Helicobacter pylori causes gastric ulcer diseases and gastric adenocarcinoma in humans. Not much is known regarding DNA replication in H.pylori that is important for cell survival. Here we report the cloning, expression and characterization of H.pylori DnaB (HpDnaB) helicase both in vitro and in vivo. Among the DnaB homologs, only Escherichia coli DnaB has been studied extensively. HpDnaB showed strong 5' to 3' helicase and ATPase activity. Interestingly, H.pylori does not have an obvious DnaC homolog which is essential for DnaB loading on the E.coli chromosomal DNA replication origin (oriC). However, HpDnaB can functionally complement the E.coli DnaB temperature-sensitive mutant at the non-permissive temperature, confirming that HpDnaB is a true replicative helicase. Escherichia coli DnaC co-eluted in the same fraction with HpDnaB following gel filtration analysis suggesting that these proteins might physically interact with each other. It is possible that a functional DnaC homolog is present in H.pylori. The complete characterization of H.pylori DnaB helicase will also help the comparative analysis of DnaB helicases among bacteria.

Systemic lupus erythematosus (SLE) is mediated by autoreactive antibodies that damage multiple tissues. Genome-wide association studies (GWAS) link >60 loci with SLE risk, but the causal variants and effector genes are largely unknown. We generated high-resolution spatial maps of SLE variant accessibility and gene connectivity in human follicular helper T cells (TFH), a cell type required for anti-nuclear antibodies characteristic of SLE. Of thẽ 400 potential regulatory variants identified, 90% exhibit spatial proximity to genes distant in the 1D genome sequence, including variants that loop to regulate the canonical TFH genes BCL6 and CXCR5 as confirmed by genome editing. SLE 'variant-to-gene' maps also implicate genes with no known role in TFH/SLE disease biology, including the kinases HIPK1 and MINK1. Targeting these kinases in TFH inhibits production of IL-21, a cytokine crucial for class-switched B cell antibodies. These studies offer mechanistic insight into the SLEassociated regulatory architecture of the human genome.

Background: Foxp3 post-translational regulation remains unclear. Results: Foxp3 is phosphorylated by cyclin-dependent kinase 2 (CDK2) and has increased stability and function without its CDK motifs. Conclusion: CDK2 is a negative regulator of Foxp3 function. Significance: Understanding how Foxp3 is stabilized could lead to new therapeutic measures for autoimmunity and transplantation.

SummaryThe mechanism of DNA replication initiation and progression is poorly understood in the parasites, including human malaria parasite Plasmodium falciparum. Using bioinformatics tools and yeast complementation assay, we identified a putative homologue of Saccharomyces cerevisiae origin recognition complex subunit 5 in P. falciparum (PfORC5). PfORC5 forms distinct nuclear foci colocalized with the replication foci marker proliferating cell nuclear antigen (PfPCNA) and co-immunoprecipitates with PCNA during early-to-mid trophozoite stage replicating parasites. Interestingly, these proteins separate from each other at the non-replicating late schizont stage, citing the evidence of the presence of both PCNA and ORC components in replication foci during eukaryotic DNA replication. PfORC1, another ORC subunit, colocalizes with PfPCNA and PfORC5 at the beginning of DNA replication, but gets degraded at the late schizont stage, ensuring the regulation of DNA replication in the parasites. Further, we have identified putative PCNA-interacting protein box in PfORC1 that may explain in part the colocalization of PfORC and PfPCNA. Additionally, use of specific DNA replication inhibitor hydroxyurea affects ORC5/PCNA foci formation and parasitic growth. These results strongly favour replication factory model in the parasites and confer great potential to understand the co-ordination between ORC and PCNA during eukaryotic DNA replication in general.

Type 2 diabetes is associated with increased mortality and progression to heart failure. Recent studies suggest that diabetes also impairs reparative responses after cell therapy. In this study, we examined potential mechanisms by which diabetes affects cardiac progenitor cells (CPCs). CPCs isolated from the diabetic heart showed diminished proliferation, a propensity for cell death, and a pro-adipogenic phenotype. The diabetic CPCs were insulin-resistant, and they showed higher energetic reliance on glycolysis, which was associated with up-regulation of the pro-glycolytic enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3). In WT CPCs, expression of a mutant form of PFKFB, which mimics PFKFB3 activity and increases glycolytic rate, was sufficient to phenocopy the mitochondrial and proliferative deficiencies found in diabetic cells. Consistent with activation of phosphofructokinase in diabetic cells, stable isotope carbon tracing in diabetic CPCs showed dysregulation of the pentose phosphate and glycero(phospho)lipid synthesis pathways. We describe diabetes-induced dysregulation of carbon partitioning using stable isotope metabolomicsbased coupling quotients, which relate relative flux values between metabolic pathways. These findings suggest that diabetes causes an imbalance in glucose carbon allocation by uncoupling biosynthetic pathway activity, which could diminish the efficacy of CPCs for myocardial repair.

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Plasmodium falciparum sirtuin, PfSir2, contains histone deacetylase (HDAC) activity that may be central to the regulation of virulence gene expression in the parasites. Although a few reports have been published recently regarding in vitro and in vivo function of PfSir2, expression of the endogenous protein (c. 30 kDa) has not been shown yet. Here we report the presence of PfSir2 in the parasite at the protein level by specific antibodies. HDAC activity of PfSir2 can be inhibited by nicotinamide, a product of sirtuin reaction. Surprisingly, we find that nicotinamide also delays parasite growth significantly in culture. These findings further our knowledge on PfSir2 and raise the possibility of using an inexpensive agent like nicotinamide as an antimalarial in combination with other antiparasitic drugs.

Although cell therapy improves cardiac function after myocardial infarction, highly variable results and limited understanding of the underlying mechanisms preclude its clinical translation. Because many heart failure patients are diabetic, we examined how diabetic conditions affect the characteristics of cardiac mesenchymal cells (CMC) and their ability to promote myocardial repair in mice. To examine how diabetes affects CMC function, we isolated CMCs from non-diabetic C57BL/6J (CMCWT) or diabetic B6.BKS(D)-Leprdb/J (CMCdb/db) mice. When CMCs were grown in 17.5 mM glucose, CMCdb/db cells showed > twofold higher glycolytic activity and a threefold higher expression of Pfkfb3 compared with CMCWT cells; however, culture of CMCdb/db cells in 5.5 mM glucose led to metabolic remodeling characterized by normalization of metabolism, a higher NAD+/NADH ratio, and a sixfold upregulation of Sirt1. These changes were associated with altered extracellular vesicle miRNA content as well as proliferation and cytotoxicity parameters comparable to CMCWT cells. To test whether this metabolic improvement of CMCdb/db cells renders them suitable for cell therapy, we cultured CMCWT or CMCdb/db cells in 5.5 mM glucose and then injected them into infarcted hearts of non-diabetic mice (CMCWT, n = 17; CMCdb/db, n = 13; Veh, n = 14). Hemodynamic measurements performed 35 days after transplantation showed that, despite normalization of their properties in vitro, and unlike CMCWT cells, CMCdb/db cells did not improve load-dependent and -independent parameters of left ventricular function. These results suggest that diabetes adversely affects the reparative capacity of CMCs and that modulating CMC characteristics via culture in lower glucose does not render them efficacious for cell therapy.