The Extracytoplasmic Domain of the Mycobacterium tuberculosis Ser/Thr Kinase PknB Binds Specific Muropeptides and Is Required for PknB Localization

The Mycobacterium tuberculosis Ser/Thr kinase PknB has been implicated in the regulation of cell growth and morphology in this organism. The extracytoplasmic domain of this membrane protein comprises four penicillin binding protein and Ser/Thr kinase associated (PASTA) domains, which are predicted to bind stem peptides of peptidoglycan. Using a comprehensive library of synthetic muropeptides, we demonstrate that the extracytoplasmic domain of PknB binds muropeptides in a manner dependent on the presence of specific amino acids at the second and third positions of the stem peptide, and on the presence of the sugar moiety N-acetylmuramic acid linked to the peptide. We further show that PknB localizes strongly to the mid-cell and also to the cell poles, and that the extracytoplasmic domain is required for PknB localization. In contrast to strong growth stimulation by conditioned medium, we observe no growth stimulation of M. tuberculosis by a synthetic muropeptide with high affinity for the PknB PASTAs. We do find a moderate effect of a high affinity peptide on resuscitation of dormant cells. While the PASTA domains of PknB may play a role in stimulating growth by binding exogenous peptidoglycan fragments, our data indicate that a major function of these domains is for proper PknB localization, likely through binding of peptidoglycan fragments produced locally at the mid-cell and the cell poles. These data suggest a model in which PknB is targeted to the sites of peptidoglycan turnover to regulate cell growth and cell division

Interaction and Modulation of Two Antagonistic Cell Wall Enzymes of Mycobacteria

Bacterial cell growth and division require coordinated cell wall hydrolysis and synthesis, allowing for the removal and expansion of cell wall material. Without proper coordination, unchecked hydrolysis can result in cell lysis. How these opposing activities are simultaneously regulated is poorly understood. In Mycobacterium tuberculosis, the resuscitation-promoting factor B (RpfB), a lytic transglycosylase, interacts and synergizes with Rpf-interacting protein A (RipA), an endopeptidase, to hydrolyze peptidoglycan. However, it remains unclear what governs this synergy and how it is coordinated with cell wall synthesis. Here we identify the bifunctional peptidoglycan-synthesizing enzyme, penicillin binding protein 1 (PBP1), as a RipA-interacting protein. PBP1, like RipA, localizes both at the poles and septa of dividing cells. Depletion of the ponA1 gene, encoding PBP1 in M. smegmatis, results in a severe growth defect and abnormally shaped cells, indicating that PBP1 is necessary for viability and cell wall stability. Finally, PBP1 inhibits the synergistic hydrolysis of peptidoglycan by the RipA-RpfB complex in vitro. These data reveal a post-translational mechanism for regulating cell wall hydrolysis and synthesis through protein–protein interactions between enzymes with antagonistic functions

CLE14/CLE20 peptides may interact with CLAVATA2/CORYNE receptor-like kinases to irreversibly inhibit cell division in the root meristem of Arabidopsis

Towards an understanding of the interacting nature of the CLAVATA (CLV) complex, we predicted the 3D structures of CLV3/ESR-related (CLE) peptides and the ectodomain of their potential receptor proteins/kinases, and docking models of these molecules. The results show that the ectodomain of CLV1 can form homodimers and that the 12-/13-amino-acid CLV3 peptide fits into the binding clefts of the CLV1 dimers. Our results also demonstrate that the receptor domain of CORYNE (CRN), a recently identified receptor-like kinase, binds tightly to the ectodomain of CLV2, and this likely leads to an increased possibility for docking with CLV1. Furthermore, our docking models reveal that two CRN-CLV2 ectodomain heterodimers are able to form a tetramer receptor complex. Peptides of CLV3, CLE14, CLE19, and CLE20 are also able to bind a potential CLV2-CRN heterodimer or heterotetramer complex. Using a cell-division reporter line, we found that synthetic 12-amino-acid CLE14 and CLE20 peptides inhibit, irreversibly, root growth by reducing cell division rates in the root apical meristem, resulting in a short-root phenotype. Intriguingly, we observed that exogenous application of cytokinin can partially rescue the short-root phenotype induced by over-expression of either CLE14 or CLE20 in planta. However, cytokinin treatment does not rescue the short-root phenotype caused by exogenous application of the synthetic CLE14/CLE20 peptides, suggesting a requirement for a condition provided only in living plants. These results therefore imply that the CLE14/CLE20 peptides may act through the CLV2-CRN receptor kinase, and that their availabilities and/or abundances may be affected by cytokinin activity in planta

Accreting Millisecond X-Ray Pulsars

Accreting Millisecond X-Ray Pulsars (AMXPs) are astrophysical laboratories without parallel in the study of extreme physics. In this chapter we review the past fifteen years of discoveries in the field. We summarize the observations of the fifteen known AMXPs, with a particular emphasis on the multi-wavelength observations that have been carried out since the discovery of the first AMXP in 1998. We review accretion torque theory, the pulse formation process, and how AMXP observations have changed our view on the interaction of plasma and magnetic fields in strong gravity. We also explain how the AMXPs have deepened our understanding of the thermonuclear burst process, in particular the phenomenon of burst oscillations. We conclude with a discussion of the open problems that remain to be addressed in the future.Comment: Review to appear in "Timing neutron stars: pulsations, oscillations and explosions", T. Belloni, M. Mendez, C.M. Zhang Eds., ASSL, Springer; [revision with literature updated, several typos removed, 1 new AMXP added

High Expression of Wee1 Is Associated with Poor Disease-Free Survival in Malignant Melanoma: Potential for Targeted Therapy

Notoriously resistant malignant melanoma is one of the most increasing forms of cancer worldwide; there is thus a precarious need for new treatment options. The Wee1 kinase is a major regulator of the G2/M checkpoint, and halts the cell cycle by adding a negative phosphorylation on CDK1 (Tyr15). Additionally, Wee1 has a function in safeguarding the genome integrity during DNA synthesis. To assess the role of Wee1 in development and progression of malignant melanoma we examined its expression in a panel of paraffin-embedded patient derived tissue of benign nevi and primary- and metastatic melanomas, as well as in agarose-embedded cultured melanocytes. We found that Wee1 expression increased in the direction of malignancy, and showed a strong, positive correlation with known biomarkers involved in cell cycle regulation: Cyclin A (p<0.0001), Ki67 (p<0.0001), Cyclin D3 (p = 0.001), p21Cip1/WAF1 (p = 0.003), p53 (p = 0.025). Furthermore, high Wee1 expression was associated with thicker primary tumors (p = 0.001), ulceration (p = 0.005) and poor disease-free survival (p = 0.008). Transfections using siWee1 in metastatic melanoma cell lines; WM239WTp53, WM45.1MUTp53 and LOXWTp53, further support our hypothesis of a tumor promoting role of Wee1 in melanomas. Whereas no effect was observed in LOX cells, transfection with siWee1 led to accumulation of cells in G1/S and S phase of the cell cycle in WM239 and WM45.1 cells, respectively. Both latter cell lines displayed DNA damage and induction of apoptosis, in the absence of Wee1, indicating that the effect of silencing Wee1 may not be solely dependent of the p53 status of the cells. Together these results reveal the importance of Wee1 as a prognostic biomarker in melanomas, and indicate a potential role for targeted therapy, alone or in combination with other agents

Gaining Greater Insight into HCV Emergence in HIV-Infected Men Who Have Sex with Men: The HEPAIG Study

OBJECTIVES: The HEPAIG study was conducted to better understand Hepatitis C virus (HCV) transmission among human immuno-deficiency (HIV)-infected men who have sex with men (MSM) and assess incidence of HCV infection among this population in France. METHODS AND RESULTS: Acute HCV infection defined by anti-HCV or HCV ribonucleic acid (RNA) positivity within one year of documented anti-HCV negativity was notified among HIV-infected MSM followed up in HIV/AIDS clinics from a nationwide sampling frame. HIV and HCV infection characteristics, HCV potential exposures and sexual behaviour were collected by the physicians and via self-administered questionnaires. Phylogenetic analysis of the HCV-NS5B region was conducted. HCV incidence was 48/10 000 [95% Confidence Interval (CI):43-54] and 36/10 000 [95% CI: 30-42] in 2006 and 2007, respectively. Among the 80 men enrolled (median age: 40 years), 55% were HIV-diagnosed before 2000, 56% had at least one sexually transmitted infection in the year before HCV diagnosis; 55% were HCV-infected with genotype 4 (15 men in one 4d-cluster), 32.5% with genotype 1 (three 1a-clusters); five men were HCV re-infected; in the six-month preceding HCV diagnosis, 92% reported having casual sexual partners sought online (75.5%) and at sex venues (79%), unprotected anal sex (90%) and fisting (65%); using recreational drugs (62%) and bleeding during sex (55%). CONCLUSIONS: This study emphasizes the role of multiple unprotected sexual practices and recreational drugs use during sex in the HCV emergence in HIV-infected MSM. It becomes essential to adapt prevention strategies and inform HIV-infected MSM with recent acute HCV infection on risk of re-infection and on risk-reduction strategies

Effects of supplemented isoenergetic diets varying in cereal fiber and protein content on the bile acid metabolic signature and relation to insulin resistance

Bile acids (BA) are potent metabolic regulators influenced by diet. We studied effects of isoenergetic increases in the dietary protein and cereal-fiber contents on circulating BA and insulin resistance (IR) in overweight and obese adults. Randomized controlled nutritional intervention (18 weeks) in 72 non-diabetic participants (overweight/obese: 29/43) with at least one further metabolic risk factor. Participants were group-matched and allocated to four isoenergetic supplemented diets: control; high cereal fiber (HCF); high-protein (HP); or moderately increased cereal fiber and protein (MIX). Whole-body IR and insulin-mediated suppression of hepatic endogenous glucose production were measured using euglycaemic–hyperinsulinemic clamps with [6-62H2] glucose infusion. Circulating BA, metabolic biomarkers, and IR were measured at 0, 6, and 18 weeks. Under isoenergetic conditions, HP-intake worsened IR in obese participants after 6 weeks (M-value: 3.77 ± 0.58 vs. 3.07 ± 0.44 mg/kg/min, p = 0.038), with partial improvement back to baseline levels after 18 weeks (3.25 ± 0.45 mg/kg/min, p = 0.089). No deleterious effects of HP-intake on IR were observed in overweight participants. HCF-diet improved IR in overweight participants after 6 weeks (M-value 4.25 ± 0.35 vs. 4.81 ± 0.31 mg/kg/min, p = 0.016), but did not influence IR in obese participants. Control and MIX diets did not influence IR. HP-induced, but not HCF-induced changes in IR strongly correlated with changes of BA profiles. MIX-diet significantly increased most BA at 18 weeks in obese, but not in overweight participants. BA remained unchanged in controls. Pooled BA concentrations correlated with fasting fibroblast growth factor-19 (FGF-19) plasma levels (r = 0.37; p = 0.003). Higher milk protein intake was the only significant dietary predictor for raised total and primary BA in regression analyses (total BA, p = 0.017; primary BA, p = 0.011). Combined increased intake of dietary protein and cereal fibers markedly increased serum BA concentrations in obese, but not in overweight participants. Possible mechanisms explaining this effect may include compensatory increases of the BA pool in the insulin resistant, obese state; or defective BA transport