The Functional Significance of Posttranslational Modifications on Polo-Like Kinase 1 Revealed by Chemical Genetic Complementation

Mitosis is coordinated by carefully controlled phosphorylation and ubiquitin-mediated proteolysis. Polo-like kinase 1 (Plk1) plays a central role in regulating mitosis and cytokinesis by phosphorylating target proteins. Yet, Plk1 is itself a target for posttranslational modification by phosphorylation and ubiquitination. We developed a chemical-genetic complementation assay to evaluate the functional significance of 34 posttranslational modifications (PTMs) on human Plk1. To do this, we used human cells that solely express a modified analog-sensitive Plk1 (Plk1^AS) and complemented with wildtype Plk1. The wildtype Plk1 provides cells with a functional Plk1 allele in the presence of 3-MB-PP1, a bulky ATP-analog inhibitor that specifically inhibits Plk1^AS. Using this approach, we evaluated the ability of 34 singly non-modifiable Plk1 mutants to complement Plk1^AS in the presence of 3-MB-PP1. Mutation of the T-loop activating residue T210 and adjacent T214 are lethal, but surprisingly individual mutation of the remaining 32 posttranslational modification sites did not disrupt the essential functions of Plk1. To evaluate redundancy, we simultaneously mutated all phosphorylation sites in the kinase domain except for T210 and T214 or all sites in the C-terminal polo-box domain (PBD). We discovered that redundant phosphorylation events within the kinase domain are required for accurate chromosome segregation in anaphase but those in the PBD are dispensable. We conclude that PTMs within the T-loop of Plk1 are essential and nonredundant, additional modifications in the kinase domain provide redundant control of Plk1 function, and those in the PBD are dispensable for essential mitotic functions of Plk1. This comprehensive evaluation of Plk1 modifications demonstrates that although phosphorylation and ubiquitination are important for mitotic progression, many individual PTMs detected in human tissue may have redundant, subtle, or dispensable roles in gene function.     

Human Circulating Antibody-Producing B Cell as a Predictive Measure of Mucosal Immunity to Poliovirus

Background

The “gold standard” for assessing mucosal immunity after vaccination with poliovirus vaccines consists in measuring virus excretion in stool after challenge with oral poliovirus vaccine (OPV). This testing is time and resource intensive, and development of alternative methods is a priority for accelerating polio eradication. We therefore evaluated circulating antibody-secreting cells (ASCs) as a potential means to evaluate mucosal immunity to poliovirus vaccine.

Methods

199 subjects, aged 10 years, and previously immunized repeatedly with OPV, were selected. Subjects were assigned to receive either a booster dose of inactivated poliovirus vaccine (IPV), bivalent OPV (bOPV), or no vaccine. Using a micro-modified whole blood-based ELISPOT assay designed for field setting, circulating poliovirus type-specific IgA- and IgG-ASCs, including gut homing α4β7+ ASCs, were enumerated on days 0 and 7 after booster immunization. In addition, serum samples collected on days 0, 28 and 56 were tested for neutralizing antibody titers against poliovirus types 1, 2, and 3. Stool specimens were collected on day 28 (day of bOPV challenge), and on days 31, 35 and 42 and processed for poliovirus isolation.

Results

An IPV dose elicited blood IgA- and IgG-ASC responses in 84.8 to 94.9% of subjects, respectively. In comparison, a bOPV dose evoked corresponding blood ASC responses in 20.0 to 48.6% of subjects. A significant association was found between IgA- and IgG-ASC responses and serum neutralizing antibody titers for poliovirus type 1, 2, 3 (p<0.001). In the IPV group, α4β7⁺ ASCs accounted for a substantial proportion of IgA-ASCs and the proportion of subjects with a positive α4β7⁺ IgA-ASC response to poliovirus types 1, 2 and 3 was 62.7%, 89.8% and 45.8%, respectively. A significant association was observed between virus excretion and α4β7⁺ IgA^- and/or IgG-ASC responses to poliovirus type 3 among immunized children; however, only a weak association was found for type 1 poliovirus.

Discussion

Our results suggest that virus-specific blood ASCs, especially for type 3 poliovirus, can serve as surrogate of mucosal immunity after vaccination. Further studies are needed to evaluate the duration of such memory responses and to assess the programmatic utility of this whole blood-based mucosal ASC testing for the polio eradication program.     

The Genetics of Bene Israel from India Reveals Both Substantial Jewish and Indian Ancestry

The Bene Israel Jewish community from West India is a unique population whose history before the 18^th century remains largely unknown. Bene Israel members consider themselves as descendants of Jews, yet the identity of Jewish ancestors and their arrival time to India are unknown, with speculations on arrival time varying between the 8th century BCE and the 6th century CE. Here, we characterize the genetic history of Bene Israel by collecting and genotyping 18 Bene Israel individuals. Combining with 486 individuals from 41 other Jewish, Indian and Pakistani populations, and additional individuals from worldwide populations, we conducted comprehensive genome-wide analyses based on F_ST, principal component analysis, ADMIXTURE, identity-by-descent sharing, admixture linkage disequilibrium decay, haplotype sharing and allele sharing autocorrelation decay, as well as contrasted patterns between the X chromosome and the autosomes. The genetics of Bene Israel individuals resemble local Indian populations, while at the same time constituting a clearly separated and unique population in India. They are unique among Indian and Pakistani populations we analyzed in sharing considerable genetic ancestry with other Jewish populations. Putting together the results from all analyses point to Bene Israel being an admixed population with both Jewish and Indian ancestry, with the genetic contribution of each of these ancestral populations being substantial. The admixture took place in the last millennium, about 19–33 generations ago. It involved Middle-Eastern Jews and was sex-biased, with more male Jewish and local female contribution. It was followed by a population bottleneck and high endogamy, which can lead to increased prevalence of recessive diseases in this population. This study provides an example of how genetic analysis advances our knowledge of human history in cases where other disciplines lack the relevant data to do so.     

Coalition Formation by Male Vervet Monkeys (Chlorocebus pygerythrus) in South Africa

Unrelated male primates frequently cohabit in bisexual groups and, despite being reproductive competitors, have been shown to cooperate in ways that are associated with reproductive success. Such coalitions between males are common in some taxa, where they can serve two primary functions – status management and improved mating opportunities – that subserve long‐ and short‐term objectives. Here, we use observational data to provide information on male coalitions in vervet monkeys (Chlorocebus pygerythrus), a guenon with a multimale group structure. We recorded a total of 62 coalitions from two troops across a 10‐mo period at Samara Game Reserve, South Africa. We found that males who were more frequently associated spatially and who had groomed one another were more likely to form coalitions and did so against higher‐ranking opponents. This was not linked to any evidence that coalitionary aggression provided either short‐ or long‐term reproductive benefits for the aggressors and coalitions were not restricted to the mating season. There was little evidence that particular individuals were targeted, reciprocation between partners was not observed, and recent immigrant males were not targeted disproportionately. Our data suggest that within‐group coalition formation between vervet males may represent something close to an ancestral state whereby males form ad hoc coalitions opportunistically, joining an ongoing dyadic contest to target an opponent without facing the possible risks of dyadic contest, such as a greater chance of injury.     

Rock geochemistry induces stress and starvation responses in the bacterial proteome

Interactions between microorganisms and rocks play an important role in Earth system processes. However, little is known about the molecular capabilities microorganisms require to live in rocky environments. Using a quantitative label‐free proteomics approach, we show that a model bacterium (Cupriavidus metallidurans CH34) can use volcanic rock to satisfy some elemental requirements, resulting in increased rates of cell division in both magnesium‐ and iron‐limited media. However, the rocks also introduced multiple new stresses via chemical changes associated with pH, elemental leaching and surface adsorption of nutrients that were reflected in the proteome. For example, the loss of bioavailable phosphorus was observed and resulted in the upregulation of diverse phosphate limitation proteins, which facilitate increase phosphate uptake and scavenging within the cell. Our results revealed that despite the provision of essential elements, rock chemistry drives complex metabolic reorganization within rock‐dwelling organisms, requiring tight regulation of cellular processes at the protein level. This study advances our ability to identify key microbial responses that enable life to persist in rock environments.     

The organelle of differentiation in embryos: the cell state splitter

The cell state splitter is a membraneless organelle at the apical end of each epithelial cell in a developing embryo. It consists of a microfilament ring and an intermediate filament ring subtending a microtubule mat. The microtubules and microfilament ring are in mechanical opposition as in a tensegrity structure. The cell state splitter is bistable, perturbations causing it to contract or expand radially. The intermediate filament ring provides metastability against small perturbations. Once this snap-through organelle is triggered, it initiates signal transduction to the nucleus, which changes gene expression in one of two readied manners, causing its cell to undergo a step of determination and subsequent differentiation. The cell state splitter also triggers the cell state splitters of adjacent cells to respond, resulting in a differentiation wave. Embryogenesis may be represented then as a bifurcating differentiation tree, each edge representing one cell type. In combination with the differentiation waves they propagate, cell state splitters explain the spatiotemporal course of differentiation in the developing embryo. This review is excerpted from and elaborates on “Embryogenesis Explained” (World Scientific Publishing, Singapore, 2016).     

A prospective observational cohort study in primary care practices to identify factors associated with treatment failure in <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> skin and soft tissue infections

Background

The incidence of outpatient visits for skin and soft tissue infections (SSTIs) has substantially increased over the last decade. The emergence of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) has made the management of S. aureus SSTIs complex and challenging. The objective of this study was to identify risk factors contributing to treatment failures associated with community-associated S. aureus skin and soft tissue infections SSTIs.

Methods

This was a prospective, observational study among 14 primary care clinics within the South Texas Ambulatory Research Network. The primary outcome was treatment failure within 90 days of the initial visit. Univariate associations between the explanatory variables and treatment failure were examined. A generalized linear mixed-effect model was developed to identify independent risk factors associated with treatment failure.

Results

Overall, 21% (22/106) patients with S. aureus SSTIs experienced treatment failure. The occurrence of treatment failure was similar among patients with methicillin-resistant S. aureus and those with methicillin-susceptible S. aureus SSTIs (19 vs. 24%; p = 0.70). Independent predictors of treatment failure among cases with S. aureus SSTIs was a duration of infection of ≥7 days prior to initial visit [aOR, 6.02 (95% CI 1.74–19.61)] and a lesion diameter size ≥5 cm [5.25 (1.58–17.20)].

Conclusions

Predictors for treatment failure included a duration of infection for ≥7 days prior to the initial visit and a wound diameter of ≥5 cm. A heightened awareness of these risk factors could help direct targeted interventions in high-risk populations.