Class III Histidine Kinases: a Recently Accessorized Kinase Domain in Putative Modulators of Type IV Pilus-Based Motility.

Histidine kinases are key components of regulatory systems that enable bacteria to respond to environmental changes. Two major classes of histidine kinases are recognized on the basis of their modular design: classical (HKI) and chemotaxis specific (HKII). Recently, a new type of histidine kinase that appeared to have features of both HKIs and HKIIs was identified and termed HKIII; however, the details of HKIII's relationship to other two classes of histidine kinases, their function, and evolutionary history remain unknown. Here, we carried out genomic, phylogenetic, and protein sequence analyses that allowed us to reveal the unusual evolutionary history of this protein family, formalize its distinctive features, and propose its putative function. HKIIIs are characterized by the presence of sensory domains and the lack of a dimerization domain, which is typically present in all histidine kinases. In addition to a single-domain response regulator, HKIII signal transduction systems utilize CheX phosphatase and, in many instances, an unorthodox soluble chemoreceptor that are usual components of chemotaxis signal transduction systems. However, many HKIII genes are found in genomes completely lacking chemotaxis genes, thus decoupling their function from chemotaxis. By contrast, all HKIII-containing genomes also contain pilT, a marker gene for bacterial type IV pilus-based motility, whose regulation is proposed as a putative function for HKIII. These signal transduction systems have a narrow phyletic distribution but are present in many emerging and opportunistic pathogens, thus offering an attractive potential target for future antimicrobial drug design.IMPORTANCE Bacteria adapt to their environment and their hosts by detecting signals and regulating their cellular functions accordingly. Here, we describe a largely unexplored family of signal transduction histidine kinases, called HKIII, that have a unique modular design. While they are currently identified in a relatively short list of bacterial species, this list contains many emerging pathogens. We show that HKIIIs likely control bacterial motility across solid surfaces, which is a key virulence factor in many bacteria, including those causing severe infections. Full understanding of this putative function may help in designing effective drugs against pathogens that will not affect the majority of the beneficial human microbiome.

Establishing the precise evolutionary history of a gene improves prediction of disease-causing missense mutations.

PURPOSE: Predicting the phenotypic effects of mutations has become an important application in clinical genetic diagnostics. Computational tools evaluate the behavior of the variant over evolutionary time and assume that variations seen during the course of evolution are probably benign in humans. However, current tools do not take into account orthologous/paralogous relationships. Paralogs have dramatically different roles in Mendelian diseases. For example, whereas inactivating mutations in the NPC1 gene cause the neurodegenerative disorder Niemann-Pick C, inactivating mutations in its paralog NPC1L1 are not disease-causing and, moreover, are implicated in protection from coronary heart disease. METHODS: We identified major events in NPC1 evolution and revealed and compared orthologs and paralogs of the human NPC1 gene through phylogenetic and protein sequence analyses. We predicted whether an amino acid substitution affects protein function by reducing the organism's fitness. RESULTS: Removing the paralogs and distant homologs improved the overall performance of categorizing disease-causing and benign amino acid substitutions. CONCLUSION: The results show that a thorough evolutionary analysis followed by identification of orthologs improves the accuracy in predicting disease-causing missense mutations. We anticipate that this approach will be used as a reference in the interpretation of variants in other genetic diseases as well.Genet Med 18 10, 1029-1036.

Joint mouse-human phenome-wide association to test gene function and disease risk.

Phenome-wide association is a novel reverse genetic strategy to analyze genome-to-phenome relations in human clinical cohorts. Here we test this approach using a large murine population segregating for ∼5 million sequence variants, and we compare our results to those extracted from a matched analysis of gene variants in a large human cohort. For the mouse cohort, we amassed a deep and broad open-access phenome consisting of ∼4,500 metabolic, physiological, pharmacological and behavioural traits, and more than 90 independent expression quantitative trait locus (QTL), transcriptome, proteome, metagenome and metabolome data sets--by far the largest coherent phenome for any experimental cohort (www.genenetwork.org). We tested downstream effects of subsets of variants and discovered several novel associations, including a missense mutation in fumarate hydratase that controls variation in the mitochondrial unfolded protein response in both mouse and Caenorhabditis elegans, and missense mutations in Col6a5 that underlies variation in bone mineral density in both mouse and human.

siRNA technology in kidney transplantation: current status and future potential.

Kidney transplantation is one of the most common transplantation operations in the world, accounting for up to 50 % of all transplantation surgeries. To curtail the damage to transplanted organs that is caused by ischemia-reperfusion injury and the recipient's immune system, small interfering RNA (siRNA) technology is being explored. Importantly, the kidney as a whole is a preferential site for non-specific systemic delivery of siRNA. To date, most attempts at siRNA-based therapy for transplantation-related conditions have remained at the in vitro stage, with only a few of them being advanced into animal models. Hydrodynamic intravenous injection of naked or carrier-bound siRNAs is currently the most common route for delivery of therapeutic constructs. To our knowledge, no systematic screens for siRNA targets most relevant for kidney transplantation have been attempted so far. A majority of researchers have arrived at one or another target of interest by analyzing current literature that dissects pathological processes taking place in transplanted organs. A majority of the genes that make up the list of 53 siRNA targets that have been tested in transplantation-related models so far belong to either apoptosis- or immune rejection-centered networks. There is an opportunity for therapeutic siRNA combinations that may be delivered within the same delivery vector or injected at the same time and, by targeting more than one pathway, or by hitting the same pathways within two different key points, will augment the effects of each other.

Uncontrolled donors with controlled reperfusion after sixty minutes of asystole: a novel reliable resource for kidney transplantation.

BACKGROUND: Organ shortage leads to usage of kidneys from donors after sudden cardiac death, or uncontrolled donors (UDCD). Ischemic injury due to cessation of circulation remains a crucial problem that limits adoption of UDCD. Our clinical investigation was to determine the applicability of kidneys obtained from UDCD and resuscitated by extracorporeal perfusion in situ after 60 minutes of asystole. METHODS: In 2009-2011, organ procurement service of St. Petersburg, obtained kidneys from 22 UDCD with critically expanded warm ischemic time (WIT). No patients were considered as potential organ donors initially. All donors died after sudden irreversible cardiac arrest. Mean WIT was 61.4±4.5 minutes. For kidney resuscitation, the subnormothermic extracorporeal abdominal perfusion with thrombolytics and leukocyte depletion was employed. Grafts were transplanted into 44 recipients. The outcomes of transplantation of resuscitated kidneys were compared to outcomes of 87 KTx from 74 brain death donors (BDDs). RESULTS: Immediate functioning of kidney grafts was observed in 21 of the 44 recipients, with no cases of primary non function. By the end of the first post-transplant year there was an acute rejection rate of 9.1% (4 episodes of rejection) in the UDCD group versus 14.2% (13 episodes of rejection) in the BDD group. The actual 1-year graft survival rate was 95.5% (n = 42) in UDCD group, and 94.6% (n = 87) in BDD group. Creatinine levels at the end of the first year were 0.116±0.008 and 0.115±0.004 mmol/l in UDCD and BDD groups, respectively. CONCLUSIONS: UDCD kidneys with critically expanded WIT could be succefully used for transplantation if in situ organ "resuscitation" perfusion is included into procurement protocol. The results of 1-year follow-up meet the generally accepted criteria for graft survival and function. In situ reperfusion may exert a therapeutic effect on grafts before procurement. This approach could substantially expand the organ donors' pool.