Proteomic Profiling of Mouse Helper T Cell Differentiation

Helper T cell differentiation is a key process in the regulation of adaptive immune responses. Here, mouse Th1 and Th2 cells are profiled using high‐throughput proteomics to increase the understanding of the molecular biology of Th differentiation to support the design of prophylactic and therapeutic intervention strategies for (infectious) diseases. Protein profiling of Th1/Th2 differentiated cells results in the quantification of almost 6000 proteins of which 41 are differentially expressed at FDR < 0.1, and 19 at the FDR < 0.05 level, respectively. Differential protein expression analysis identifies a number of the expected canonical Th differentiation markers, and gene set analysis using the REACTOME database and a hypergeometric test (FDR < 0.05) confirms that helper T cell pathways are the top sets that are differentially expressed. Additionally, by network analysis, many differentially expressed proteins are associated with the Th1 and Th2 pathways. Data are available via PRIDE database with identifier PXD004532.     

The association between sociodemographic,hormonal, tubo-ovarian factors and bacterial count in Chlamydia and Mycoplasma infections with infertility

Aim: To determine if there is an association between the Chlamydia and Mycoplasma infections with socio-demographic and clinical factors, and also with infertility. Methods: We conducted a study on 100 infertile married women and 100 control group, and collected data on the socio-demographic, hormonal and tubo-ovarian factors. The results of the endocervical swabs were analyzed for Mycoplasma and Chlamydia infection, the bacterial counts were also determined. Results: The percentage positivity to infection was significantly more among the infertile group compared to the control group, and also significantly more among the age group <30 years old. The positivity for infection with Chlamydia and/or Mycoplasma was significantly correlated with age, history of irregular menstruation, and history of previous abortion. Further sub-analysis of the infertile group showed that positivity to Chlamydia and/or Mycoplasma infection was significantly correlated to hormonal factors, ovarian factors, irregular menstruation, and previous abortion. Regression analysis showed that hormonal, ovarian factors, and irregular menstruation were the most significant factors in the positivity to Chlamydia and Mycoplasma infection. Bacterial count was significantly correlated with age, history of irregular menstruation, and history of previous abortion. Conclusion: Infection to Chlamydia and Mycoplasma is associated to younger age (?30 years old), and occurs in the infertile women. There is an interplay between infection in younger women, irregular menstruation, hormonal, and tubo-ovarian factors with infertility. Bacterial count was significantly correlated with age, history of irregular menstruation, and history of previous abortion.     

Stringent response leads to continued cell division and a temporal restart of DNA replication after initial shutdown in Vibrio cholerae

Vibrio cholerae is an aquatic bacterium with the potential to infect humans and cause the cholera disease. While most bacteria have single chromosomes, the V. cholerae genome is encoded on two replicons of different size. This study focuses on the DNA replication and cell division of this bi‐chromosomal bacterium during the stringent response induced by starvation stress. V. cholerae cells were found to initially shut DNA replication initiation down upon stringent response induction by the serine analog serine hydroxamate. Surprisingly, cells temporarily restart their DNA replication before finally reaching a state with fully replicated single chromosome sets. This division‐replication pattern is very different to that of the related single chromosome model bacterium Escherichia coli. Within the replication restart phase, both chromosomes of V. cholerae maintained their known order of replication timing to achieve termination synchrony. Using flow cytometry combined with mathematical modeling, we established that a phase of cellular regrowth be the reason for the observed restart of DNA replication after the initial shutdown. Our study shows that although the stringent response induction itself is widely conserved, bacteria developed different ways of how to react to the sensed nutrient limitation, potentially reflecting their individual lifestyle requirements.     

Strikingly high levels of heterozygosity despite 20 years of inbreeding in a clonal honey bee

Inbreeding (the mating between closely related individuals) often has detrimental effects that are associated with loss of heterozygosity at overdominant loci, and the expression of deleterious recessive alleles. However, determining which loci are detrimental when homozygous, and the extent of their phenotypic effects, remains poorly understood. Here, we utilize a unique inbred population of clonal (thelytokous) honey bees, Apis mellifera capensis, to determine which loci reduce individual fitness when homozygous. This asexual population arose from a single worker ancestor approximately 20 years ago and has persisted for at least 100 generations. Thelytokous parthenogenesis results in a 1/3 of loss of heterozygosity with each generation. Yet, this population retains heterozygosity throughout its genome due to selection against homozygotes. Deep sequencing of one bee from each of the three known sub‐lineages of the population revealed that 3,766 of 10,884 genes (34%) have retained heterozygosity across all sub‐lineages, suggesting that these genes have heterozygote advantage. The maintenance of heterozygosity in the same genes and genomic regions in all three sub‐lineages suggests that nearly every chromosome carries genes that show sufficient heterozygote advantage to be selectively detrimental when homozygous.     

The gap junction protein connexin32 interacts with the Src homology 3/hook domain of discs large homolog 1

Scaffolding of membrane proteins is a common strategy for forming complexes of proteins, including some connexins, within membrane microdomains. Here we describe studies indicating that Cx32 interacts with a PDZ-containing scaffolding protein, Dlgh1 (Discs Large homolog 1). Initial screens of liver lysates using antibody arrays indicated an interaction between Cx32 and Dlgh1 that was confirmed using coimmunoprecipitation studies. Yeast two-hybrid complementation determined that the Cx32 bound via interaction with the SH3/Hook domain of Dlgh1. Confocal microscopy of liver sections revealed that Cx32 and Dlgh1 could colocalize in hepatocyte membranes in wild type mice. Examination of levels and localization of Dlgh1 in livers from Cx32 null mice indicate that, in the absence of Cx32, Dlgh1 was decreased, and the remainder was translocated from the hepatocyte membrane to the nucleus with some remaining in cytoplasmic compartments. This translocation was confirmed by Western blots comparing Dlgh1 levels in nuclear extracts from wild type and Cx32 null murine livers. Using SKHep cells stably transfected with Cx32 under the control of a tet-off promoter, we found that acute removal of Cx32 led to a decrease of membrane-localized Dlgh1 and an increase in the nuclear localization of this tumor suppressor protein. Together, these results suggest that loss of Cx32 alters the levels, localization, and interactions of the tumor suppressor protein Dlgh1, events known in other systems to alter cell cycle and increase tumorigenicity.     

A model of bacterial intestinal infections in Drosophila melanogaster

Serratia marcescens is an entomopathogenic bacterium that opportunistically infects a wide range of hosts, including humans. In a model of septic injury, if directly introduced into the body cavity of Drosophila, this pathogen is insensitive to the host's systemic immune response and kills flies in a day. We find that S. marcescens resistance to the Drosophila immune deficiency (imd)-mediated humoral response requires the bacterial lipopolysaccharide O-antigen. If ingested by Drosophila, bacteria cross the gut and penetrate the body cavity. During this passage, the bacteria can be observed within the cells of the intestinal epithelium. In such an oral infection model, the flies succumb to infection only after 6 days. We demonstrate that two complementary host defense mechanisms act together against such food-borne infection: an antimicrobial response in the intestine that is regulated by the imd pathway and phagocytosis by hemocytes of bacteria that have escaped into the hemolymph. Interestingly, bacteria present in the hemolymph elicit a systemic immune response only when phagocytosis is blocked. Our observations support a model wherein peptidoglycan fragments released during bacterial growth activate the imd pathway and do not back a proposed role for phagocytosis in the immune activation of the fat body. Thanks to the genetic tools available in both host and pathogen, the molecular dissection of the interactions between S. marcescens and Drosophila will provide a useful paradigm for deciphering intestinal pathogenesis.     

Flight initiation distance and escape behavior in the black redstart (Phoenicurus ochruros)

There are many anti‐predatory escape strategies in animals. A well‐established method to assess escape behavior is the flight initiation distance (FID), which is the distance between prey and predator at which an animal flees. Previous studies in various species throughout the animal kingdom have shown that group size, urbanization, and distance to refuge and body mass affect FID. In most species, FID increases if body mass, group size or distance to refuge decreases. However, how age and sexual dimorphism affect FID is rather unknown. Here, we assess the escape behavior and FID of the black redstart (Phoenicurus ochruros), a small turdid passerine. When approached by a human, males initiated flights later, that is allowing a closer approach than females. Males of this species are more conspicuous, and therefore, may exhibit aposematism to deter potential predators or are less fearful than females. Additionally, juveniles fled at shorter distances and fled to lower heights than adults. Lastly, concerning escape strategy, black redstarts, unless other passerine birds, fled less often into cover, but rather onto open or elevated spots. Black redstarts are especially prone to predation by ambushing predators that might hide in cover. Hence, this species most likely has a higher chance of escaping by fleeing to an open spot rather than to a potentially risky cover.