Amanda N. Wavrin Week 11

Vocabulary

 * 1) Electrophoresis-separation of ionic molecules, (principally proteins) by the differential migration through a gel according to the size and ionic charge of the molecules in an electrical field. High resolution techniques normally use a gel support for the fluid phase
 * 2) Gavage-Forced feeding by stomach tube
 * 3) Enumerate-To count; to reckon; to ascertain the units of
 * 4) Chemotaxis-a response of motile cells or organisms in which the direction of movement is affected by the gradient of a diffusible substance. Differs from chemokinesis in that the gradient alters probability of motion in one direction only, rather than rate or frequency of random motion
 * 5) Inoculation-The process of introducing an antigenic substance or vaccine into the body to trigger immune response against a specific disease.
 * 6) Dissemination-The act of dispersing or diffusing something
 * 7) Homogenization-The process by which a material is made homogeneous
 * 8) Chromogenic-Producing colour, a chromogenic colony is a pigmented colony
 * 9) Murine-Of, relating to, a member of the rodent family muridae, including rats and mice
 * 10) Shine-Dalgarno sequence-A short stretch of nucleotides on a prokaryotic mRNA molecule upstream of the translational start site, that serves to bind to ribosomal RNA and thereby bring the ribosome to the initiation codon on the mRNA

Dictionary used: biology-online.org dictionary

Outline

 * It is unclear as to what factors enhance the transission of pathogens during epidemic spread
 * Vibrio Cholera is a water-borne diarrhoel disease
 * Cholera was studied in its natural habitat in Dhaka, Bangladesh
 * Stool samples were collected and tested for V. cholerae O1 Inaba El Tor from patients at the International Centre for Diarrhoeal Disease Research
 * These strains were used in murine infection studies by mixing an in vitro strain with the stool V. cholerae and using the mixture to inoculate mice
 * The mice were euthanized after around 24 hours and the bacteria was collected from the mice and plated.
 * out put ratios of stool-sample V. cholerae to the in vitro strain were corrected and represent the competitive indices
 * A CI above one indicates enhanced infectivity
 * A CI below one indicates decreased infectivity
 * Human shed V. cholerae consistently showed enhances infectivity
 * However, when the V. cholerae strain was purified and cultured in vitro this competitive advantage was lost
 * These results suggest that passage through the human gastointestinal tract increases infectivity of the cholera bacterium
 * They tested to see whether or not the human shed V. cholerae maintained its hyperinfectious state after being dispersed back into the environment
 * After being infected into mice, they found that the hyperinfectious state remained
 * They proposed that human passage enhances the infectivity of V. cholerae by lowering the infectious dose in secondary individuals
 * They then conducted transcriptional profiling of human shed V. cholerae by using DNA microarray
 * The DNA microarray contained around 87 percent of the ORFs of the Bangladeshi O1 El Tor reference strain
 * stool samples were collected from three patients in the ICDDR
 * The samples were filtered and frozen
 * The samples showed the 'rice water' appearance typically found with V. cholerae
 * The contained around 100 million V. cholerae per millilitre and showed few contaminating bacteria
 * The stools were analyzed using gel electrophoresis
 * One microgram of RNA from each sample was used for DNA synthesis, labelled with Cy5 and hybridized to the microarray with a Cy3 labelled reference strain- an expotentially growing O1 Inaba El Tor strain
 * The relative flourescent intensities were determined using an Axon scanner and the data was quantified, normalized, and corrected
 * Using the Statistical Analysis for Microarrays program, they were able to identify significant differences in the intensity ratios
 * An in vitro strain was used as class I and each individual stool sample as class II
 * They obtained the following results: 237 genes were differentially regulated, 44 genes were induced, and 193 genes were repressed in human shed V. cholerae
 * The transcriptomes of the stool derived V. cholerae and the strain of DSM-V999 were similar
 * The transcriptome in consistent with bacterial growth in conditions similar to those found in rice water stools
 * Hierarchical clustering of stationary phase, expotential phase, and human shed V. cholerae shows that the transcriptome bears traits of both growth phases
 * Results suggest that before it is shed, V. cholerae turns off the expression of particular genes, genes that are needed for successful infection of humans and mice
 * This indicates that increased expression of these genes is not necessary for increased infectivity
 * In vitro induction of the acid toerlance response enhances the infectivity of V. cholerae
 * The role of chemotaxis in the infectivity of V. cholerae in uncertain
 * Most of the genes for chemotaxis were repressed durinf infection
 * The results suggest that motile bacteria are non-chemotactic when being shed
 * These results shed some light on the infectivity of the cholera bacterium
 * Passage through the human gastrointestinal tract increase the infectivity of V. cholerae
 * Humans help prepare the cholera bacterium for infection of other hosts
 * These findings may help with the understand of the epiemic spread of other microorganisms
 * This work could also aid in the development of a vaccine to prevent infection

Methods
Competition assays were done by mixing DSM-V984 grown overnight with stool bacteria in a ratio of 1:10
 * patients at the ICDDR had cholera and rice water stools
 * Fresh stool samples were collected in beakers, filtered through cheese cloth, and frozen at 80 degrees celsius
 * Protocols were reviewed and approved by the Research Reviem Committee, the Ethical Review Committee, and the Institutional Review Board
 * It was given to 3-5 day old mice by gavage
 * The mice were then euthanized and the small intestines removed
 * Output ratios were corrected
 * The PH of the two pond water samples used were 7-7.5

Microarray analysis

 * ORFs were found using and ORF-finding program and portions of the ORFs were amplified by polymerase chain reaction and spotted onto slides
 * V. cholerae RNA was collected from stool samples and DSM-V999 strain was grown overnight in vitro
 * DNAse treatment to remove DNA contamination was carried out
 * Equal concentrations of each test RNA and common reference RNA were used for reverse transcription reactions
 * Labelling reactions were done on two separate days which resulted in quadruplicate arrays for each strain
 * Control arrays were also hybridized to identify potential affects of freezing the stools

Figures

 * Figure 1a: Fresh stool samples were collected from six patients. Figure one a is showing that human shed V. cholerae are hyperinfectious and show enhanced infectivity (having a CI above 1) *Figure 1b: Fresh stool samples were collected from six patients. The samples in figure 1b were collected and then incubated in local pond water before being used for the infection of animals.  The hyperinfectious state of the bacterium remained.
 * Figure 2: Fifure two is a cluster diagram of V. cholerae collected from three patients (A,B,C). There are both technical replicates and biological replicates. Fold changes were calculated using the arrays on the left. Red indicated a minimum two fold increase, green represents a minimum two fold reduction. The arrows are pointing to representitive genes.

Merrell Journal Club
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