 100 years -HIV has two "species," each with many strains: HIV-1 (M-P) and HIV-2 (A-H) -Different virulence and transmission characteristics -Most human infections are by HIV-I M, from chimpanzee -Emerged in humans due to changing demographics in Africa? Why Did HIV Strains Emerge in the 20Th Century? -Humans have hunted monkeys and apes for many millennia --Plenty of opportunity for infection --None emerged until recently --Why? -Changes in human behaviour and ecology have occurred beginning in the late 19th century in the Congo --Cities grew for an export economy --Gold, diamonds, copper, wood, etc -Emergence factors: Economic development, travel. Prostitution in new crowded urban areas Has HIV-M Adapted to Humans? -In culture, HIV-1 M replicates best in human T cells, and SIVcpz best in chimp T cells -There are 10 fixed amino acid sequence differences between SIVcpz and HIV-1 M -SIVcpz Gag 30 mutation Met -> Arg or Lys in HIV-1 M isolates -Mutation of HIV-1 M Arg to back to Met reverses tropism -Evolution towards more or less virulence? --Want to cut transmission to lead to less virulence Tracking the HIV Epidemic in the USA -Index cases were 5 deaths in young men due --Pneumocystis pneumonia in LA. (June 5, 1981 MMWR) Pneumocystis carinii (jurovecii) --Also had other opportunistic infections -Many features suggested sexual transmission --Multiple sexual partners --More males than females in USA (but not elsewhere) -Intravenous drug use also common factor -Consistent with transmission by sex and by blood --Suggested control by using "safe sex" practices that minimize fluid exchange --Also avoid needle sharing HIV Antivirals -High mutation rate - single drugs ineffective due evolution of resistance within patients --Prob of mutation to resistance for one drug ~ 10^-4 --For three drugs, ~10^-12 -Combination therapy, "cocktail" of several --7 classes of inhibitor --Toxicity is an issue -- maintenance doses with fewer drugs (e.g., Cabenuva) Driving HIV Towards Extinction -Globally, incidence has been decreasing for about 20 years, so Re >1 --90-90-90 Program (http://www.unaids.org/en/resources/909090) --Drugs to suppress HIV levels infected patients --Condoms, Pre-exposure Prophylaxis (PrEP) for those at risk ---Truvada and Descovy (2 RT inhibitors), Dapivirine Ring (IUD RT drug) --Male circumcision decreases probability of hetersexual transmission by 60% -Complete elimination may require a vaccine --Numerous active vaccines have failed stimulate production of broadly neutralizing antibodies (bNAb) in uninfected humans --Yet, HIV-infected patients do produce bBNb --New passive recombinant vaccine based on human bNAB in clinical trials! Lessons on Emergence from Studies on HIV-1 M -RNA virus with high mutation rate that went from Stage 2 or 3 to Stage 5 by adapting to humans -Spilled over from animal reservoir and circulated for decades before detection --Changes in land use and intensity of interactions with animals? --Changes in social system including prostitution --Lack of health care and public health infrastructure allowed spread without detection -Do these factors exist in parts of the world today? Other Emergent Infectious Diseases -E. coli O157 (cattle-associated) -Lyme Disease -West Nile in the USA -Ebola -Dengue, Zika in the Western hemisphere -Acute Flaccid Myelitis (Polio-like disease) in the USA West Nile in the USA -Discovered in Uganda in 1937. Entered USA in 1999 -Populations of many birds initially plummeted but have since recovered --Enzootic in birds—humans are terminal hosts --Discovered by a veterinarian: noticed dead birds just prior to human epidemic in NYC -In 1999, the USA had ~16,000 diagnosed cases with 660 deaths. --Recently, seroprevalence was ~2.2% of US population in 2019 (~ 7.2 million people) -Most cases inapparent with age being important for disease severity -Vaccine now in clinical trials -Emergence factors: travel, trade in exotic birds? EBOLA EPIDEMIOLOGY-1 -Discovered in 1976 in infected monkeys from Uganda used in vaccine research in Europe --Produced severe illnesses in workers who handled tissues --Occasional further outbreaks in Western labs -Most outbreaks sporadic in Central Africa died out quickly due to low population density -Higher densities, altered land use, and regional travel contributed to expansion of the 2014 - 2016 epidemic -Reservoir --Non-human primates an unlikely reservoir because Ebola is so lethal for them -Many populations are dying off in part due to Ebola outbreaks --Almost certainly fruit bats -Index case thought to be an 18-month old boy, who may have contacted bat feces (see Ebola by David Quammen) 2014-2016 Epidemic -WHO recorded 28,646 cases with 11,323 deaths (~40% death rate) -Emergence factors: deforestation for palm plantation brought human settlements in contact with forest animals including fruit bats. Poverty, burial rites, lack of health care EBOLA EPIDEMIOLOGY-2 -Transmission by bodily fluids into wounds or onto mucous membranes -Infectiousness: --High viral titers (concentrations) in blood, feces, vomit. --Less virus in urine, semen and breast milk. --Saliva, tears and sweat may contain low concentrations. --Can transmit on aerosolized droplets but not if they dry out in air. --Can survive on surfaces. -Health care workers and those who handle bodies are most at risk. -Funerary practices were a major transmission factor in 2014-2016 epidemic. EBOLA PATHOGENESIS -Kills APC immune cells (macrophages, dendritic cells) --Avoids specific adaptive immunity -Massive cytokine production (cytokine "storm") --May produce a superantigen --Large vessel vasodilation and increased vascular permeability -> shock --Clotting induced by infected macrophages -> Disseminated Intravascular Coagulation (DIC) -Blood pH decreases contributes to clotting --Clotting further impairs circulation --Damaged vessels become leaky -> hemorrhaging -Necrosis in many organs --Organ failures EBOLA CONTROL -Put out the fires in Africa! --Stopping sparks that land in US and Europe is relatively easy -Higher population densities and travel have contributed to the current epidemic -Isolate those who are infected and use great precautions -Educate the public to handle bodies with extreme caution -Look for evidence of adaptation to human hosts --Variants might be more difficult to control -Be vigilant for future outbreaks Arboviruses -Arthropod-borne-viruses --Flaviviruses -- named for "yellow fever" ---Yellow Fever ----Formerly a huge killer. Now controllable by vaccine ----Walter Reed showed that it was transmitted by mosquitoes ---West Nile ---Dengue ---Zika Dengue, Zika -Dengue and Zika very closely related --Zika originated in Africa spread to Southeast Asia in the 20th century -Dengue may have occurred in Asia first, and then spread to Africa in ancient times --All became endemic in Caribbean and equatorial Americas -Reservoirs include non-human primates and humans --Stage 4 emergence --No vaccine, although one may become available -Most cases in USA are in travellers -Only Zika infects neurites --Impairs brain development Zika Virus -Discovered in Uganda in 1947 in a "sentinel" monkey --Used to detect viruses that infect our close relatives -Now has global tropical distribution --Reached Brazil as recently as 2014 --Endemic in Central America, northern South America and the Caribbean -Transmitted by mosquitoes that thrive in human habitats (Aedes mosquitoes, primarily) Cases Declining in the USA -Most cases associated with travel. Some due to sexual transmission. -Knowledge is power! Mechanism for Neurite Infection by Zika? -Dengue and Zika infect many cell types, but only Zika infects neurites, developing neurons -One difference between Zika & Dengue is a mutation in prM (S139N) (serine at 139 mutated to asparagine) -prE is the envelope protein and is important for tropism -prM is inside virion but interacts with prE -S139N interaction with prE may extend tropism? Globalization of Infectious Diseases Since 1492 -Tremendous exchanges of peoples, crops, animals and infectious diseasesInfectious diseases die out in small populations"New" infectious diseases came primarily from animals, especially domestic animals --Diseases that caused massive mortality in Native AmericansSmallpox, Measles, Flu, probably Streptococcus pyogenes from EuropeYellow fever and malaria from Africa --Diseases that transmitted from the Americas to Eurasia and AfricaSyphilis (probably) and Lyme disease (possibly) -Emerging infectious diseases that could affect developed societies today are likely to come from wild animals -- Zoonoses that Spillover

Notes 01/20/2022

Both gram-negative and positive has a cell membrane, outer membrane (distinction below, not necessarily), and a cell wall Gram negatives are red, gram positives are blue/purple Endospores form inside of cells, will eventually become free spores Light Has Wavelike Properties -Wavelength is related to color =Waves spread and bend as it passes object edges ---Diffraction -Also bends as it changes medium (air to glass or water) ---Refraction -Diffraction and refraction can blur images (!!!) Magnification and Diffraction -Magnification using convex lenses -Diffraction problematic when object size is close to wavelength ---For visible light, the limit of resolution is ~0.2 microns (~200 nm; or ½ wavelength of blue light) ***LOR-When can you no longer distinguishes 2 things as 2? (Looks like one) ---Most bacteria are micron-ish in size, so we can resolve cells but not subcellular contents Refraction and "Immersion Oil" -The speed of light changes when it enters a medium with a different "refractive index." ---That causes changes in direction that depend on wavelength ---Refractive index = ~1 for air, ~1.34 for water, ~1.52 for glass -Refraction effects can be minimized by using immersion oil ---Same refractive index as glass ---Minimizes passage through interfaces with materials that have greatly different refractive indices Brightfield Microscopy -Use the 100x for the best views -Staining used to provide contrast Cell Shapes -Coccus or sphere (cocci) -Rod or "bacillus" -Spirillum (spirilla) -Spirochete -Appendaged, coryneform, stalked, etc. ***Appendage increase surface area for more nutrient absorption (common where nutrients may be scarce) -Filamentous - fusiform ***typically multiple cells that just have not separated very well Gram Stain -Staining provides contrast -Gram-positives have a thick cell wall and no outer membrane -Gram-negatives have a thin wall and an outer membrane -You can see Gram reaction, cell shape, cell-cell interactions ***Procedure on slide! Arrangements of Cells -Streptococci: chains of cocci -Staphylococci: "grape clusters" -Diplococci -Chains of rods -Arthrobacter -- "Jointed rods" -Rods with Endospores Electron Microscopy -Electrons have shorter wavelengths and EM can resolve down to about 1 nm, so limit of resolution can be in the sub-nm range! -Subcellular features can sometimes be visualized Eukaryotic vs Prokaryotic Strategies -Eukaryotic cells: --Bigger with more genes, more complex cell structures ---Extensive cytoskeleton important for mitosis, movement and endocytosis ---Greater variety of cellular phenotypes ---Cell type diversification in multicellular organisms -Prokaryotic cells: --Smaller cells, smaller genomes & simpler cell structures ---Rapid growth ---Strains specialize -Multicellular "consortia" contain multiple species --More flexible? Smaller Can Mean Faster Growth -Growth rate depends on rate that nutrients are supplied to cytoplasm -High S/V ratio means faster nutrient supply Surface/volume (S/V): 4πr^2 ________ = 3/r 4/3πr^3 ***as cell gets larger, S to V ratio gets smaller, take longer to bring nutrients in ***as r gets large, the ratio gets smaller ***bacteria have rapid generation times due to being smaller in this way (easier to take nutrition in and process it) DNA Replication Strategies ---Replication/division cycles must provide complete copies of genomes for daughter cells ---Eukaryotic and prokaryotic chromosomes replicate with similar enzymologies but use different strategies for duplicating chromosomes ---We'll look at replication strategies here Eukaryotic Chromosome Duplication and Segregation -G phase to S phase where genome duplicates and then separation occurs and cell splitting Eukaryotic Chromosome Replication -Replication time depends on number of origins, not chromosome length Bacterial Chromosome Replication -Chromosomes are circular -Replication proceeds from the single origin to the terminus region -When the forks meet, chromosomes separate -Replication time depends on chromosome length Replication Time Can Exceed Generation Time -Growth rate depends on the medium -During rapid growth, second round of replication begins before the first one is completed -Replication has a "head start" for the next generation -Replication time can be longer than cell cycle time -Prokaryotes specialize in being small, with small genomes Bacterial Plasma Membrane -Membrane critical for nutrient import, waste export, motility, and for Energy Metabolism -Fluid Mosaic. Cytoskeleton. Proteins involved in ATP synthesis, motility, nutrient transport, protein export, others... The Proton Gradient -Metabolic reactions generate a proton gradient (cation gradient) across the plasma membrane (often greater proton conc outside than in) --Chemiosmotic potential --delta P, ΔP, dP -Used to perform several kinds of work ---Transport in (symport) (proton and nutrient come in) and out (antiport) (proton in, nutrient out) ---Flagellar rotation (used for ATP synthesis) ---ATP Synthesis Chemiosmotic Transporters in Action -"Chemiosmotic", "Simple Transport" or "Permease type" transporters -Symporters and Antiporters -K+ enters through a "uniporter" via Facilitated diffusion ***one thing straight in, nothing out ATP Synthase -Delta-P and the intracellular ATP pool are interchangeable storage forms of energy ***energy of the chemiosmotic potential is used to make ATP ***use each energy for each other (cell might need to make delta-P for transport so ATP hydrolysis will be used for this.) Neisseria meningitidis -Plasma and Outer membranes --Periplasm (large fragments brought in from porins are digested down to monomers which can later enter and pass through the inner membrane) -Symport and Antiport -ATP-dependent System (ABC) -Antiport systems for drug resistance (something comes in, drug comes out, common in antibiotic resistance) -Outer membrane porins ***allow things to pass through, 10000 daltons is the size boundary, must be smaller than this The Membrane Is Valuable Territory -Membranes play critical roles in transport, ATP synthesis, and motility, respiratory metabolic reactions -Strains can vary in how much space they devote to each of these activities

Notes 03/22/2022 (Bacterial Pathogenesis)

Controlling infectious diseases has made a lot of progress over the years!!! A Well-Defended Nutrient Source-We are a wonderful medium for microbes, something that they can exploit. We are well defended though! Terms and Concepts -Infection --Organism present --Not necessarily a disease state --Examples are our microbiota -Infectious Disease --Organism present and causing symptoms --Symptoms result from a combination of parasitic and host activities (immune system) --An example is disease caused by E. coli O157 in our intestines -Microbial Toxicity --The disease is caused by a toxin, not the organism's growth in the host (organism can be, but not necessarily present) --An example is botulism Our Normal Microbiota -All surfaces have characteristic microbiota or microflora --Differ among individuals and over time --Their growth and metabolism can affect our health -Vaginal microflora --Lactobacillus usually predominates ---Glycogen secretion by host feeds Lactobacillus (glycogen increases pH making person more susceptible) --Lactic acid decreases pH, inhibiting pathogens --Progesterone decreases glycogen in vagina ---DOI:10.1371/journal.pone.0153553 -Intestinal microbiota --Produce many of our metabolites --Short Chain Fatty Acids critical for our nutrition --Numerous other metabolites of unknown impact Intestinal Microbial Metabolism -Fermentations, syntrophies and methanogenesis span the upper portion of the Redox Tower -Nitrate Reduction (NR) allows more ATP per organic substrate --Greater range of redox tower plus complete oxidation of organic substrate --High NO3 foods like Beet Juice allow the usually minor NR group to bloom --But, NR blooms can cause gas, cramping, diarrhea -Separately, recent work shows that inhibiting nitrate reductase with Tungstate can alleviate IBS symptoms in mice --doi:10.1038/nature25172 --Patented in 2018: US10092596 Pathogenicity and Virulence -Pathogenicity is a qualitative term. -Virulence has two quantitative definitions: 1. Number of organisms required to cause symptoms. (Omicron more virulent in this case, requires less particles) 2. Severity of symptoms (Delta more virulent in this case) Interaction Surfaces -Skin vs. mucus membranes -Penetration --Adherence important! ---Wounds ---Endocytosis through mucous membranes Infectious Processes -All steps must be completed to cause disease -Virulence Factors identified as genes responsible for symptoms --Likely that "normal" flora have some that are not dangerous for healthy hosts ---Often on mobile elements --Gene transfers may create new pathogens -Virulence Factor gene expression varies among stages --Genes activated by environmental cues --For detecting the intestinal environment: ---Two-component gene regulation ----Lack of O2 ----Presence of bile ---Thermosensor ----Body temp opens mRNA structures ***exposure->adherence->invasion->colonization and growth-> invasiveness and/or toxicity->tissue damage, disease Adherence -Proteins: Fimbriae, pili, flagella --Some are on mobile elements -Capsules: primarily polysaccharide -E. coli strains differ: --Intestinal pathogens: Type 1 fimbriae and CFA pilus (Colonization Factor Antigen) --UTI pathogens: P- fimbriae (Pyelonephritis), and K antigen (capsule) Invasion Using Type III Secretion "Injectosomes" -Type III Secretion Systems (T3SS) -Injected Effectors can cause uptake and passage through cells -Various other effectors inhibit or kill our phagocytic white blood cells -Genes expressed and/or proteins activated by host environment --Pathogen must be able to distinguish intestine from submucosa Invasion by Shigella (E. coli Relative) -Left panel: --Two-component system binds bile to activate a transcription factor that results in the activation of T3SS and the IcsA adhesin -Right panel: --T3SS secreted Effectors trigger Endocytosis --Other effectors cause Actin fibers to push bacteria through cells --Shigella thus invades submucosa Colonization, Growth, Further Invasion -Nutrients: Monomers can be limiting --Secrete enzymes that digest extracellular polymers --Kill and lyse cells --Use Siderophores to bind Fe, a critical metal for all cells -Growth and Spread: --Digestive enzymes that degrade extracellular matrix --Phagocyte inhibition ---Capsules ---Effectors and exotoxins (exotoxins are things that can go out and kill other cells) ---Other extracellular surface molecules inhibit immune defenses ---Shedding LPS may confuse phagocytosis --Adherence to distant target tissue Capsules and Phagocyte Inhibition -Capsules (can mask things on the surface) --Extracellular polysaccharide (mostly) --Adherence and protection against phagocytosis -Images of Bacillus anthracis --Biohazard by Ken Alibek Exotoxin Types -Several major types, each with many examples --T3SS Effectors -- modulate host cell physiology --Cytolytic toxins -- damage or destroy host cell membranes --AB exotoxins -- kill cells --Superantigens -- misdirect the immune system Membrane-Destroying Exotoxins -Cytolytic exotoxins act on host membranes --Digest membrane lipids to lyse cells --Form holes in membranes A-B Exotoxins Enter Host Cells -A-B polypeptides linked by disulfide bond (Pep1-S-S-Pep2) -B component binds cell receptor and causes cell entry by endocytosis pVacuole acidification by V-ATPase triggers A component exit to cytosol -The A component is an enzyme that modifies a host protein or RNA Functions of A Components -"A" components kill host cells by inactivating key enzymes or ribosomes -Many are ADP-ribosylases --Attach ADP-ribose to a specific amino acid of target enzyme --Inactivates the enzyme -Other A component types: --Highly specific Proteases that digest target proteins --Highly specific Nucleases that cut rRNA, inactivating ribosomes Diphtheria Toxin -Diphtheria toxin, an "ADP-Ribosylase" -Inactivates EF-2, an essential translational factor --Protein synthesis is inhibited, killing the cell -The diphtheria vaccine is a toxoid -- inactivated toxin Botulism Toxin -Clostridium botulinum -Botox Blocks acetylcholine release -SNARE proteins mediate vesicle/PM fusion -A components are proteases that cleave SNARE proteins -LD50 ~ 10-7 g. Tetanus Toxin -Cuts SNARE proteins in inhibitory interneurons --Blocks Glycine release -Overactivation of muscular contraction Enterotoxins -Exotoxins that function in the gut -Cholera toxin: An A-B toxin -ADP-ribosylates a G protein, locking it in active form (prevents from hydrolyzing GTP, so always active!) --Causes overactivity of adenyl cyclase -Too much cAMP blocks Na+ uptake -Too much Na+ in intestine causes anions to flow out -High intestinal ion concentration causes fluid to rush into the intestinal lumen Superantigens -Exotoxins made by many bacteria and viruses -Non-specific and massive T-Cell activation -Misdirects the immune system and causes tissue damage -Systemic activation of T-cells can cause shock Classic Exotoxin Examples -Chart in Slides Salmonella typhi Virulence Factors -In slides Salmonella enterica, var. typhi Genome -Pathogenicity Islands -Virulence Plasmids -Rings 1&2 are S. typhi, Rings 3&4 are shared with E.coli. -Rings 5&6 are S. typhi specific. -VF genes are dark blue. Mobile element genes are pink MOBILE ELEMENTS ON A S. TYPHI PLASMID -In slides

Notes 04/19/2022 (Bacteria & COVID)

Overview of Coronaviral Cellular Infectious Process -Envelope Spike protein binds receptor (ACE2, Angiotensin Converting Enzyme 2) -gRNA (genomic RNA) translated to make replicase from ORF-1a and ORF-1b --Requires programmed frameshift! -Continuous synthesis produces 'anti-genomes' -Discontinuous synthesis produces (-) strands with common 5' and 3' ends and deletions -Copying (-) sg-RNAs produces other sg-mRNAs -Virions assemble in Golgi-derived vesicles --Cleavage of Spike protein during exit -Vesicle fuses with PM to release virion Programmed Frameshifting -... THE CAT ATE THE BIG RAT ... ... HEB IGR ATX ... -Translation of gene 1 (replicase gene) --Without the frameshift => protein 1a --With a frameshift => 1b --Together, they make the replicase enzyme -Programmed Frameshift site is a Slippery run followed by pseudoknot Transcription of the gRNA Template -Complete synthesis reproduces 'anti-genome' -Discontinuous synthesis produces Minus-strand sgRNAs -Transcription of sgRNAs produces sg-mRNAs Spike Cleavage Important for Membrane Fusion -Amino-terminal receptor binding (rb), and carboxy-terminal fusion part of same Spike polypeptide -Following binding, rb must get out of the way to allow for conformational changes that fold carboxy termini and promote fusion -Cleavage activates Spike SARS-CoV-2 Relatedness to Other Viruses -Similar to viruses from bat (mostly) and pangolin -Unique feature at the spike cleavage site -Probably not bioterror related --Not close enough to any known virus to be an engineered version -Could it be an accidental the release of a new virus, or a "natural" spillover? Variation in Spike Cleavage -Processing differs between SARS-CoV and SARS-CoV-2 --CoV-2 efficiently cleaved by a wider range of human processing proteases --Could covid infect more easily than SARS? Did SARS-CoV-2 Come From a Lab or a From Wet Market? -Recently, five samples of SARS-CoV-2-related viruses detected in the Wuhan wet market --Photos show that the stall that tested positive had recently housed Raccoon Dogs, a species sold in the market that can host CoV-2 -In another article, CoV-2 strains A and B in China are too different to be related to the same spillover -Simplest explanation is that CoV-2A and CoV-2B viruses separately transferred from bat to Raccoon Dogs that were sold in the market -Bats are likely reservoir, Raccoon Dog is likely "amplifier" or "intermediary" host What Determines Mutation Rate? -Deleterious mutations are more common than beneficial mutations -Mutation rate should be at an "acceptable" level, determined by evolution -Number per genome is an important factor: ~ one per new genome -Long genomes must have repair systems to fix errors and damage Error and Damage Repair Systems -Proofreading during DNA synthesis --Exonuclease removes errantly incorporated nucleotides -RNA viruses do not proofread and have high mutation rates Coronaviruses Proofread! -Relatively long RNA genomes, 3 X 10^4 bases -But, they have an exonuclease activity (remove bases from an end) -Errant bases get removed and new RNA synthesized in its place -Coronavirus mutation rate is lower than that of the flu What Might This Mean? -Most chain-terminating antivirals do not effectively terminate coronavirus replication, probably because they are removed by proofreading -But Remdesivir might escape proofreading?? -Molnupiravir doesn't stop replication and is not proofread, but makes it error-prone -Paxlovid is a protease inhibitor An Explosion of Bacterial Phyla! -Massive sequencing projects are uncovering many new groups, most of whom have never been cultured (OP1, 2, etc. from "Obsidian Pool") -Also, some phyla have been divided, based on sequence data Not Just Organism Names But Genes -Most work to date done using 16S rRNA gene as a tag to get the name of the organism --Organisms identified by comparing sequence to large database of 16S sequences from known organisms -But genomes can be highly variable even within "species" --Genus-level IDs may be more meaningful -Characterizing genes has the potential to greatly improve our understanding of the roles of microbes in environments Microbial Diversity -Selected members of the Pseudomonadota (aka Proteobacteria), Gram Positives, a few others -Bacteria that are either commonly encountered in this course's lab, or that have some considerable importance to biology Phylum 1: The Pseudomonadota -Aka the Proteobacteria, aka "Purple Bacteria and their Relatives." -This large, diverse phylum subclassified into α, β, γ, δ Proteobacteria Pseudomonas Genus -G- rods, oxidase +. Respiratory. Nutritionally versatile. Ubiquitous. -Entner-Doudoroff (ED) pathway. -Spoilers of meat. -Cystic fibrosis patients and burn victims. -Xenobiotics. ***Famous for making fluorescent pigments Acetic Acid Bacteria -Acetobacter (overoxidizers) --Vinegar, gluconate, vitamin C -Gluconobacter (underoxidizers) --Kombucha -Cellulose & surface Redox Series for Carbon -Ethanol->Acetic Acid ***Most reduced to most oxidized state Neisseria and Other G- Cocci -Gram negative cocci or "coccobacilli." -Oxidase positive, penicillin sensitive: --Neisseria (always cocci), Moraxella, Kingella. Neisseria meningitidis, N. gonorrhoeae ---Gram (-) diplococci -Roseomonas -- pink colonies, "secondary metabolites" --Colonize mucous membranes -Oxidase negative, penicillin resistant: Acinetobacter. --Reservoirs in soil and humans/animals. --Colonize skin --Associated with reduced allergies. Early childhood exposure may be protective (GGBG) The Enterobacteriaceae (Enterics) -G -, oxidase -, facultatively anaerobic rods -Nutritionally versatile Fermentative Three Phyla of G+ Bacteria -Bacillota (formerly Firmicutes): --Lactic acid bacteria --Staphylococci --Spore formers -Actinomycetota: --Mycobacteria --Coryneforms and jointed bacteria --Actinomycetes -Mycoplasmatota: --Wall-less bacteria - Mycoplasmas Arrangements of Cocci -Strepto- -Diplo- -Staphylo-cocci -Sarcina (cubes of cocci) Glycolytic Pathways -Homofermentative: --Streptococcus, Lactococcus, Enterococcus, Some Lactobacillus -Heterofermentative: --Leuconostoc, Some Lactobacillus -Bifidobacterium (not classified as LAB, but ecologically related) Characteristics of the Lactic Acid Cocci -α-hemolysis (greening) -β-hemolysis (true lysis) -γ-hemolysis (no lysis) Staphylococcus -Staphylococcus, Micrococcus (non-fermentative), Stomatococcus (slimy) --Salt tolerant -S. epidermidis vs. aureus --Carotenoid pigment and coagulase -Macrococcus - similar to Staph but with large cells and Oxidase +, no commensal Bacillus -G+, aerobic, sporeforming rods, ubiquitous in soil. --Various strains are sources of extracellular hydrolases used in industry and household. Many produce antibiotics. Other Bacilli -Widespread, most strains eat dead organic matter -Have important roles in the rhizosphere, modifying soil and making inorganic nutrients available to plants (subtilis, cereus) -Some are pathogens (anthrax, thuringiensis) -Many are commonly cultivated in undergraduate microbiology labs (LOL) B. thuringiensis Toxin -Bt spores used as insecticides in garden ponds to kill mosquito larvae -Bt toxin gene spliced into crop plant genomes to kill insect larval pests Clostridial Fermentations -Obligate anaerobes. Widespread in soil, aquatic habitats, animal intestines -Saccharolytic - ferment sugars to a variety of organic alcohols and acids --Contribute to intestinal VFAs -Proteolytic -- ferment amino acids, producing a variety of foul-smelling acids, amines and mercaptans --Associated with intestinal dysbiosis Actinobacteria: Mycobacterium -Mycolic acids and "Cord Factor" --"Acid Fast" stain Acid Fast Stain for Mycobacterium -Red = Acid-Fast, Blue = Something Else

Notes 02/17/2022 Syntrophies and Phototrophies

Syntrophies are the linked metabolisms of anaerobic species (two organisms that are very close to each other and require each other, ICM) Phototrophies are important processes for large groups of microbes as well as for plants Interactions Among Anaerobes -Anaerobes often specialize in one small section of the redox tower -End product of one organism is the food for another --Lactic acid bacteria (LAB) and Propionic acid bacteria in Swiss cheese is one example --Syntrophy is a very close kind of metabolic interaction Acetogenesis and Methanogenesis -Converts CO2 to Acetic Acid (Acetogens) and Methane (Methanogens) (Use H2 as electron source and CO2 as electron sink) --Important processes in Anoxic environments Acetogenesis and Methanogenesis -Acetogenesis: 4 H2 + 2 CO2 => CH3-COOH + H2O CH3-COOH also produced by syntrophs -Methanogenesis: 4 H2+ CO2 => CH4 + H2O CH3-COOH => CH4 + CO2 (Acetoclastic methanogenesis) --All of these processes are favorable. All are coupled to proton pumping. --These processes consume H2 -- they require an H2 source H2 Production by "Syntrophs" -NAD and FAD are regenerated by making H2 -Acetyl-CoA thioester is phosphorylyzed to make an anhydride (SLP) --Then used to make ATP --The acetyl group becomes acetic acid -Can work only if H2 is consumed in a syntrophic relationship Syntrophic reaction coupling (THROUGH PARTNERSHIP) makes reactions much more favorable!!! Syntrophy Summary -Coupled growth of two organisms that have complementary metabolisms -H2 production: "Syntrophs" produce H2 from fatty acids and alcohols --Unfavorable under standard conditions -H2 consumption: Syntrophs partner with H2 consumers like acetogens, methanogens, nitrate reducers or sulfate reducers --H2 + CO2 → CH3-COOH (Acetogenesis) --H2 + CO2 → CH4 (Methanogenesis) --H2 + NO3 → NH3 (Nitrate reduction) H2 + SO4 → S, H2S (Sulfate Reduction) -All of these linked "metabolisms" are favorable Organotrophies in Anoxic Communities 1. Extracellular hydrolysis 2. Fermenting "pioneers" make VFAs from sugars and amino acids (acidogenesis), FAs ignored ***VFA=Volatile Fatty Acids 3. Syntrophs degrade FAs and VFAs, partnered with H2 consumers (=> CH4, acetate) 4. Acetoclastic Methanogenesis from acetate ***2 CH2O => CH4 + CO2 (Can be harvested as "Biogas") Rumen Ecosystem -Rumen Digestion occurs in stages -Some of the VFAs will become carbon nutrition for the animal -Nitrogen nutrition obtained by digestion of microbes in the acidic stomach (abomasum) -Cattle on grain diet --Grain (starch) causes LAB bloom, acidifying rumen and beyond --Favors E. coli O157 ***Inside Rumen -Hydrolysis of polymers -Fermentation of sugars and amino acids -Syntrophic Acetate and Methane production -Acetoclastic Methanogenesis ***Abomasum, the acidic chamber Herbivores with a Large Cecum -Herbivores that lack a rumen -Cecum positioned after the acidic stomach, so Nitrogen nutrients not harvested --Some animals eat feces to acquire N -Human gut has no rumen and a vestigial cecum (appendix) Lean vs. Obese Flora in Mammals -http://www.nature.com/news/bacteria-from-lean-cage-mates-help-mice-stay-slim-1.13693 (2013) -Flora from lean and obese twins transplanted to axenic (germfree) mice -Energetic considerations and obesity --Lean flora more rich (more species, more metabolic pathways) --Not just flora but diet important, too. ---Lean flora can rescue obese mice but only if on a normal or low-fat diet --No simple link to amount of VFAs ---Lean flora better at carbohydrate fermentation and VFA production ---Obese flora better at amino acid fermentation --Other data shows that VFAs affect our own fat metabolism The Microbiome and Health -Can we use the microbiome to promote health? -Biomarkers for diseased states -- Dysbiosis --Associations between specific organisms (or genes) and diseases --Not necessarily causative, but biomarkers for diagnosis or to monitor treatment --High Prevotella-to-Bacteroides (P/B) ratio predicts weight loss for dieters -Treat the microbiome to change patient outcomes --Changes in diet -- High Fiber Diets, less processed foods ---Recent Review: https://dx.doi.org/10.3390%2Fnu12030859 --Fermented Foods and Probiotics --Fecal transplants Probiotics and Fermented Foods? -"Fermentation" is perhaps the oldest method of food preservation --Many involve Lactic Acid Bacteria, Acetic Acid Bacteria, Bifidobacteria or Yeast --See "Foods" in 3.Topical Resources on our Team site -Probiotics do not need FDA approval --Most are LAB or Bifids --Recent Review: https://doi.org/10.3389/fcimb.2019.00454 Fecal Transplants Delivery by Intubation or a set of "Crapsules" -Considered experimental and to be done as a last resort for serious diseases such as Crohn's disease or C.diff (Clostridium difficile) infections -Unanswered Questions: --Potential pathogens in the transplant? --Might someone with serious GI disease be harmed by implanting an entire flora adapted to someone else's GI tract? --Unanticipated interactions between original and transplanted microbiota? The Phototrophic Lifestyle -Light for making ∆P / ATP --Low energy electrons in photopigments capture photons, boosting their energy levels --High energy electrons enter ETC for proton pumping -May or may not be autotrophic --Autotrophy vs Heterotrophy --Depends on environment and organism -Two general types, oxygenic and anoxygenic PS --Some types of PS are more amenable to autotrophy than others --Several subtypes of anoxygenic PS Photosynthetic Reaction Centers -Specialized chlorophylls that gather energy -Have specific places on the Redox tower --For example, P870 at +0.5 for purple bacteria ***Cyclic electron flow in anoxygenic PS Anoxygenic PSIn Purple Bacteria -Photoheterotrophy -- Assimilate fixed carbon --Cyclic electron flow can be used to make ATP -Photoautotrophy -- CO2 fixation --Inorganic compounds can serve as e- donors --Partially inhibit ATP synthase and transport? -Many Purple Bacteria can use Aerobic Resp using organics or H2 as e-sources --Use PS if O2 or e- donor absent Oxygenic PS -1st photosystem can reduce NADP. Can also operate cyclically to make ∆P and ATP. -2nd photosystem uses H2O as a source of e-. Also, produces O2 as a byproduct. -Frees cell from reliance on specific inorganic e- donors. Earth History -Oxygenic PS originated somewhere between 2.4 - 2.9 bya --"Great Oxidation Event" -Massive Cyanobacterial radiation -Fe precipitation (red bar) from oceans delays O2 buildup Banded Iron Formations -Aka "Red Bed Rocks" -Formed 2.4 to 1.8 bya -Iron oxides that precipitated from oceans -Caused by O2 and resulted in oceanic iron depletion Types of Energy Metabolism -Organotrophies: --Fermentation (ABE) --Aerobic Respiration (ABE) --Anaerobic Respiration (AB) --Syntrophies -Lithotrophies: --All are respirations --N,S,Fe,H2 oxidation (AB) --Acetogenesis (B) --Methanogenesis (A) -Phototrophies: --Anoxygenic PS (AB) --Oxygenic PS (CyanoB,E) Freshwater Systems -Organic pollution: Aerobic respiration of the organic matter occurs first, algal blooms follow, etc. -Inorganic nutrient pollution: Algal bloom occurs first, then aerobic respiration, etc. -Eutrophication: classically inorganic nutrients feed algal blooms followed by O2 depletion. --"Dead zones" in near-shore environments --Some algae can also produce toxins (Red tide)

Notes 03/29/2022 (Clinical Micro)

CLINICAL MICROBIOLOGY -Accurate diagnoses are critical to health delivery -Important for research into future treatments Several Routes -Microbiological assays for organisms --Culturing methods --Antibody reagents --Detect DNA or RNA -Immunological or "serological" route looks for antibodies to a pathogen --Certain chronic infections (e.g., TB, HIV, hepatitis viruses) --Past exposures (epidemiological research) Sampling and Culturing Approaches -Collect samples using swabs, urinalysis cups, syringes -Place either directly on Petri dishes or into liquid enrichment broths, and then Petri dishes -Media is typically "Selective" and/or "Differential" --Many media are partially selective, and differential among those organisms that do grow Types of Media for Different Samples -CHART IN SLIDES -Selective: Inhibitors like salts, metals, dyes, bile, specific antibiotic mixtures -Differential: pH indicating dyes -Blood Agar: Enzymatically Digested Soy Extract plus 5% sheep's blood -Selective and differential -7.5% salt selects G+ cocci -pH indicator plus Mannitol. Mannitol fermenters turn agar yellow --Coagulase test: Mix cells with plasma, look for clotting MacConkey-Lactose Agar (MC) -Used to detect "fecal coliforms" -Selective and differential -"Peptone" with salts, bile, lactose and a pH indicator --Bile inhibits non-Enterics --pH indicator detects acids from lactose fermentation 1. Enterobacter VP fermenter (Mostly alcohols) 2. Escherichia Mixed Acid fermenter 3. Staphylococcus Inhibited by bile 4. Salmonella Lactose negative Tests for Further Identification -Gram stain --Color, shape, arrangements of cells --Presence/absence of spores -Test kits for groups of organisms --Enterotube for those that grow on an enteric agar -A battery of additional tests --Oxidase test for Cytochrome C --Catalase test for ability to breakdown H2O2 -E. coli is a Gram - rod, oxidase -, catalase +, acid and gas anaerobically, acid from lactose under aerobic conditions Tests For Gonorrhea Neisseria gonorrhoeae -Gram (-) diplococci from a urethral or vaginal swab -Presumptive -Modified Thayer-Martin (MTM) Agar. --CA with vancomycin, colistin, trimethoprim, nystatin --Inhibits almost everything except Neisseria -Oxidase reagent turns purple if cells have Cytochrome C -Confirmatory for Neisseria gonorrhoeae Antibiotic Disk Diffusion Assay -Filter disks impregnated with known concentrations of specific antibiotics Antibody-Based Tests -Culture-based methods have distinct advantages --Tried and true methods --Access to pure cultures allow further characterization ---Antibiotic resistance, tracking outbreaks in hospitals, research on pathogens, etc --But culturing is slow, and diagnosis should be fast -Antibodies can be highly specific for surface molecules --Many tests are easy to perform and give rapid results --Many can be automated --Do not give information on antibiotic resistance, etc -Monoclonal antibodies --Permanent sources of antibodies that all have the same antigen-binding characteristics Monoclonal Antibodies -Antibody production in an animal is "polyclonal" -Key is to create permanent cell lines that produce individual, "monoclonal" antibodies -"Hybridomas" that can be maintained indefinitely Fluorescent Antibody or Immunofluorescence Tests -Direct --Antigen with surface receptors will bind to an antibacterial antibody that is labeled with fluorescent dye. -Indirect --A bacterial cell with surface antigens is reacted with a monoclonal antibody made in a rabbit, it binds and that reacts with a secondary antibody that is anti-rabbit labeled with fluorescent dye. ***Monoclonal antibodies typically made in some sort of animal -Made in Rabbit -Made in Goat --Syphilis, autoimmune diseases Rapid Antibody-Based Strep Throat Test -Insert swab into port -Add dye-labeled antibody in solvent -Unlabeled antibodies in the stripes capture "analytes" bound to labeled antibody -Takes 5 minutes Covid Antibody Test -Same principle as for the Strep test -IgM (early antibody) and IgG (later, blood form) captured at distinct stripes Direct ELISA Enzyme-Linked Immunosorbent Assay (ELISA) -Antibody to the pathogen fixed to a microtiter plate. --Captures antigen. -Detect with antibody to the pathogen that has an enzyme attached to it. --Enzyme makes a colored product -Very sensitive because each enzyme processes many substrate molecules -Hepatitis viruses, rotavirus Indirect ELISA -Attach antigen to the surface of the microtiter plate wells --Capture antibody from patient's serum -Detect antibody with anti-human antibody linked to an enzyme --Colorimetric assay -Rapid screen for anti HIV antibodies -An example of the "Immunological" or Serological Route because it is a test for antibody Western Blot -Run serum from patient on a gel -Detect pathogen-specific protein with an antibody linked to either a radioactive or enzymatic tag -Confirmatory for HIV infection New HIV Tests -"Bifunctional antibody" to both Red Cells and HIV HIV-containing samples agglutinate Red Cells Now used to screen donor blood -Binds HIV -Binds RBC -There are home test kits that can detect IgA (secretory AB) in saliva Agglutination Principle -Bifunctional antibody can bind HIV and RBCs to cause the mixture to agglutinate Nucleic Acid Hybridization Assays -Can detect toxin and antibiotic resistance genes! -Some assays, like PCR, are sensitive but slow expensive --Covid-19 tests rarely complete in under a day -Research seeks to make assays more reliable, and cheap -Some assays allow "multiplexing", the use of multiple probes Isothermal "Strand Displacement" Amplification -Nonspecific amplification of any DNA -One temperature instead of thermocycling (usually between 50-72o) -A small amount of DNAse added to create "nicks" -"Strand-displacement" DNA synthesis by Bst (Bacillus stearothermophilus) DNA polymerase at nicks -Can amplify genomic materials in a few minutes -Can also be made specific by using primers "SANDWICH HYBRIDIZATION" PLATFORM -Have a biotin-labeled (detection probe) probe and a dye-labeled probe (detection probe) -Have a pathogen target DNA sequence -Streptavidin magnetic bead binds Biotin and whatever is bound to it (biotin, pathogen DNA, and dye-labeled probe) --WASH WITH A MAGNET Emerging DNA-Based Tests -Rapidly evolving technologies --Luminex "Verigene" system: --Automated nucleic extraction and isothermal amplification --Hybridization to probes in an array ---"Multiplexed:" Allowing simultaneous screening for multiple pathogens Smart Bandages -Bandage reacts when bacterial "hole punching" exotoxins release dyes in tiny capsules in the bandage -Capsules can contain dye (signal) and/or local antibiotic (treatment) -Could minimize replacing bandages on burn victims "Humanized" Monoclonal Antibodies -Antibodies from animals can make great chemotherapeutic (animals that have a humanized immune system, so you can use them indefinitely with many uses) -Our immune systems will develop immune responses to antibodies from other animals --After several doses, we can undergo anaphylactic shock when receiving another dose --New technologies greatly reduce that risk -Humanized AB made by splicing human AB gene clusters into animals --Monoclonal antibodies developed from such animals are humanized --MAB drugs (e.g., adalimumab = Humira) --Also used to make Immunologic therapeutics - "mab" suffix drugs. -Adalimumab (Humira -- blocks TNF-α) -Trastuzumab (Herceptin -- blocks HER2) Humanized MABs as Passive Vaccines -Against SARS-CoV-2 (REGENERON) -Against HIV Cancer Immunotherapies -Humanized MABs that block PD mediated immunosuppression (closes this door and allows the T cell to kill the Tumor Cell, autoimmune issues is a risk of this) -PD-1 = "programmed death"; PD-L1 = PD Ligand -Examples are pembrolizumab (Keytruda) and nivolumab (Opdivo) Survival Rates Increasing -PD-1 blockade is either anti-PD-1 or anti-PD-L1 mab -Ipilimumab blocks CTLA-4, thereby up- regulating Tc cells -X is a variety of "targeted therapies", depending on specific mutations in cancer cells

Notes 02/08/2022

place thin layer of cells on petri dish and wait, place Filter paper infused with antibiotics, if organism is sensitive, the antibiotics will inhibit it... (wider zone, smaller concentration of antibiotic required to inhibit it) HEAT STERILIZATION (causing death) -"D" is the time required to kill 90% -Depends on species and medium -N = No * 10-D -Scientific notation: For 1 billion cells treated for 6D, 109 * 10-6 = 103 ***Semi-log scale so never approaches 0, Exponential decrease line, higher temperature is the faster they die Steam Pressure Sterilizer "Autoclave" *Heat to 121 degrees Celsius and hold for many D -Canned goods are commonly treated for 12D -Suppose the initial number of Clostridium botulinum spores is 10^3: 10^3 * 10^-12 = 10^-9 1 can in 10^9 (a million) may have a surviving spore To multiply, add the exponents: 10^12 cells x 10^-5 = 10^7 10^2 x 10^7 = 10^9 PASTEURIZATION -Designed to kill pathogens and to extend shelf-life of milk --TB, salmonella, typhoid, diphtheria, giardia, norovirus, others --Filtration now becoming common -- very long shelf life -Raw milk: --About 1% of milk consumed in USA is raw milk --Federal law bans interstate sales and states enact their own laws on consumption ---NC allows use for pet foods but not for human consumption --To promote removing federal restrictions, in 2016 several elected officials got sick after drinking raw milk ELECTROMAGNETIC RADIATION -D10, or "decimal reduction dose" --Organisms can differ in sensitivity --Deinococcus -- extensive DNA Repair -UV light in laboratory hoods --Can protect against pathogens --Protects against DNA contamination in labs using PCR --No longer recommended by CDC, and not permitted for new labs at NIH --Airflow and filtration systems can provide safer ways to prevent release ***UV LIGHT IS DAMAGING TO DNA! -X-rays and Gamma rays --Ionizing radiation (OH-dot radical) --Medical supplies, foods ***Powerful enough to just blast molecules into ions! IRRADIATION PRACTICES -Foods can also be irradiated, but are not well accepted by the general public --Listeria in packaged meats would be a great target -Irradiation does not make foods radioactive! FILTRATION -Depth filters (for Clarification), Membrane Filters (for sterilization), Nucleopore Filter (For Sample Prep with Electron Microscopy) CHEMICAL CONTROLS -Disinfectants --Lysol, clorox ***Harsh, so use on inanimate surfaces -Antiseptics --60 - 80% alcohol --Hydrogen peroxide ***Can use on skin!!! -Chemotherapeutics: --Target molecular features unique (or nearly so) to pathogens --Antibiotics --Synthetics ---"Growth Factor Analogs" -Classifications --Prefix: Bacterio-, fungi-, viri- --Suffix: -static (stops growth), -cidal (kills them, cells remain the same but exponential decrease in viable cells), -lytic (causes them to lyse) --Bacteriostatic, for example CHEMOTHERAPEUTIC TARGETS -Most examples here are antibiotics -Growth factor analogs: --Quinolones bind topoisomerases --Sulfonamides resemble para-aminobutyric acid (PABA), required for folic acid synthesis CHEMOTHERAPEUTIC SPECTRA -Not every antibiotic is active on every type of organisms --Different spectra speak about the activeness of antibiotics on certain organisms β-Lactam Antibiotics -Penicillin, cephalosporins and carbapenems are "β-lactams --Penicillins have the thiazolidine ring at "X" --Cephalosporins and Carbapenems have different "X" rings -Natural and semi-synthetic antibiotics --Vary the "R" group --Make acid tolerant (-> oral drug) --Combat resistance --Extend spectrum (broad spectrum with some R groups, other more narrow) --Change tissue specificity (some R groups will cross blood-brain barrier, some will not) -β-lactamases! (Bla, for short) β-Lactams Inhibit Cell Wall Transpeptidases -4-membered Beta-lactam ring -Beta-lactams mimic the transition state of the reaction -Permanently binds transpeptidase active sites Mycobacterium and "Mycolic Acid" -Mycobacterium tuberculosis is the most famous species, but strains are widespread in nature -MA is a very thick waxy coat outside of the peptidoglycan wall -Strong barrier to many chemicals, increasing ability to survive being phagocytosed -If mycolic acid synthesis is blocked, our white cells can kill the cells -Lipoarabinomannan (LAM) is a lipoglycan that inhibits bacterial killing by phagocytes and is an antioxidant Growth Factor Analogs Inhibit Synthesis of Critical Metabolites -Used to treat Tuberculosis --Mycobacterium tuberculosis --NAD required for extensive Mycolic Acid synthesis --Isoniazid inhibits NAD synthesis Antiviral and Antibacterial NUCLEOSIDE ANALOGS -Terminate chain elongation -Can be toxic to hosts --Nuclear and mitochondrial polymerases -Remdesiver for Coronavirus? ANTIBIOTIC RESISTANCE -Methicillin Resistant Staph Aureus, MRSA CA-MRSA and HA-MRSA Strains: USA300, etc. VISA (Staph Aureus that is partially resistant to vancomycin) and VRSA (Staph aureus that is fully resistant to vancomycin) in HA-MRSA RESISTANCE MECHANISMS -Outer membrane can be a barrier to some antibiotics (common in gram negatives) -Inactivation of antibiotic β-lactamase (Bla) (acetylases attach chemical group that target it for destruction or do not allow it to bind to its target) -Alteration of target (Mutation that might no longer bind something like RNA polymerase) -Development of Resistant Biochemical Pathway (PABA, blocking folic acid synthesis, organisms resistant to this acquire a gene for a symporter where they can bring folic acid in so they no longer have to synthesize it) -Efflux (pumping out of cell) (ANTIPORT) ISOLATION OF ANTIBIOTIC PRODUCING STRAINS -Spread thin film of Staph aureus -Also spread soil extract -Isolate soil bacteria that inhibit Staph -Spread thin film of isolate on lower part and allow it to grow -Then, streak test strains up to the isolate -Allow growth, look for inhibition NEW CHEMOTHERAPEUTIC AGAINST FtsZ? -PC190723. Identified in a large chemical library -Causes FtsZ to be randomly distributed throughout cell -Protects mice against lethal doses of Staph aureus -Haydon et al. (2008). Science 321:1673 Resistance mutations found during drug development. Current research seeks derivative molecules that act on resistant strains. RATIONAL DRUG DESIGN -Find binding pockets based on structures and inferred binding properties -HIV protease inhibitors COMBINATION THERAPIES -Take multiple drugs at the same time -For HIV, use combinations known to be currently effective 1 drug: (10^9 virions *10^-5 = 10^4 resistant virions) 3 drugs: (10^9 virions *10^-5 * 10^-5 * 10^-5 = 10^-6 virions) --It is unlikely that organisms will simultaneously develop resistance to multiple drugs -For bacteria, however, some plasmids can carry genes for resistance to multiple antibiotics -Augmentin --Clavulanic acid, a Bla inhibitor, plus Amoxicillin Antibiotic Development in Decline -Future Strategies: --Target toxins rather than organisms? -Decrease selective pressure for resistance --Strain-specific drugs? -Minimize damage to microbiome -Would requires better diagnostic tests --Now under development (more later)

Notes 02/22/2022

More distant things are on the phylogenetic tree, the more sequence differences that will be present! ***archaeans small bush is interpreted to mean that they have not evolved much (they are very ancient) (extremophiles-extreme environments is how they were though to originate) Earth's History ---Endosymbiosis: mitochondria and chloroplasts ---Origin of Life and early events Dating Ancient Rocks -"Parent-Daughter" isotope ratios -Potassium 40 decays to Argon 40 with a half-life of ~1.25 billion years --Used to "bracket" sedimentary rocks -U-Pb dating of zircons (ZrSiO₄.) within sedimentary rocks gives precise dates (URANIUM LEAD DATING) ***Old lava that has solidified (only potassium remains due to gaseous argon) (old lava has a greater ratio of argon to potassium) (POTASSIUM ARGON DATING) Evidence for Early Life -Stromatolites (formed from cyanobacteria which are photosynthesizers) (often close to shore) ( they were photosynthesizers, but not oxygen photosynthesizers, so progenitors to cyanobacteria) -Microfossils -Chemical "signatures" of life in keragen (sugars do not survive that length of time but converts to a substance known as keragen) The Science of Life's Origin -"Bottom Up" Approach --Environments that have high energy compounds like ---H2, and have CO2 for carbon fixation --Spontaneous formation of organic compounds --Deep sea volcanic "vents" have such properties -"Top Down", Molecular Fossils: --Genome, molecular biology and biochemistry Life Began Near Volcanic Vents? -Top Down --Molecular Fossils" Approach to Look Back in Time --LUCA = Last Universal Common Ancestor --"RNA World" -Bottom Up Approach --spontaneous organic compound formation --H2, H2S, CO2, spontaneous thioester formation (R-S~CH=O} --"Bottom Up" Approach to Deduce Pathways for Pre-life to Life ***simple things to organic compounds Primordial Soups vs. Autocatalytic Cycles -Spontaneous assembly of macromolecular structures from components from a "Primordial Soup" --"Order out of chaos" (THIS MODEL IS UNLIKELY) --Assemblies inherently unfavorable and, therefore, very low probability (BUT OVER TIME THEY HAPPEN) -Autocatalytic Cycles of energetically favorable reactions that build concentrations of nutrients that spontaneously assemble into protocells --"Order out of order out of order" (energetically favorable pathways) --Requires an environment(s) that provide energy-rich building blocks that self-assemble -- oceanic volcanic vents! (spontaneous formation of thioesters at volcanic vents) --Autocatalytic cycles "Autocatalytic" Cycle -Side reactions mean that less that two "A" produced per cycle -So, overall efficiency must be > 50% to build cycle -Finally, collection on a cationic surface, perhaps a clay, could facilitate intracycle interactions. -A side reaction product(s) might catalyze a specific reaction in the cycle --X stimulates B ->C -Because it is cyclic, stimulating one reaction elevates all intermediates Modified Reverse TCA as an Autocatalytic Cycle for OAA -Energy source is thioesters that drive C-C bond formation -Wächtershäuser: this occurred on mineral surfaces near volcanic vents "Vitalizers" (Protoenzymes) -Anions accumulate on cationic surfaces in vents Membrane -Fatty acids could result from a reversal of Beta-Oxidation in an FeS world -Can assemble into vesicles with drying, rehydration -What size fatty acid will function as a semi-permeable membrane suitable for a protocell? --PNP is polynucleotide phosphorylase. It synthesizes RNA from NDPs. -14-Carbon fatty acids function well in this assay. (ADP Can get in, but protease can not, so RNA can form inside these vesicles!!!) ***Cell membrane keeps good stuff in and transports bad things out (semi-permeable) RNA World Hypothesis -"Top Down" or "Molecular Fossils" Approach --Life passed through a stage in which RNA molecules served as genomes and as enzymes --Protein and DNA came later... DNA <==> RNA ---> Protein DNA --In DNA, deoxyribose increases genome stability --Allows for larger genomes --DNA metabolism is a branch of RNA metabolism ---Fraction of NDP pool reduced to make dNDP pool RNA -RNA may have preceded both protein and DNA -RNA can serve as both genome and enzyme --RNA viruses --Ribozymes --Ribosomes -RNA plays central roles in molecular biology: --mRNA, tRNA, rRNA PROTEIN -Protein enzymes more stable and better catalysts than ribozymes -Protein replaces most of most of enzyme structures, except for catalytic roles that amino acids cannot perform --Nucleotide cofactors: NAD, FAD, ATP, CoA-SH (energy part still done by RNA!, these were in an RNA world!!! If a protein world, amino acid would just completely do the job) (rely on metals or nucleotide cofactors to do catalytic things when amino acids cannot) RNA-Based Enzyme Cofactors -NAD, FAD as e- carriers -ATP as energy carrier -Co-A with an SH for thioester formation -S-adenosylmethionine (SAM) for transfer of methyl groups RNA First, Then DNA too -Simple "prebiotic" chemistry makes ribose, not deoxyribose -RNA is unstable, DNA is not -DNA nucleotides derived from RNA nucleotides, never vice versa RNA => DNA -Cells have enzymatic pathways to make ribonucleotides (ATP, UTP, CTP, GTP) -A subset of NDP pool is converted to dNTP for DNA synthesis --Never the other way around -Conclude that DNA metabolism is an add-on to RNA metabolism Origins of Mitochondria and Chloroplasts -Endosymbiont hypothesis --Cytoskeleton and endocytosis -Mitochondria acquired once to create modern eukaryotic cells --Lost from many euk cells... -Chloroplasts acquired multiple times in different photosynthetic organisms Gene Exchanges -Gene transfers among organelles and nucleus --Most mito and Cp proteins now encoded in nucleus, proteins imported into organelle --Chapter 8 of "I contain Multitudes" -Not all euks have mitochondria --All euk cells have nuclear genes derived from mitochondria -Aerobic Euk cells have mitochondria --perform Aerobic Respiration -Anaerobic Euks have organelles derived from mitochondria --Perform tasks other than Aerobic Respiration -One of many copies of an organelle lyses -Some of its DNA becomes incorporated into nuclear genome -Signal sequences for mito or cp import evolve -Also some transfer to mito Hydrogenosomes in Anaerobic Protists -Many polyphyletic anaerobic protozoa have them, suggesting multiple derivations from mitochondria -These organisms may live syntrophically with H2 consumers like methanogens. E.g. in the guts of cockroaches and termites Endosymbiotic Events Among Phototrophs -Primary endosymbiosis --Cyanobacteria enters protist to become Alga with Chloroplast with two membranes --Occurred at least 3x -Secondary endosymbiosis --Protist engulfs algal cell, which becomes chloroplast with three membranes -Tertiary Endosymbiosis --Secondary Alga enters Protist to become Cp with four membranes Model for Eukaryotic Evolution - the Big Picture -Proto-eukaryotic cell may --Have lived by engulfing other cells by endocytosis -An associated (engulfed?) respiratory bacterium became endosymbiotic giving rise to the mitochondrion --This permitted a radiation of organelle types, some of which lost respiratory functions ---Hydrogenosomes, mitosomes -Chloroplasts were (are?) derived from primary and secondary endosymbioses.

Notes 01/13/2022

No asymptomatic cases of original SARS, so did not spread as much A virus with spokes out, G16022A (going to be Europe variant) (call it A.1.1.155) (variant of interest would be B.1), A412G (going to be Austrailian variant), etc. (Most changes will probably just be neutral mutations, but these built up over time) (some will be detimental) ---site of emergence always has the greatest number of variety in mutations ---variant of interest if it beings to have deleterious effects on the spread (more contagious) ------track the variant of interest, if it is truly not due to chance, then it becomes a variant of concern (VOC) (given greek letters for naming, delta etc.) R0 also known as R naught is the reproductive number-average number of new cases per singular case. (Tells you about the rate of spread!) Re (R sub e, or sometimes just R), R naught happens when no one is immune in a population and it is spreading at highest rate... Re happens when people begin to become immune but sometimes people still just use R naught. Re is the effective R at a given moment. New Cases over Time graph -R naught=1 leads to a flat line -When R is even a little greater than 1 it is an exponential increase, higher the number is the faster rate of increase -For public health, want to reduce R naught to below 1, here it will begin to die out (slope down). -How many people to vaccinate to get R naught to 1? 1-(1/Rnaught)=Fraction of the pop vaccinate ---Initial COVID strain had an R naught of 3, but delta variant was greater than or equal to 6, for omicron it is estimated to have an R naught of 12-18 (measles is 18) Doubling Rate (assumes R is greater than 1 so cases are increasing) -Plot New cases over Time (Days) (Incidence Plot) --N=Nnaught*2^n... when n=10, one case becomes 1024 roughly. (little n is the number of doublings) ---Doubling time is important! Second month, that 1024 becomes over a million (10^6) (two months one case if it doubles every 3 days) ----next would be 10^9 in another month (so the whole world!) -----short doubling times mean a very rapid spread (exponential) Transmission is important for a pathogen! A successful variant is one that transmits effectively. THE MOST IMPORTANT!!! -What factors affect transmission? --Better replication within an organism, produces more particles that get into the air, infecting more people (ADAPTATION TO NEW HOST) (likely first kind of mutation, ability to bind to receptor (want to be tight)) (increased transmission leads to selection, outcompeting original) ---Next comes Immune Avoidance mutations (once people start getting vaccinated, this is a way of mutating to avoid the antibodies) (antigenic drift and people lose some of their natural immunity) Virulence (number of particles required to cause a symptom) -More common DEFINITION is the seriousness of the illness (how many people are hospitalized, etc.) THIS IS THE DEFINITION --Diseases tend to become less virulent over time as they evolve because that facilitates their transmission. Transmission is the key to selection, not virulence. (not guaranteed to become less though, mutation can cause to be more virulent and transmissable) ---Virulence makes people less willing to transmit, something like HIV does not need to become less virulent because it is stealthy, COVID is even stealthy with asymptomatic. ----Host behaviors can decrease virulence (masking, etc.) Eradication of the Virus -Mass vaccinations over a short period of time (did this for smallpox) (no non-human reservoirs) --worry about COVID going into another host species ---Everyone gets it and builds immunity ***locations of receptors not in the lungs (lower respiratory system) is a hypothesis for omicron (only in upper respiratory system)

Notes 02/15/2022 Microbial Respiration

Respiratory Metabolism -Inorganic electron sources (H2, HS, NH3, Fe2+, etc) (typically on outside of the cell) -Organic electron sources (sugars, fatty acids, amino acids, etc) (typically on inside of the cell) Electron Flow in Energy Metabolism High-energy organics like Sugars, also H2 NAD (electron carrier) Low energy organics like Pyruvate ***FERMENTATIONS ARE THE ABOVE STEPS Sulfur compounds, CO2 Nitrogen compounds Iron (at low pH) Oxygen ***ELECTRON ACCEPTORS IN RESPIRATIONS COVER THESE STEPS ***As electrons (H) flow downhill, ATP gets made Energy Metabolism -- Respirations -Metabolism requires electron donors and acceptors equally --Every e- removed from a donor must eventually find an acceptor -NAD+ regeneration in fermentations: --Place H on carbon products of metabolic pathways; pyruvate, for example ---Limited oxidation of carbon sources --No external e- acceptors required to regenerate NAD from NADH -Respiration is an alternative way to regenerate NAD+ from NADH --All types require some abundant, exogenous electron acceptor ---O2 in surface or aerated subsurface environments ---NO3, SO4, CO2, (& others) in other environments ---Complete oxidation of carbon sources to CO2 possible --Not all environments contain suitable acceptors Respirations Require an Electron Transport Chain (ETC) -The Proton Motive Force (PMF, Proton gradient, Chemiosmotic potential, ΔP) is created by (1) proton pumping and (2) proton consumption on the inside -ΔP used to perform work: ATP synthesis, nutrient import, waste export, flagellar rotation ***We want proton consumption occur on the inside of the cell so that the cell can generate ATP, do not want the proton acceptor to be on the outside! ***Cytochrome C OXIDASE: Test to divide the bacterial group into two major subgroups: Fermenter or not? Proton Gradient Functions -Small molecule import (symport) and export (antiport) -Flagellar rotation -ATP Synthesis Respiration and Carbon Oxidative Pathways -Oxidative pathways produce NADH and FADH2 for the ETC -Glycolytic pathway produces 2 NADH per glucose -Because pyruvate not needed as an e- acceptor, it can be fully oxidized to CO2 --Thus, pyruvate is oxidized by the TCA Cycle, producing a lot of NADH and FADH2 -Also, fatty acids can be oxidized --β-oxidation of fatty acids also produces a lot of NADH and FADH2 Important Dehydrogenation Reaction Series Type 1DH: RCH2-CH2R + FAD ⬄ RCH=CHR + FADH2 OH H | | 2. Add H2O at the double bond: RCH-CHR 3. Type 2 DH: OH O | || RCH-CH2R + NAD ⬄ RC-CH2R + NADH2 Decarboxylation Produces a Thioester Coenzyme A contains a thiol: CoA-SH Oxidative decarboxylation of α-keto acids: (in slides photo) High energy thioesters can do two things: -Substrate Level Phosphorylation -Form C-C bond by transfer of the carbonyl to another organic coumpound TCA or Citric Acid Cycle -3 Carbons in, 3 CO2 out -NADH and FADH go into electron transport chain -> ∆P -Also produces a GTP from each pyruvate -Total of 38 ATP per glucose (FAR MORE THAN FERMENTERS DUE TO FULLY OXIDIZING THE PYRUVATE, FERMENTER ONLY YIELDS 2) -Fermenters DO NOT use TCA for energy production ***GREAT FOR RESPIRING ORGANISM AND TERRIBLE FOR A FERMENTER Beta-Oxidation of Fatty Acids (convert a lot of fatty acid into hydrogen, end product is acetyl coA which can go into TCA cycle) -Fatty acids and alcohols, and hydrocarbons -NADH and FADH dump e- into ETC to make ∆P -Acetyl groups from acetyl-CoA go into TCA for further oxidation -> more ∆P -Fermenters cannot do this except under special circumstances Overview -In respiration: --Carbon can be fully oxidized to CO2 ---Glycolysis and TCA for sugars ---FA oxidized via β-oxidation --NADH2, FADH2 oxidized to NAD, FAD by dumping H into the ETC --Protons pumped as electrons flow downhill towards the final acceptor -> dP Fermentative vs Respiratory Lifestyles -Is fermentative growth slower than respiratory growth? (YES, but not in the short run!) --Membrane space: nutrient transport vs. electron transport --Fermenter can quickly change an environment. E.g., reduce pH --Respirers more fully exploit resources if they have access to suitable e- acceptors -Thus, dominance can be determined by e- acceptor availability (HERE IS THE HUGE POINT) -How about partitioning carbon into energy and growth? --Respiration, roughly 1:1 (get a lot of energy out of every carbon molecule, every sugar, that goes in) (one sugar goes into energy and one goes into biomass) --Fermentation, typically >10:1 (Not nearly as efficient!!!) --No large organisms are known to survive by fermentation alone -How do fermenters generate proton gradients? (USE ATP SYNTHASE A RUN IT BACKWARDS FROM WHAT WE TYPICALLY THINK OF) (In respiring organisms, metabolism generates the deltaP and uses that to power respiration) Types of Energy Metabolism -Organotrophies: --Fermentation (ABE) --Aerobic Respiration (ABE) --Anaerobic Respiration (AB) -Lithotrophies: (also respirations just not using organics) --N,S,Fe,H oxidation (AB) --Methanogenesis (A) ---All are respirations) -Phototrophies: --Oxygenic PS (CyanoB,E) --Anoxygenic PS (AB) Aerobic vs Anaerobic Respiration -E- acceptors must be oxidized compounds --I.e., must be able to accept e- --O2, NO3, SO4 work well. H2O, NH3, H2S can't work (REDUCED COMPOUNDS CAN'T WORK) -O2 is lower on the e- tower than any other sink, therefore it allows the greatest energy production (O has higher electronegativity, allows it to be lower on e-tower and for aerobic to produce greater respiration) --In direct competition, aerobic typically wins -Anaerobic respiration occurs in the very many environments that lack O2 --The type of anaerobic respiration that can occur depends on the availability of suitable acceptors --All else equal, in direct competition the acceptor that is lower on the tower typically wins --Acceptor abundance also shapes outcomes among competitors (having a lot more sulfate than say, nitrate, could cause a better outcome in this competition) Examples of Anaerobic Respirations -Electrons come from donors farther up the Electron Tower like organics or H2 --Intestines, complex organic (CO2 is oxidized acceptor and CH4, and Acetate is the Final reduced product) (Organism is Methanogens, Acetogens) --Seawater, Terrestrial Aquatic systems, --Terrestrial --Intestines (NO3 is oxidized acceptor and NH3 is the final reduced product) (Organisms are Enterics like E. coli) Nitrate Reduction & Denitrification -E Coli can grow through respiration, but does not have Cytochrome C (so will be negative for this on test) ***Nitrate Respiration also known as Nitrate Reduction... Denitrification occurs in soil, this is bad because when you put nitrate out for plants to use, they convert it to N2 which plants can not use) Electron Flow in Sulfate Respiratio High-energy organics and H2->Low energy organics like Pyruvate->Sulfur compounds ***Occurs in lots of environments at the bottom of the seafloor ***SO4 activated with ATP, then accepts 6 e- from ETC (and 8 H+) to produce 3 H2O and an H2S Fermentation can occur in aerobic or anaerobic environments, use organic molecule as e- acceptor, and make an organic molecule such as pyruvic acid Aerobic Respiration occurs in aerobic conditions with the final hydrogen being molecular oxygen (O2)... the reduced product formed is H2O. SUMMARY SLIDE!!! Travel farther down the electron tower with more ATP being produced!!! Donor should always be above the acceptor so that energy can be generated as you travel downhill (cannot go uphill!) Lithotrophic Metabolism -Usually external electron donor=Inorganic molecule -Terminal Electron Acceptor is usually O2 And CO2, SO4, NO3 under anoxic conditions Lithotrophies -"Respiratory" because its uses an Electron Transport Chain -> ∆P --Litho- uses inorganic compounds as e- sources -We need to consider autotrophy (carbon fixation) (sometimes facultative) to produce the fixed Carbon needed for growth --Autotrophy requires CO2, ATP and "reducing power" (Hydrogen) ---NADH, NADPH, reduced ferredoxin (FdH2) ***obligate autotrophs have to fix carbon Using H2 as electron donor, can use almost any compound as electron acceptor! Electron Donors and Acceptors -Electron donors must be reduced compounds --H2, H2S, NH3, Fe2+ -Electron Acceptors must be oxidized --O2, Fe3+, NO3, SO4 --There may be many intermediate compounds ... ---NO3, NO2, NO, N2O, N2 H2 Oxidation (H2 => 2e- + 2 H+) -Occurs at interface between anoxic and oxic environments -Some H2 produced in sediments diffuses into water column Fe Oxidation (Fe2+ => Fe3+) -pH is a log scale, so 4 pH units is 10^4-fold difference in H+ concentration! -Extreme H+ concentration gradient drives proton pumps backwards, with H coming out of NADH Dehydrogenase onto NAD => NADH --Reverse electron flow -Example of non-standard energetics ***very strong deltaP so that some protons will literally flow backwards, if they go forward it allows for proton consumption -------this is where electrons go uphill to get to NAD (EXCEPTION TO DOWNWARD ELECTRON TOWER TRAVEL) Summary -- So Far! -Fermentation -- anaerobic, chemoorganotrophic processes --Colonize newly available, organic-rich habitats -Respirations --Occur for many organotrophies and all lithotrophies --Aerobic. Dominate surface environments because of high energy yield --Anaerobic. Very common where NO3, SO4, etc. occur in anoxic systems. Energy yield depends on how far e- travel down the e- tower -Lithotrophies --All are respiratory and can be aerobic or anaerobic --Common where high-energy inorganic materials available. ---Erosion of rocks, volcanoes, release of NH3 during organotrophies, ---In many organic-rich, anoxic systems where H2 is produced!

Notes 03/31/2022 (Epidemiology)

Infectious Disease Control and Prevention -Understanding and controlling transmission TERMINOLOGY -Sporadic (something not always present, but comes up from time to time) -Endemic (always cases of this disease, usually low prevalence though) -Outbreak (a cluster of something, could extend to the next term) -Epidemic (continues to spread) -Pandemic (on multiple continents) --For diseases in animals: Zoonoses, Enzootic, etc. Incidence Plot -For tracking rates of spread ***Short Times Intervals: Weeks, Months Cumulative Incidence Plot -For tracking total victims ***Short Times Intervals: Weeks, Months, Prevalence Plot -For tracking improvements or setbacks Modes of Transmission and Epidemic "Signatures" -Common Source Epidemic: Food toxicity (very short) Food Infections (days or weeks) -Direct Person-to-Person: Respiratory acquired STDs Epidemic Disease Seasonality and Annual Periodicity -Winter, spring: Respiratory acquired (Measles, Scarlet Fever) -Summer, fall: Vector-borne (Lyme disease), water-borne (polio) -Multi-year cyclicity: Population resistance differs among years Transmission During Phases of Acute Infectious Diseases -Incubation -Prodromal -Illness -Decline -Convalescence Reservoirs, Transmission Modes, Interventions -Reservoirs: Soil, Water, Animals (Zoonoses), Human Carriers (acute and chronic) --Vaccines, Water & Wastewater treatment, Monitor herds, Education, Quarantine --Food preparation laws (Certified Food Protection Manager system in NC) -Transmission modes: Exit, travel, entry --Direct Person-to-Person: Respiratory acquired, STD's ---Vaccines, behavioral interventions, education. Handwashing, condoms ---MMR, DTaP, HBV, HPV, & other vaccines --Indirect Person-to-Person ---Fomites ----Education, washing, disinfectants, hand sanitizers, etc. ---Fecal-oral route. Vehicles: Food, Water ----Water and sewage treatments, food preparation laws ---Arthropod vectors ----Insecticides, drain swamps, eliminate standing water near homes, etc. -The 7 "F"s: Feces, food, fluids, fingers, flies, fomites, fornication The Host Community -Natural immunity --Genetic variation among and within host species ---Population-level genetic variation within species --Variation in: ---Cell receptors for bacteria and viruses ---Cytokines, receptors for cytokines ---MHC (HLA) molecules -Population size, social parameters --Size --Density ---Crowded schools, dorms and dining halls, barracks -Herd Immunity --Dependent on the level of past exposure either to pathogen or vaccine ***Large populations lead to more transmissions, as new individuals are being born in and crowded areas form Coevolution of Hosts and Parasites -Parasites: Successful parasites MUST spread to new hosts --Variants that spread more readily can replace parent strains -Hosts: Host must survive and reproduce despite infection --Selective pressure to resist, eliminate or adapt to pathogens -Parasites grow, mutate, and thus evolve more quickly, than hosts do --If increased virulence increases transmission, that is selected --If decreased virulence increases transmission, that is selected --How might virulence affect transmission?? Host Behaviors that Affect Transmission Influence Parasite Virulence -Inverse correlation is for diseases caused by different pathogens -Also evidence between strains of a given pathogen species. E.g., E. coli, Staphylococcus aureus in hospitals -See Evolution of Infectious Diseases by Paul Ewald (1994). Nosocomial Infections -Ecology is different from General Community --Pathogens evolve to exploit the hospital environment -Hospital personnel acting as reservoirs and vectors --Patient resistance low --Invasive procedures -Surveillance --Screen personnel flora --Test for Antibiotic Resistance --Genomic analyses Human Ecology Can Affect Transmission Cycles -Clean water -> impaired transmission. Parasite adapts to cause low-intensity, chronic infections -Contaminated water, -> active transmission cycle. EMERGENCE of high-impact, virulent strains ***Genetic changes modulate virulence within potential pathogen populations ***If you make transmission easy, you show favor more virulent strains! Predicting Host-Parasite Coevolution -Reducing transmission favors low-impact, chronically infecting strains --If you can't move to a new house, don't destroy the old one! -Easy transmission can favor highly virulent strains --Don't worry about destroying your current house, you're moving! -So, we can reduce pathogen virulence by reducing transmission --Can we apply that to Covid-19? -Hosts can also adapt through evolution, but evolution is a painful process! (more susceptible genotype is killed off, others live and resistance is obtained) ***more transmission=more virulence (damage), because it is easy to transmit! Parasite-driven Evolution of the Human Genome -There have been strong selective pressure for genes that confer disease resistance --In our past, ~½ of all children died before reproducing -~100 genes known to affect susceptibility in humans --Many but not all are in immune system genes --Often, there are trade-offs: increased childhood survival but decreased lifespan or quality of adult life ---Autoimmune and other diseases ***Reproduction for transmission matters, as long as this stays the same, the host can die soon! (WE JUST WANT TO REPRODUCE) What Will Happen to SARS-CoV-2 (Covid-19 Virus)? -Unlikely to just disappear -The highly lethal 1918 pandemic flu was outcompeted by less virulent strains -Could SARS-CoV-2 evolve to a less virulent form? --Reduce probability of transmission ---Isolate the symptomatic ---Masks, social distancing etc. ---Vaccinate ---Should favor variants that are more easily transmissible and less likely to cause symptoms CDC and US Public Health -Notifiable Diseases. Dangerous, highly communicable, controllable, common -Investigation may be required to locate sources, reservoirs, vehicles or carriers --EIS (Epidemiological Information Service) -- "Epi Aids" --Contact Tracing. Track and control epidemics. Often help overseas. -Interventions aimed at eliminating source, education, vaccination, etc. -Research to identify pathogen and determine mode of transmission SLEUTHING: E. COLI IN SPINACH, 2006 -Of the first 6 cases, 5 were associated with raw spinach -Further reports also linked to spinach (retrospective: associative, hypothesis forming). -Look for E. coli in spinach samples (prospective: hypothesis testing). -Track contaminated spinach back to producers. --Recall Spinach. -Test farm soils and look for sources of contamination. -Manure use is regulated. In NC, land cannot be planted for 120 days after application. MODERN PUBLIC HEALTH MEASURES -Clean water and food --Potable water plants --Wastewater treatment --Food preparation laws -Vaccines and policies on their use --Threat: Anti-vax movement -Antibiotics and policies on their distribution --Threat: Overuse/misuse -Access to health care -Pandemic preparedness Water as a Source of Contagion -Role of water as reservoir not understood until mid-19th century Cryptosporidium - a Protist -Reservoir is cattle --Gets into surface waters -Not killed by standard chlorination treatment -About 1 outbreak per year in in the US -1993 outbreak in Milwaukee infected ~400,000 people, > 100 deaths -In Forsyth County (includes Winston-Salem): --Our water drawn from the Yadkin River and stored in small reservoir --Cattle farms upstream --Forsyth County does not collect water following heavy rainfall

Notes 02/10/2022

How organisms get energy from the environment and use it for growth! Plants are obligate autotrophs... they have to synthesize their own carbon! (fix carbon) Periodic Table with Electronegativities -Electronegativity reflects "affinity" of atoms for electrons in bonds --Functional groups can also differ in their affinity for electrons -For example, electrons will "flow" from Carbon to Oxygen -The fundamental basis of metabolism -- electron flow from a low affinity to high affinity group can be used to make ATP ***Fluorine has the highest electronegativity, makes it difficult to remove from a bond Electron Flow 1. High-energy organics like Sugars, also H2 (low electronegativity) 2. NAD (electron carrier) 3. Low energy organics like Pyruvate 4. Sulfur compounds 5. Nitrogen compounds 6. Iron (at low pH) 7. Oxygen ***As electrons (H) flow downhill, ATP gets made (starts at 1 and flows down to 7) ***Low EN at the top, Increasing EN as e- flow downward, High EN at the bottom Nicotinamide Adenine Dinucleotide, NAD+, and Redox Reactions -Loss of e- = oxidation; Gain of e- = reduction. Oil-Rig, Redox Reactions -NAD+ participates in "dehydrogenation" (DH) reactions: ***accepts H -Reduced forms are NADH + H+, NADH2, NADH -Oxidized forms are NAD+, NAD Dehydrogenases and Redox RXNs -NAD must be regenerated after it is used to oxidize a compound! ***two enzymes, one that reduces NADH and one that oxidizes this for back to NAD+ Energetics and Redox Rxns ***pyruvic acid->lactic acid is an essential reaction -Is the reaction favorable from left-to-right or vice versa? -Consider this as two half-reactions... ***Whichever one has a higher e- is where electrons are going to want to flow! (that tells you which way is favorable) Which has a higher EN, NAD or pyruvate? Said another way, a which e- donor, NADH or lactic acid have higher energy e-? The E- Tower and Redox Rxns -Half-reactions organized according to energy of the electron(s) -Oxidized form on left, reduced form on right -These values were determined for standard conditions -Mix-n-match metabolism! -Electron acceptors are as important as electron donors! ***ELECTRONS WILL WANT TO FLOW OFF OF NADH AND GO ON TO PYRUVATE ***NADH2 has a HIGHER ENERGY ELECTRONS than lactate Types of Energy Metabolism Organotrophies: Fermentation (ABE) Aerobic Respiration (ABE) Anaerobic Respiration (AB) Lithotrophies: N,S,Fe,H oxidation (AB) Methanogenesis (A) (All are respirations) Phototrophies: Oxygenic PS (CyanoB,E) Anoxygenic PS (AB) Electron Flow in Fermentation 1. High-energy organics like Sugars, Amino Acids 2. NAD 3. Low energy organics like Pyruvate ***H moves onto NAD, Then onto pyruvate ***Does not involve O2 or an Electron Transport Chain ***The organic food substance (sugar, amino acid, etc.) is both the electron donor and, after processing, the electron acceptor Substrate Level Phosphorylation (SLP) Series 1. Oxidation of aldehydes makes high-energy thioesters: 2. Phosphorolysis of thioesters (SLP) to make acid anhydrides: 3. Phosphotransfer to make ATP phosphoanhydrides: NAD Regeneration and Fermentation Products -Volatile Fatty Acids and Alcohols (VFA) -Short Chain Fatty Acids and Alcohols (SCFA) -End product of one metabolism can be the e- source for another ***Used in our gut to generate energy... Dairy Microbiology -Lactic Acid Bacteria (LAB) --Streptococcus, Lactobacillus, Lactococcus, others... --Mucous membranes --Colonize mammary glands and inoculate milk -Fermentation generates many desirable flavors --Yogurt --Cottage cheese --Aged cheeses ---Secondary fermentations --Diacetyl, acetoin Other Useful Fermentation Products -Our intestinal microbiome --Volatile (Short Chain) Fatty Acid production (VFAs, SCFAs) --Clostridiales (and others) in our intestines --Butyrate, propionate, acetate products are nutrition for us -Alcoholic fermentation by yeast --Pyruvate + NADH => CO2 + ethanol --Note: yeast have mitochondria and can also respire under oxic conditions Redox Series for the Carbon Atom Reduced forms on the left Oxidized forms on the right ***Methane is the most reduced form of carbon, carbon dioxide is a very oxidized version of carbon Oxidation liberates energy (can make ATP)-> Carbonyls Aldehydes are on or derived from sugars, α-keto acids from amino acids Atomic Balance -Some carbons become more oxidized, others more reduced -Carbohydrates and some amino acids work very well --Intermediately oxidized carbonyls -Atomic Balance: Ratios of all elements are conserved Where Do Fermentations Occur? -In environments that: --Have complex organic mixtures, especially if anoxic (no O2) --Check out the "Winogradsky Columns" in the lab Our intestines --Produce VFAs like acetate, propionate, butyrate --Hundreds of other small molecules, most of whose human significance is not yet known ---~40% of our metabolites are made by our microbiota --Clostridium botulinum in lake sediments (and potentially in canned goods) -As we will soon see: --Fermenters can rapidly exploit an environment --"Pioneer" species like the Lactic Acid Bacteria (LAB) ---Act very quickly, converting sugars to acids ---Modify their environments, inhibiting competitors ---Many practical implications, in cheesemaking or pickling, as examples Energy Metabolism -- Respirations -Electron acceptors are as important as electron donors! -NAD+ regeneration in fermentations: --Place H on carbon products of metabolic pathways; pyruvate, for example ---Limited oxidation of carbon sources --No external e- acceptors required to regenerate NAD from NADH -Respiration is an alternative way to regenerate NAD+ from NADH --All types require some abundant, exogenous electron acceptor ---O2 in surface environments ---NO3, SO4, CO2, (& others) in other environments ---Complete oxidation of carbon sources to CO2 possible --Not all environments contain suitable acceptors

01/18/2022 Lab Notes

***Did not go to lab today, but general overview ***Create Team Folder with a Google Doc and take picture of Petri Dishes, take through microscope lens, etc. First Goal -- Single Colony Isolation Always keep Plates "Agar-Side Up" Always Label the Top or Agar Side ***PUT LID ON BOTTOM, LABEL AGAR SIDE Good Streaking Technique! 1. Fan cells over a wide area ---Sterilize loop in flame; cool in agar 2. Cross previous streak once and fan 3. Repeat..... Not So Good Technique... ---Only lift lid up a tad while working ---Make sure you sterilize!!! ---Do not waste space!!! Incubate for 24 - 48 Hours Only! ***24 is IDEAL! -After cells have grown to form dense colonies, cells begin to die -The higher the temperature, the greater the death rate -They should survive well at room temp but not necessarily at 37 degrees -Leave your plates in a stack in your aluminum Team Tray Brightfield Microscopy -Staining used to provide contrast Sukaina and Ellie are my teammates

Notes 03/15/2022 (Molecular Biology & Gene Regulation)

-Cells respond to: -Carbon/energy sources --Glucose is preferred -Nutrient availability --Amino acids, vitamins, etc -Environmental cues --"Quorum Sensing" --Host environments ---Temperature ---O2 concentration ---Bile (presence is in the gut, absence of oxygen would also mean in the gut) Proteins Bind and Slide -Protein domains recognize sequence using weak bonds --Proteins are often "dimers", so sites are inverted repeats -Proteins bind and slide -- tight binding for regulation depends on sequence -Regulation is often on a spectrum -- not "on or off" (repressors can turn down ability to bind) Searching for Sites -1D searching is faster than 3D searching --Bind and Slide --But proteins can be somewhat limited to search small regions --Intersegmental transfer and hopping -Together, these strategies allow rapid site binding with minimal protein Information Theory -How large should a regulatory site that occurs once per genome be? -Large enough to be unique, and no more. --Probability of a site appearing by chance = 1 / 4^n nucleotides, where n = number of nucleotides --Bacterial genomes range from 0.5 - 10 million bp, with an average of a few million. 4^10 = 1,048,576 4^12 = 16,777,216 -So, in general regulatory sites found once per genome have the same info as 10 to 12 specific nucleotides --Can have more nucleotides when some are not specific -Sequences that regulate multiple gene systems (regulons) have less information (TYPICALLY FOR MAJOR THINGS LIKE DNA REPAIR, AEROBIC RESPIRATION, ETC.) --Shorter, and/or some positions partially specific (not too small because would be binding wrong places, not too large so there is no selective pressure) -Bottom line: just enough information to ensure that they are not too common Promoters -Promoters recognized by the sigma factor -The more closely a promoter matches the consensus, the more likely it is to be recognized -Genes vary greatly in their transcriptional activity (Transcription factors are expressed at low levels, some other genes are expressed at high levels) Polycistronic Messages and Operons -Genes often clustered together with the same function, often have a common promoter, regulatory regions near the promoter, and operons (cluster of genes all co-regulated) and regulons ***all have similar structures and respond to the same regulatory signal Translation -Initiation occurs at "Shine/Dalgarno", spacer, then AUG ...GGAGGAxxxxxxAUGxxx... The S/D must be "unstructured" (can not have a hairpin structure that includes this sequence) -Aminoacyl-tRNA concentrations, and this amino acid concentrations, can determine translational rate Polysomes -Multiple ribosomes on the same message -- polysome -The average message is translated ~90 times ***This is the rate-limiting step! Making mRNA is not very expensive, but the mass production of the protein is expensive!!! There is definitely some regulation at the translational level. Chaperones and Protein Folding -Most proteins fold on their own, others need chaperones ***Increase in temperature, Increase in denaturation, TURN ON GENES FOR CHAPERONES Protein Secretion Mechanisms -Several systems that use different signals and membrane channels -Type I: Signal Recognition Particle, SRP, for integral membrane proteins -Type III secretion systems for translocation into other cells (taking protein that is synthesized in the cytosol of bacterial cell and shoves it into the cytoplasm of another cell, potentially an animal cell) Type III Secretion Systems -Used to inject proteins into other cells --Toxins that can kill cells --Effectors that modify host signal transduction pathways -Free-living bacteria inject toxins to kill predatory protists -Pathogens inject proteins into host cells that can kill or modify activities ***Effectors = Toxins, Signal Transduction Modulators ***Affect host cells in various ways Gene and Enzyme Regulation -examples on slides Allosteric Enzyme Regulation -End product feeds back to inhibit the first "committed" step -Step that consumes ATP and/or valuable carbon molecule ***might not bind at active site, but instead at allosteric site to regulate allosterically Regulation of Transcriptional Initiation -Negative regulation (repression) --lac operon --Induction or "derepression." --overlap the promoter or bind downstream of it -Positive regulation (activation) --mal operon --upstream of promoter Catabolic (Degradative) Systems -Operon-specific repressor and/or activator (e.g. lac or mal operons). -No feedback enzyme regulation. -Catabolite repression (activation). "Glucose effect." (next slide) Catabolite Repression (Activation) -"Glucose effect" (both glucose and lactose present) -Catabolite Activator Protein (CAP) binds to cAMP to act as a transcriptional activator -Glucose inhibits cAMP synthesis, preventing activation Positive and Negative Regulation of the lac Operon -Full expression requires both induction (derepression) and activation by the CAP-cAMP complex. -No Lactose and No Glucose allows ++ activation -Plus Lactose and No Glucose is ++++++++++++ activation -No Lactose and Plus Gluclose is + activation -Plus Lactose, Plus Glucose is ++ activation Summary of Catabolic System Controls -Operon-specific repressor and/or activator (e.g. lac or mal operons) --Repressors generally bind downstream, activators upstream, of promoters --Regulators can bind elsewhere and function through DNA looping -Catabolite repression (activation). "Glucose effect" --Glucose inhibits cAMP synthesis --cAMP required for catabolite activation -No feedback enzyme regulation Anabolic (Biosynthetic) Systems -Operon-specific repressor -Feedback allosteric enzyme regulation -No catabolite repression -Attenuation -Riboswitches The trp Attenuator -mRNA has four sections, 1 - 4 --Two Trp codons (UGG) in section "1" -Tryptophan starved: Slow translation of UGG codons results in 2:3 structure, which is not a transcription terminator (stem-loop followed by a run of U's is a terminator) -Adequate Tryptophan: Rapid translation of UGG results in 3:4 transcription terminator structure ***transcription and translation simultaneous in prokaryotes ***this is regulation at the transcriptional level, trigger is how fast translation is occurring Riboswitches -The mRNA folds into two different structures (allosteric structures) depending on the availability of the metabolite -In the absence of the metabolite, the mRNA is translated ***Translational regulation! TPP Riboswitch Regulates Translation -Thiamine pyrophosphate (TPP) regulates translation of TPP biosynthetic genes -In the absence of TPP, the S/D is accessible and translation occurs. -TPP changes the structure (allostery) so that the S/D is blocked. Catabolic and Anabolic Systems -Catabolic --System-specific Repression/activation --No Attenuation --Catabolite repression (activation) --No allosteric enzyme regulation -Anabolic --System-specific Repression --Attenuation, Riboswitches --No Catabolite Repression --Allosteric enzyme regulation RNA Thermal Sensors -Proteobacteria -Listeria, Yersinia, Vibrio -Neisseria ***Shine Delgarno must be in unstructured region at low temperature, increased temperature unfolds the hairpin structure that is already there... ***Chaperones needed under a new temperature regime, they can sense this temp change and turn on a new package of genes to exploit the host, genes often to accomplish a specific purpose Two-Component Regulatory Systems -ArcB and ArcA for O2 -GlnL and GlnG for NH3 -Many others... ***membrane-bound sensor kinase that responds to an environmental cue, response is activated upon binding and conformational change, phosphorylates response regulator, this response regulator goes and does a job of repression/activation/etc., ... phosphatase activity is constant to reset this action so that it can happen again Global Control Networks -Regulons - a set of co-regulated operons related to the same function -Pathogenic E.coli uses two-component systems to sense lack of O2 and bile, and a thermosensor to detect body temp to turn on a suite of virulence factor genes

Notes 01/11/2022

-Richer microbiomes are healthier in terms of people -Relate material to your life in some way when it comes to Slides Presentations (100 words or less)! (EACH MODULE HAS REFLECTION QUESTION) ---Don't just regurgitate lecture material. ---1 topic from each book ---final is on a personally chosen topic! -Topics available in the drive! -Slides also in the drive! -Microorganisms play absolutely essential roles in Earth's geochemical systems -Microbes and their control are critical for our personal and public health -One of the world's largest scientific organizations is the American Society for Microbiology -Microbes Built Modern Earth ---Aerobic respiration did not exist for a very long time!!! -Origin of cyanobacteria (oxygenic phototrophs) changed the world forever. ---About half of the produced oxygen is produced by microbes -Types of Energy Metabolism ---Phototrophs ---Chemotrophs ------Chemoorganotrophs-Aerobic Respiration -Fermentation ---Our muscles can do it, but we can live and do it constantly Deep Sea Thermal Vents -No light, so no photosynthesis at the depths of the ocean, but there can be volcanic activity that brings energy often used to fix carbon (lithotrophs doing lithosynthesis) ---Lithotrophs are the primary producers. Lithosynthesis instead of photosynthesis ---H2, H2S, Fe++, CH4, NH3, CO as energy sources ---These communities rival tropical forests in biomass! Survivorship Throughout History -Increased life expectancy by controlling infectious diseases Microbiomes -Produce 40% of blood metabolites -Affects glucose and fat utilization and storage -Affects production of hormones that regulate hunger and satiety -Affects immune system -Affects serotonin production and vagus nerve activity -Linked to anxiety level

Beginnings (ORFs) 04/28/2022

AUG is generally the start codon! -triplet pattern is probably a gene Peak of G and A (T and C relatively low here) before the start codon A is less prone to be involved in secondary structures, and G would be the most prone! All genomes show biases for A-richness and G-poorness for the ten nucleotides downstream of start codons, relative to their average nucleotide frequencies (FOR THE THREE BACTERIA IN SLIDE) Sawtoothed patterns suggest selection for specific codons and/or amino acids Bias for codons with two A's, which promotes A-richness. -preference for a rich sequences (preference for the aaa triplet more than one would expect All species have a preference for the AAA triplet -Bias most pronounced at second codon but occurs in all reading frames. The AAA triplet promotes openness better than expected for the independent effects of individual nucleotides Possible preference for lysine? About 10% of genes start with a non-AUG codon Non-ATG genes do not compensate for weaker codon: anticodon base pairing with greater... Functional problems, if any, are not compensated by downstream codon bias Hydrophilic amino acids near amino termini contribute to expression or function? -More hydropathic means more hydrophobic! --Low hydropathy means relatively hydrophilic Only amino acids that can be encoded by A-rich and/or G-poor codons are favored Weak E site codon: anticodon interactions od not increase frameshifting ***Basepairing does not seem to lead to frameshifting There are biases for both nucleotide and amino acid usage -Initiation Region Openness: --Start sites are A-rich and G-poor, and C-richness used in the GC-rich Pae genome --The AAA triplet may be especially effective in promoting openness -Formyl-Methionine removal: Reading frame establishment and retention:

Notes 01/25/2022 Prokaryotic Cell Biology

Bacterial plasma membranes do not have sterols in them (they are important for stiffening a membrane) ---although it is sometimes possible to get them from host ------sterols are a marker for eukaryotic organisms A capsule is primarily polysaccharide If motile, has a flagellum, can swim... Cell Wall -- Peptidoglycan -Peptide and glycan chains form a grid-like mesh over cell --Walls give cells their shapes Structural support against --hypoosmotic (dilute) environments -Does not protect against hyperosmotic (salty, sugary, dry) environments --Does not prevent plasmolysis --Food preservation ---Salted vegetables (pickles), salted meats, some cheeses, jams --Honey -Internal Compatible Solutes: --Sugars (phototrophs) --Amino acid derivatives --Ions other than Na+ (K+) Peptidoglycan Chemistry -Penicillin blocks transpeptidase enzymes that crosslink peptide chains -Lysozyme hydrolyzes the bond between M and G sugar (add to a bacteria in a hypoosmotic environment and water rushes in and the cell explodes) ***N-Acetylmuramic acid (M) is a signature molecule for bacteria (not archaea, etc.) ***crosslinks between strands is common in cell walls Cell Wall Destroyers -Lysozyme destroys existing walls --All cells explode in a hypoosmotic environment, whether growing or not -Penicillin blocks crosslinking --Only growing cells explode in hypoosmotic environment --Cultures that are not growing (because they lack nutrients) are not penicillin sensitive Gram Positive Covering -Nutrients diffuse through wall, transporters in plasma membrane take in -Ribitol is an open chain sugar alcohol derived from ribose ***Have a plasma membrane, no sterols, have transporters, have dozens of layers of peptidoglycan sheet for structural support, teichoic acid is an analog of RNA and are there to provide adherence ***diffusing nutrients must be small (can't get through peptidoglycan Gram Negative Covering -Transporters are in plasma membrane -Thinner peptidoglycan layering (less layers), an outermembrane outside of the peptido walls that along with the thin wall provides the structural support, also have porins through which waste can get out and nutrients can get in (have some chemical specificity, but not rigid, nothing big gets in), zone. between the membranes is the periplasm that has two membranes with enzymes that further digest small things into smaller things which are taken in by the transporters. Outer membrane, aka Lipopolysaccharide LPS, endotoxin, lipid A... -PAMP: Pathogen-Associated Molecular Pattern -PRR: Pattern Recognition Receptors (Toll-like receptors, TLRs) --TLR4 binds LPS to trigger inflammation --Local inflammation is good; systemic is very bad -MAMP: Microbe-Associated Molecular Pattern -From ICM book: Bacteroides fragilis Polysaccharide A (PSA) may bind TLR2 to induce tolerance ***Bind PAMPs with PRRs... MAMP for not everything is a pathogen!!! sometimes used by an organism for doing something good in our guts (same organism that causes an illness in a wound does not in our gut). E. coli O157 -The "Hamburger E. coli"; now also associated with raw vegetables -O157 refers to "O-specific" lipopolysaccharide detected with an antibody reagent in the clinical lab -Associated with very serious intestinal disease that can also cause kidney failure -Can proliferate in cattle intestines, especially if corn fed Relationships of Cell Structure to Nutrition -Extracellular hydrolysis of polymers (can not taken in large things) -Partially digested polymers (oligopeptides, oligosaccharides [or "dextrins"]) enter through porins in the LPS of G- cells, or though thick peptidoglycan wall on G+ cells -Further digestion to trimers, dimers and monomers in periplasm -Plasma membrane has transporters for small molecules Extracellular Features and Functions -Flagella -- Motility and adherence -Pilus (pili) -- Hair. Few, long. Adherence and gene transfer -Fimbriae -- Fringe. Numerous, short. Adherence -Polysaccharide "slime" --Capsules -- Adherence, avoid phagocytosis (covers the handle that phagocytes want to grab on to) --Biofilms -- Create favorable microenvironments Bacterial Flagella -Structurally and functionally different from eukaryotic flagella and cilia (don't beat but they spin, spinning is done by chemiosmotic potential not ATP hydrolysis, proton entry causes the conformational change enabling the propeller to spin, they can change direction of the rotor (counter vs clock wise), also much thinner than eukaryotic flagella) -Rotation driven by proton entry (dP) ) (deltaP) Swimming and Tumbling E.coli -Counterclockwise swims or runs -Clockwise tumbles ***uses swimming in tumbling to sample different environments Random walk! If you elongate lines generally moving upward then the arrowhead moves up the page (detecting nutrient, changes concentration) (focus on highest concentration) Fimbriae and Pili -Fimbriae -- short, numerous -Pili -- long, few --Involved in DNA exchange Capsules and Biofilms -Primarily extracellular polysaccharide --Can contain proteins, etc. Promote cell-cell adherence -Inhibit phagocytosis (PAMPs would be covered so no phagocytosis) -Biofilms are made from large populations making a lot of polysaccharide Biofilms -Primarily polysaccharide -Modify microenvironment -Protection from predators and even antibiotics Medical devices like catheters allow for colonization of a biofilm (BAD)! Our typical cells are good at preventing biofilm formation. Internal Cell Contents -Inclusion bodies --Energy and carbon stores -Bacterial Endospores PHA Inclusion Bodies -Poly-β-HydroxyAlkanoate -β-hydroxybutyric acid -PHB is a polymer, linked via ester bonds PHA Products for Convenience Foods Packaging -Products used in bulk, but not worth recycling -Fast Food containers -Medical supplies --PPE, bedpans and other containers -Agriculture --Netting or ground covers ***BIODEGRADABLE PLASTIC NET WHEN USING PHA Bacterial Endospores -Environments that are highly variable can select for cells that form durable spores to safely 'wait out' harsh periods -Bacillus: G+, aerobic, endospore-forming rods -Clostridium: G+, anaerobic, endospore-forming rods ---mostly in our gut system, many are not tolerant of oxygen but actually harmed by it Sporulation -Nutrient depletion triggers sporulation -Mother cell feeds Forespore -Hard Cortex shell and dehydration confer extreme durability --Disulfide-crosslinked protein -Mother cell may make a proteinaceous toxin Endospore-forming Strains with Potent Exotoxins -Bacillus thuringiensis, an insect pathogen. Aerobic Can be ingested on leaves ***Toxin crystal inside mother cell -Clostridium botulinum, fish and waterfowl pathogen. Anaerobic Lives in mud, can be ingested by animals that eat aquatic plants

Notes 03/24/2022 (Immunology)

Immunology -Physical and Chemical barriers -Innate or Constitutive Immunity -Acquired or Adaptive Immunity (vertebrates with jaws have this, it is built on top of the innate immunity) Antibodies ~35-40% of protein in blood -Four polypeptide chains, two identical heavy chains, two identical light chains -Blue boxes are constant within an antibody class, red boxes are highly variable -Fab, Fc fragments released by protease Cells -Antigen Specific cells: --B-Cells produce antibodies --T-Cells regulate immune cells -Nonspecific cells: --Phagocytes ---Macrophages ---Neutrophils ---Dendritic cells --Digest invaders -APCs, Antigen Presenting Cells --Certain phagocytes, and B-type cells --Present antigens to T cells to stimulate responses Summary of the Innate System -Not Antigen Specific. Designed to stop invaders quickly, regardless of previous exposure --Depends on tissue damage and PAMPs (Pathogen Associated Molecular Patterns, next slide) to signal pathogen invasion --Also depends on Blood Clotting and Complement (discussed soon...) --Phagocytes engulf and kill pathogenic cells ---Inflammation -- recruits more immune cells & factors ---Present antigens to T-cells to activate the Acquired Immune System PAMPs and PRRs -PAMP = Pathogen Associated Molecular Pattern --LPS is the prototypical PAMP -Immune cell PRRs Pattern Recognition Receptors (aka TLRs) bind PAMPs --PAMPs can serve as handles for phagocytosis --PAMP binding can initiate inflammatory response Complement, Slide 1 -System of ~30 proteins that kills cells and viruses and makes them easier to phagocytize -Abundant -- a few percent of the protein in serum --Activation involves Cleavage of Precursor Proteins (next slide) --Large fragments assemble into complexes on pathogens --The little fragments chemoattract more immune cells -Assembled activated complement: --Punches holes in membranes --"Opsonizes" cells -- handles for phagocytosis Complement, Slide 2 -Multiple activation pathways -Precursor cleavages and assembly on cell or virus -MAC = Membrane Attack Complex -Opsonization Handles for phagocytes -Small fragments can chemoattract more immune cells Phagocytosis -Opsons: PAMPS recognized by PRRs Antibody Fc and complement recognized by specific receptors Overview of the Innate System -Inflammation: PAMPs, complement, clotting factors, phagocytosis cause vasodilation and attract more immune cells -Phagocytes --Kill pathogens --Secrete Cytokines (hormones) that attract more phagocytes --Present antigens to stimulate the acquired immune system Bacterial Defenses Against Phagocytes -Capsules, certain flagella, etc. can inhibit phagocytosis -Fc receptors can orient antibodies outward -Leukocidins, hemolysins, T3SS Effectors kill phagocytes -Carotenoid pigments, other defenses prevent killing by phagocytes. Lymphatic System -MALT for "Mucosa-Associated Lymphoid Tissue" -"Peyer's patches" in the gut system for sampling gut contents Antibody Classes -Class switching -IgG, A, D -IgM, E (all starts with IgM, potential progression into other types) Antibody Gene Rearrangements (Deletions) -200 V x 50 D x 4 J = 40,000 combinations. -Combine these with a large number of possible light chains. -Unstimulated B cells make one specific antibody of the IgM class. ***B Cell undergoes this, which is a permanent change!!! B Cell Activation -B Cell with IgM on surface presents antigen to T Cell -If T Cell recognizes antigen, B cell induced to proliferate (BODY AND T CELL BOTH HAVE TO RECOGNIZE ANTIGEN, ONLY TINY AMOUNT OFTEN DO THIS) (T Cell secretes cytokine to tell B cell to proliferate) -Class switching can occur during proliferation ***Naive B cells must have IgM on surface ***Class of antibody (IgM, IgG, et.c( is determined by Constant-region genes (M, then D, etc.) Antibody Responses Summary Primary Response First Exposure: (results in Memory Cells of IgM) -Mostly IgM -Some Class Switching -Memory Cells have IgM Secondary Response (IgM Memory Cells proliferate and result in IgG) Later Exposures: -Lots of Class Switching -Mostly IgG -Memory Cells have IgM Antibody Response Timecourse -IgM to IgG (Antigen injection the first time is IgM, Second injection results in IgG at a lot higher antibody quantity in total!!!) (Class Switching) Specificity, Memory and Tolerance -Memory B cells for previously encountered antigens --IgM on surface --T cells also have memory (these can also undergo gene rearrangements to produce different receptors) -Tolerance --Self-tolerance - self-reactive T and B cell precursors killed during maturation ---Minimizes autoimmune disorders --Induced tolerance ---Parasites can block immune response ---Antigens encountered via the gut induce tolerance? (gut ingestion and not injection can produce a true tolerance?!?) Other IGS Proteins Class I -All nucleated cells Class II -APCs ***"MHC" or "HLA" Antigen Presentation to T Cells Activated T Cells ***Tc Cytotoxic Cell Linked to autoimmunity leading to type I diabetes, possibly MS ***Th1 Cell attacks bacteria, protozoa, fungi. Can increase inflammation ***Th2 T-Helper Cell has CD4 which is linked to asthma and seasonal allergies ***Also stimulates attack on pathogens like helminth worms Overview of Immune Response -Microbes in submucosa or subderma activate innate immune response (pamps, complement, clotting) -These components trigger inflammation including recruitment of phagocytes, T cells, B cells -Complement and antibody opsonize pathogens (handles making them easier to digest), which are engulfed, digested and presented on APC's -T cells scan cells and activate antigen-specific responses. TC for cytotoxic effects on infected cells, TH1 for increased phagocytosis, TH2 for antibody production -Memory for future exposures --Memory B cells for future antibody production --Memory T cells for rapid activation of phagocytes, B cells, and cell killing in future responses Superantigens -Exotoxins made by many bacteria and viruses -Non-specific and massive T-Cell activation -Misdirects the immune system and causes tissue damage -Systemic activation of macrophages can cause shock Septic Shock or Severe Sepsis -Can be a consequence of Bacteremia -Downward spiral: --Vasodilation of large vessels (drops blood pressure) --Impaired circulation lowers pH --Disseminated Intravascular Coagulation (DIC) (blood clotting initiates all over!) --Leaky capillaries ---Subcutaneous hemorrhaging ---Bleeding from orifices -Other kinds of shock can produce similar symptoms --Hemorrhagic viruses like Ebola MHC and Population Diversity -Three genes each for Class I and Class II --Because we each have two alleles, each of us has up to six alleles Class I and Class II MHC protein -Highly polymorphic --Highly diverse within human populations --Contribute to differences in immune responses --Probably related to exposures of our ancestors to diverse pathogens --Some data in humans (in epidemiology section) -Responsible for tissue rejection following transplants (T Cell may recognize transplanted tissue and say, this is not normal, and attack it!!!) --Self antigens presented by foreign Class I molecules may appear foreign --Cheetahs Immunization or Vaccination -Active vaccines -- Antigens: --Attenuated strains (many generations produced in culture, ends up losing its virulence) --Toxoids --Component (parts of cells) --Killed pathogens (dead pathogens) --Recombinant viruses ---Express antigen in yeast ---HPV vaccine is an "empty capsid" (no genome, so can't call an illness) -Passive vaccines -- Antibodies: (GIVE ANTIBODIES, COVID WOULD BE REGENERON OR CONVALESCENT SERUM AS ANTIBODIES) --Patient already exposed --Hepatitis A --Strep B for preemies --HIV and SARS-CoV-2 Active Vaccines -- Inject Antigens (or can inject mRNA and we can make the antigen) -Inflammation important --APCs activate Th2 cells -> B cells -> memory cells --Too little inflammation and patients don't become immune --Too much inflammation produces side effects -Conjugates and adjuvants increase local inflammation --Conjugate (chemically attach) antigens to mutant (inactive) diphtheria toxoid (MAKES A RESPONSE MORE LIKELY) --DT is exceptionally immunogenic -Adjuvants are coinjected irritants (e.g., aluminum salts, LPS) (CAUSES IRRITATION THAT WILL TRIGGER IMMUNE RESPONSE) --Act at PRRs or by enhancing tissue damage to cause inflammation

Notes 01/18/2022

Important Historical Points -John Snow's "Ghost Map" from London's cholera epidemic of 1854 ---Father of modern epidemiology (connecting cholera to water) (tie source of water to cholera and then convinced public health authorities to remove handle from pump so that no one could use it... cases went very far down!) Leeuwenhoek's Microscope -Late 17th century -Spherical bead, ~1.6 mm in diameter -Saw small objects that we now recognize as bacteria -Spontaneous generation? ***Marks begninng of microbiology, as now can see some microbes Pasteur's "Swan neck" flask expt Cells produced by growth of other cells, not of "vital force" enlivening organic matter ***Microbes come from microbes! Germ Theory of Disease -Proposed often but without credible evidence -In 19th century, many observations consistent with theory --Pasteur showed that diseased silk moth eggs associated with bacteria could transmit disease to entire clutches --In hospitals ---Ignaz Semmelweis promoted handwashing to prevent childbirth fever --John Snow linking cholera to water ---Pandemic, Sonia Shah ---The Ghost Map, Steven Johnson -Proof required new experimental technology... Pure Cultures -Agar as a solidifying agent with various nutrients added --Allowed for: ---Isolation of strains ---Tests of growth properties -Different strains of microorganism had different growth properties -When re-plated, colonies gave rise to reproducible phenotypes -Microorganisms had the same reproductive qualities as the other classes of organism Koch's Protocol -If you can perform these you can link a specific bacterium to a specific infectious disease --different animal, same disease and same symptoms, same bacterium, proves it somewhat ***Association is not enough, causation or correlation? Sergei Winogradsky -Father of Microbial Ecology -Discovered "lithotrophy." The use of inorganic chemicals as energy sources (nitrogen compounds, sulfur compounds, etc.) -Winogradsky columns widely used to study organisms with various needs for O2 and other compounds ***Layers of metabolic activity (plenty of oxygen on top gets lower in concentration as you go further down the column) Reverse Citric Acid Cycle for Carbon Fixation ---Incorporate CO2 and Hydrogen instead of getting these out Biochemical Functional Groups -Hydroxyl -Carbonyl (aldehydes and ketones) -Carboxyl (COOH or COO-) -Amino -Sulfhydryl -Phosphate (PO4 or HO-PO3 Carbonyls: Aldehydes and Ketones -Acetaldehyde --Functional groups: methyl & aldehyde -Pyruvate --Functional groups: methyl, keto & carboxyl -Pyruvate is an "α-keto acid" --Can be produced by exchanging amino group on amino acids for a keto group --Energy sources for microorganisms ***Pictures on slide 13 for history Sugars -Carbonyl carbon = Anomeric Carbon -Changes at the anomeric carbon lock configuration --Important for the regular structure of DNA, for example Glycosidic bonds and Polysaccharides -The anomeric Carbon --α and β "anomers" ***down is alpha configuration, must be anomeric to get this classification --Amylases and cellulases ***Amylases recognize alpha 1,4 linkage and cut it... do not recognize cellulose... cellulase recognizes the beta 1,4 glycosidic bond and cut it. -Different polysaccharides (glycans) can have kinds of sugars and linkages: --Glucans, fructans, xylans, mannans, galactans, etc --"Mutan" and dental plaque: α-1,3 linkages -Microbiologically important saccharides --Hundreds of plant fibers --Breast milk has ~200 oligoglycans --Bacteria and viruses can bind our cell surface 'oligosaccharides' Sucrose -Disaccharide: Glucose (α-1, β-2) fructose --Transport form of sugar in plants --Linkages are at the anomeric carbons of both monosaccharides --Decreases reactivity (Doesn't have the oxidative stress that glucose does) -Can induce dental plaque formation --Streptococcus mutans --"Mutan" has glucose in α-1, 3 linkages, and branches at α-1, 6 linkages Lipids -unsaturated (double bond on carbon) vs saturated fatty acid -Glycerol -Terpenoid Fatty Alcohol Membranes of Archaeans and Bacteria -Bacteria and Euks: Ester bonds between glycerol (OH) and fatty acids (COOH) -Archaea: Ether bonds between glycerol (OH) and isoprenoid fatty alcohols (OH) -Crosslinking through the membrane creates tetraether monolayers ***Typically, two fatty chains within the membrane and a polar group in the solvent ***Ethers are more heat and acid resistant than esters Tetraether Monolayer Lipids even more heat resistant Metabolism and Redox Rxns -Catabolism and Anabolism --Breakdown of compounds to get energy (ATP) (CATABOLISM) --Building molecules often requires ATP (ANABOLISM) -Redox reactions and catabolism --The removal of electrons, often in H atoms, form a high energy molecule is oxidation --The addition of electrons, often in H atoms, to a low energy molecule is reduction --"OIL RIG" oxidation is loss, reduction is gain of electrons --Many reactions they we will examine use"NAD", Nicotinamide Adenine Dinucleotide ---NAD + XH2 ⇔ NADH2 + X ---NADH2 + Y ⇔ NAD + YH2 Energy "Flows" Downhill -A and B may have different affinities for electrons. In this case, e- (on H) 'flow" from A to B. Periodic Table Showing Electronegativity -Electronegativity reflects "affinity" of atoms for electrons in bonds ---Functional groups can also differ in their affinity for electrons -For example, electrons will "flow" from Carbon to Oxygen -The fundamental basis of metabolism -- electron flow from a low affinity to high affinity group can be used to make ATP ***Electronegativity reflects affinity for electrons (Oxygen has highest affinity that is relative to us for electrons) (electrons will flow toward oxygen in reactions!) The Redox or "Electron Tower" Low to High Electronegativity -High-energy organics like Sugars, H2 (Low EN) -NAD (electron carrier) -Low energy organics like Pyruvate -Sulfur compounds, CO2 -Nitrogen compounds -Iron -Oxygen (High EN) ***As electrons flow from low EN donors towards high EN acceptors, ATP gets made ***Low EN indicates "high energy" High EN indicates "low energy"

Notes 04/26/2022 (Infectious Diseases)

Respiratory-Acquired Diseases -Most particles stick in the upper respiratory system, but smaller particles can reach the lungs -Lungs protected by the "mucociliary blanket" -Outer mucus layer (gel layer) is thick and filled with antibodies, phagocytes -Inner layer (sol layer) is more fluid and allows for movement of mucus up and out of lungs --Move at ~1 mm/minute -- Complete clearance of lungs ~ 24 hrs Streptococcus pyogenes -S. pyogenes (Group A Strep or "GAS"). -Prevalence ~ 15-20% in children. Responsible for ~40% of sore throats -At least 80 virulence factor variants. Differ mainly in their spe (streptococcal pyrogenic exotoxin) A,B,C,F and M, and other toxins. Most are superantigens. A-F phage-encoded -Adherence pili and capsule. Capsule also blocks phagocytosis -Blocks complement and kill phagocytes with hemolysins (Streptolysin) -Destroys chemotactic peptides that would otherwise recruit phagocytes Superantigens -Non-specific and thus massive activation of T-cells --Cytokines activate phagocytes, B cells and other cells -Can help pathogen avoid specific attacks by adaptive immunity -Localized: lesions like impetigo -Systemic: rashes and in severe cases, shock Group A Strep Syndromes 1 -"Suppurative Syndromes:" (Acute symptoms) --Upper respiratory: throat, ear & sinus infections ---Pili, capsule, "M" for adhesion and invasion ---Various superantigens ---Hemolysins -- "Streptolysins" ---Streptokinase - plasminogen activation --Erysipelas ---Invades lymphatic vessels -> erysipelas --Scarlet fever "erythrogenic toxins" (speA,B,C superantigens) become systemic ---Can lead to Toxic Shock-Like Syndrome Massive, systemic inflammatory response -GAS can also enter through wounds in skin --Local inflammation - impetigo --Necrotizing fasciitis, "Flesh-Eating Bacteria." Severe local inflammation -> tissue destruction Group A Strep Syndromes 2 -Delayed Sequelae, "Nonsuppurative Diseases": --Due to our immune response --Rheumatic fever. Autoimmune. ---Reaction to hyaluronic acid ---Kills 280,000 kids every year ---This number is steadily decreasing --Acute glomerulonephritis (in high concentrations, can cause clogging in the kidneys due to this) --Arguments for rapid application of antibiotic treatment to avoid strong active immune response Diagnostic Tests -Rapid antibody tests as well as culture-based tests --Neither can tell which if any superantigens might be available --Clinical diagnosis can identify them --Future Nucleic Acid based tests might look for virulence factor genes Streptococcus pneumoniae -Adhesive pili and pneumolysin, a pore-forming exotoxin -Capsule and Invasion -Mortality rates from 10-30% -Most problematic for elderly and very young -Drug-resistance becoming a problem. --Multidrug resistance is now a reportable disease -Vaccines includes capsular material from many serotypes --Prevnar20 has 20 capsules 'conjugated' to diphtheria toxoid --Pneumovax 23 has capsules from 23 strains and no conjugate Human Papillomavirus -Papilloma = wart -HPV > 150 strains, ~40 are sexually transmitted -Sexually transmitted strains are highly prevalent, with ~20M active infections, and 80% of women become seropositive by age 50 -Some strains cause cervical cancer. Can also cause penile and oral cancers, but they are much rarer HPV-Induced Tumors -Proteins E1, E2, E6, E7 (and others) regulate expression of both virus and host genes -E6, E7 promote cellular replication => warts ---Block p53 and pRB tumor suppressors, promoting "S" phase and blocking apoptosis -E1, E2 down-regulate E6, E7 (and promote viral replication) -In normal infection results in the papilloma, from which infections virions can transmit -Cancer occurs following integration of the E6, E7, but not E1, E2 genes into the host genome. --Unregulated cell growth, absence of apoptotic control -> cancer ***E6, E7 promote entry into S phase, leading to cell division and production of the wart. Pap smear detects abnormal cells from cervix HPV Vaccines -Original Gardasil by Merck protects against the three most common cancer-causing strains (~70% of cervical cancers) plus the most common wart-causing strain. It also protects against anal, vaginal and vulval cancers. It is also approved for males. --Without vaccinating males, R < 1 won't be possible --Cervarix protects against cervical cancer but not warts and is not approved for males in the USA --Gardasil now replaced by Gardasil 9, which protects against 9 HPV strains ---People vaccinated before June 2017 may have protection against only the original 4 strains -Vaccine recommended for kids before they are sexually active (~ ages 11-12). Nationally, ~2/3 of adolescents have received at least 1 dose, and about 50% received 3 doses. Vaccination rates are increasing for both girls and boys. RI, VA and DC now require it. -Since introduction, cancer strain infections and are down ~ 85% Rabies 1 -ssRNA, complementary to mRNA (Class V). Globally, ~55,000 human deaths/year -Enzootic in many animal species -Essentially 100% fatal in humans; a few patients have survived following induced coma, but most suffer severe brain damage -Replicates in muscle, then enters nerves at neuromuscular junctions and moves retrograde to CNS where it further replicates --Interferes with neurotransmitter release and binding in synapses -Travels down nerves to salivary glands for transmission Rabies 2 -Symptoms that drive transmission: --Replicates in salivary glands --Inability to swallow -> "foaming at the mouth" ---Hydrophobia --"Furiosis" -- restless/biting ---Paralytic form common, but may not transmit -Reservoir in wild animals with domestic animals serving as "amplifiers" -Prevention by vaccination of domestic animals and people who work with animals -~40k people received PEP (Post-Exposure Prophylaxis) annually in the USA. --In USA, prior to vaccination > 100 deaths per year. Now 0-3 deaths/yr Yersinia pestis - Bubonic Plague -Three major plague episodes in recorded time: --Justinian's plague, 541-542 and subsequently through 750 AD --Medieval plague, 1347-1351 and onward. ---A Distant Mirror: The Calamitous 14th Century by Barbara Tuchman --Hong Kong plague of the 1890s though the 1920s -All caused major, highly mortal pandemics -Plague is enzootic in many parts of the world and causes ~1-2k cases and ~100-200 deaths/yr Virulence Factors -Capsule polysaccharide and embedded proteins inhibit phagocytosis -T3SS effectors cause phagocyte apoptosis --Murine toxin triggers apoptosis --Other effectors block phagocytes from signaling to immune cells --So, phagocytes die without sending distress signals -Plasminogen activation facilitates spread (via clot busting) -Endotoxin triggers severe sepsis ***Injected effectors include apoptosis-triggering proteins, which kill phagocytes Plague Genomics and Evolution -Yersinia pestis Genome Derived from Y. pseudotuberculosis -Gain of genes needed for flea-borne transmission and for virulence (Rings 5&6) -"Pseudogenes", mutant genes needed for growth in rodent gut (rings 3&4) -Also has two virulence plasmids Nucleotide Biases -GC content --Average GC content varies among organisms --"Biased mutation pressure" --Horizontal Gene Transfer can introduce "islands" with different GC content -GC Skew --Leading strand tends to be G-rich and C-poor (also slightly T-rich, A poor) --Changes in skew can be caused by "inversions" and by HGT

Notes 04/14/2022 (Public Health)

Vaccination, and Public and Global Health -MMR vaccination rates among kindergartners --Vaccination rate required to reach Re = 1 is 1- 1/Ro Measles has an Ro = 18, so 1- 1/18 = 94.4% Vaccination -Variola (smallpox) --Probably emerged from camel pox virus in antiquity -At times it killed more people than any other cause --In 18th century Europe: ---Killed ~500,000 people per year ---Responsible for ~10% of all deaths ---Responsible for ~33% of deaths in children -Variolation practiced for centuries --First documented in China ~ 1000 CE -Vaccination --In 1776, Edward Jenner "vaccinated" James Phipps with cowpox (vaccinia) and then showed that he was resistant to variolation -Eradicated by 1978. See: House on Fire: the fight to eradicate smallpox (2011) by William Foege Bifurcated Needle Used for Vaccination Eradication Program More Vaccination History -Pasteur and vaccine development in 19th century --Vaccines for domestic animals --Rabies in dogs and in a boy who had been mauled by a rabid dog ---Vaccine made by passaging the disease-causing agent (viruses not yet discovered) through rabbits and using the spinal cord -1960's and 70's saw explosion of new vaccines --Polio, Measles, Mumps, Rubella (MMR), many others -Vaccines provide tremendous savings in lives --Programs expanding globally at a rapid rate --2 - 3m lives saved globally --Vaccination costs are much lower than the costs of disease --~1.5m vaccine-preventable deaths still occur --Public health infrastructure can provide additional benefits Transmissibility Within A Population -Vaccination programs designed to protect individuals and populations -Re depends on pathogen, behaviors, population parameters, and the level of acquired immunity -Vaccination programs designed to reduce Re < 1 --"Herd Immunity" --In USA, occurs when vaccination rates are above 70% (flu), 80% (polio, diphtheria) to 95% (measles) ***R ≡ "Reproductive Rate" = Number of new cases per current patient Fraction of Population that Must be Vaccinated to Achieve Herd Immunity -Fraction = 1 - (1/Ro), where Ro is the reproductive number without the vaccine --Assumes vaccine is highly effective -With Covid's initial Ro ~3.5, herd immunity requires ~71% to be vaccinated. --Omicron and BA.2 may have Ro closer to 20, requiring ~95%! -Natural covid infection also confers immunity --~95% of recovered patients have durable antibody ***Higher Rnaught means must vaccinate more people Vaccination Or Immunization Policies -Vaccines developed by pharmaceutical companies --FDA approval if drugs are "safe and effective" -CDC convenes a panel, "Advisory Committee on Immunization Practices," or ACIP --Mostly MDs (mostly pediatricians and immunologists), researchers and bioethicists ---One factor is whether herd immunity can be achieved -Protects populations -Protects those who can't be immunized for medical reasons -Protects those who receive vaccine but do not become immune -CDC makes recommendations, states make policy NC Immunization Practices -Recommendations from CDC, states set own policies. NC... --Requires vaccination against 12 pathogens to attend school and/or daycare (MMR, Varicella, DTaP, Hib, HepB, Polio, Pneumococcus, Meningococcus). --Exemptions: Parents may refuse for medical or religious reasons -Resistance due to different views on personal rights and public responsibility -AntiVax movement exacerbates resistance --The HPV (cervical cancer), influenza, hepatitis A, and rotavirus vaccines are recommended by CDC but not required in NC. (DC, RI and VA require HPV) -NC requires health insurance plans to cover vaccination. NC also receives vaccine from the CDC's "Vaccines for Children" program so that all children can be covered. --Providers may charge an administrative fee (typically $25-30) --In NC, vaccines that are recommended but not required are also supported Vaccines for Children (VFC) Program -CDC uses federal funding buys vaccines at bulk discount and distributes to states -States distribute vaccine to public and private clinics -Eligible children receive vaccine for free ***Total CDC Budget is only ~7b / year -Multiple doses ensure strong, long-lasting immunity -Variation in immune system development -Boosting to drive class switching, refresh antibody pools and develop more memory -Maternal antibody can interfere with vaccination Vaccinations for Expectant Mothers -Maternal antibody can protect infants --Recommended that expectant moms get the Tdap in the third trimester --Protects infants from whooping cough (pertussis) --Covid-19 vaccination -Ongoing research on maternal vaccines for other infectious diseases --Streptococcus Group B (common flora in adults, dangerous for preemies) --RSV (respiratory syncytial virus) --Zika Measles Rubeola "Red Pox" -Came to settled human populations from Rinderpest, a virus that infects cattle -Ro ~ 18 in naive populations -Respiratory-acquired, primarily infects immune cells (T-cells, B-cells, phagocytes) --Infected T-cells redistribute to subderma; inflammation causes rash --~5% develop fatal pneumonia --Acute and Subacute Sclerosing Panencephalitis kills or permanently disables --Temporary immunodeficiency and Immune Amnesia leads to other infections -Old World peoples have considerable natural immunity, but it still has high mortality in undeveloped environment Measles in the USA -"Love is like the measles; we all have to go through it." Jerome K. Jerome -Annually, in the USA in the 1950's: --3-7 x 10^5 severely infected --~350-700 died --Several thousand with disability -With vaccination programs: --No longer endemic in the USA --Adverse reaction rate: ~3 per 100k --For comparison, the risk of death of 11 year olds from all causes: 1 in 10k -* 2nd dose recommended after 1992 Recent Measles in the USA -Most cases in 2014 were within an Amish community that now embraces vaccination -Disneyland-related outbreak in 2015 began to spread widely -The 2018-2019 outbreak had > 1280 cases, mostly in NY Measles Vaccination Rates -Measles is very highly transmissible --Slight decreases in vaccination can result in epidemic -If vaccination rates continue to decrease, epidemics of other diseases will appear Global Measles Toll -In 2000 > 500k deaths/yr --Now, ~85% of kids under age 5 get the vaccine --Deaths now < 150k/yr -Estimated that measles vaccine saved 15.6m lives between 2000-2013 Rubella ("Little Measles") -Less contagious and generally milder than measles --Epidemics occurred every 6 - 9 years in pre-vaccination era -Link between birth defects and rubella made after WWII in Australia --Ophthalmologist noticed a large number of blind children --Comorbidities include deafness, cognitive impairment, heart defects and high rates of childhood death -Retrospective Study (association, hypothesis building): Blindness linked to maternal rubella infection in 1st trimester --Cause not widely accepted until prospective studies done -Prospective Study (hypothesis testing): Follow pregnant women who contract rubella and observe high rates of miscarriage and congenital diseases Rubella-2 -Last epidemic in the USA occurred in 1964 (births in 1964-65) --12.5m people got the disease (~7 % of the population) --Nationally, there were 20,000 cases of "Congenital Rubella Syndrome" plus 6250 miscarriages and stillbirths and 5000 therapeutic abortions --Rubella was not a reportable syndrome, so these numbers are underestimates -Live, attenuated vaccines approved in 1969 --Nearly eliminated it from developed countries --Girls and women should be vaccinated prior to pregnancy Bordetella pertussis - Whooping Cough -Most significant problem for infants. Globally, 50m cases, 300k deaths/yr -Pertussis is endemic in the US, with epidemics occurring every few years --~10% mortality for infants in the USA -Several known virulence factors --Adherence: "Filamentous hemagglutinin" and fimbriae --Toxins: Pertussis toxin, tracheal cytotoxin, and lipopolysaccharide (PAMP) -Detected by Agglutination, or Fluorescent Antibody test, or PCR --Early antibiotic treatment for infants Bordetella pertussis - Epidemiology -Ro for naive populations ~15! -3-5 year cycles of disease -Adult reservoir -Why the resurgence? --Incomplete vaccination in the past --Immunity wanes over ~ a decade -Recommendations are for DTaP in childhood, Tdap in adolescence and Td every 10 yrs for adults --Recently, one Tdap for adults Whooping Cough Vaccine -DTP, DTaP, Tdap --"D"iphtheria and "T"etanus toxoids --"P" = killed Bordetella pertussis cells -High rate of side effects --"aP" = external antigens (a = acellular) --DTaP may be less effective than DTP but has fewer side effects -Current recommendation: --Expectant mothers to get a Tdap booster between 27 - 36 weeks of pregnancy --Adults receive one Tdap in place of Td -Vaccine ads that target parents and grandparents --Ads are effective Strategies for Disease Eradication -Reservoirs, tracking, culture and politics -Smallpox - "Ring vaccination" --Killed 300m people in 20th century --Acute human cases obvious and only reservoir --Politics an issue, but not insurmountable -Measles and polio - targeted mass "blanket" vaccination in affected areas in Africa and West Asia --Measles epidemiology similar to smallpox --Great success in Africa, less in Pakistan -Polio down to a handful of countries --Carrier state common, but blanket vaccination working --Political hurdles: The Antivax Movement: How Long Does Collective Memory Last? -From April 20th, 2019, "The Economist": How soon does a community rebuild in a high-risk area after a natural disaster? --Only about one generation. Without first-hand knowledge, people discount risk. --My parents' generation knew all about measles, polio, etc. --Many but not all in my generation know --The next generations? Modern AntiVax Movement Began in 1998 -Paper, now retracted, claimed link between MMR and autism --Claimed behavioral and developmental problems occurred within days of vaccination --Based on recall and belief of a small set of parents --Also claimed that MMR altered gut physiology, triggering autism -Since then, a dozen large studies on many populations show no MMR link to autism -Later speculation that the MMR preservative (Hg-based Thimerosal) might be the cause --Removal of Thimerosal in 2005 had no effect on autism rates -More recent data suggesting that autism begins before birth --There is now linkage between autism and microbiome -Persistence of the movement highlights challenges in communicating science and statistics to the public Use of Humor to Reach the Broader Public