Abstract The S100s are a large group of Ca2+ sensors found exclusively in vertebrates. Transcriptomic and genomic data from the major radiations of mammals were used to derive the evolution of the ...mammalian S100s genes. In human and mouse, S100s and S100 fused-type proteins are in a separate clade from other Ca2+ sensor proteins, indicating that an ancient bifurcation between these two gene lineages has occurred. Furthermore, the five genomic loci containing S100 genes have remained largely intact during the past 165 million years since the shared ancestor of egg-laying and placental mammals. Nonetheless, interesting births and deaths of S100 genes have occurred during mammalian evolution. The S100A7 loci exhibited the most plasticity and phylogenetic analyses clarified relationships between the S100A7 proteins encoded in the various mammalian genomes. Phylogenetic analyses also identified four conserved subgroups of S100s that predate the rise of warm-blooded vertebrates: A2/A3/A4/A5/A6, A1/A10/A11/B/P/Z, A13/A14/A16, and A7s/A8/A9/A12/G. The similarity between genomic location and phylogenetic clades suggest that these subfamilies arose by a series of tandem gene duplication events. Examination of annotated S100s in lower vertebrates suggests that the ancestral S100 was a member of the A1/A10/A11/B/P/Z subgroup and arose near the emergence of vertebrates approximately 500 million years ago.
The host structural maintenance of chromosomes 5/6 complex (Smc5/6) suppresses hepatitis B virus (HBV) transcription. HBV counters this restriction by expressing the X protein (HBx), which redirects ...the cellular DNA damage-binding protein 1 (DDB1)-containing E3 ubiquitin ligase to target Smc5/6 for degradation. However, the details of how HBx modulates the interaction between DDB1 and Smc5/6 remain to be determined. In this study, we performed biophysical analyses of recombinant HBx and functional analysis of HBx mutants in HBV-infected primary human hepatocytes (PHH) to identify key regions and residues that are required for HBx function. We determined that recombinant HBx is soluble and exhibits stoichiometric zinc binding when expressed in the presence of DDB1. Mass spectrometry-based hydrogen-deuterium exchange and cysteine-specific chemical footprinting of the HBx:DDB1 complex identified several HBx cysteine residues (located between amino acids 61 and 137) that are likely involved in zinc binding. These cysteine residues did not form disulfide bonds in HBx expressed in human cells. In line with the biophysical data, functional analysis demonstrated that HBx amino acids 45 to 140 are required for Smc6 degradation and HBV transcription in PHH. Furthermore, site-directed mutagenesis determined that C61, C69, C137, and H139 are necessary for HBx function, although they are likely not essential for DDB1 binding. This CCCH motif is highly conserved in HBV as well as in the X proteins from various mammalian hepadnaviruses. Collectively, our data indicate that the essential HBx cysteine and histidine residues form a zinc-binding motif that is required for HBx function.
The structural maintenance of chromosomes 5/6 complex (Smc5/6) is a host restriction factor that suppresses HBV transcription. HBV counters this restriction by expressing HBV X protein (HBx), which redirects a host ubiquitin ligase to target Smc5/6 for degradation. Despite this recent advance in understanding HBx function, the key regions and residues of HBx required for Smc5/6 degradation have not been determined. In the present study, we performed biochemical, biophysical, and cell-based analyses of HBx. By doing so, we mapped the minimal functional region of HBx and identified a highly conserved CCCH motif in HBx that is likely responsible for coordinating zinc and is essential for HBx function. We also developed a method to produce soluble recombinant HBx protein that likely adopts a physiologically relevant conformation. Collectively, this study provides new insights into the HBx structure-function relationship and suggests a new approach for structural studies of this enigmatic viral regulatory protein.
•A CODEHOP PCR based screening method was employed for type III polyketide synthase gene identification in fungal endophytes.•By this approach, partial type III PKS genes from eight fungal endophytes ...were amplified and sequenced.•FiPKS gene from Fusarium incarnatum BMER1, an endophyte of Bacopa monnieri was cloned and functionally characterized.•FiPKS produced pyrones and resorcinols with the highest catalytic efficiency of 7.6 x 104 s-1 M-1towards stearoyl CoA.
Endophytic fungi provide benefits to host plants by producing a diverse class of secondary metabolites (natural products). Arrays of polyketide natural products are synthesized by specific classes of polyketide synthases (PKS I, II and III) in host organisms. In the present study, we attempt to screen and identify type III PKSs in culturable fungal endophytes isolated from the ethno medicinal plants including Arbus precatorius, Bacopa monnieri,Citrus aurantifolia and Datura metel to detect the genetic potential of endophytic fungi in producing bioactive compounds. A total of seventeen endophytic fungal strains belonging to eight genera were identified using fungal morphology and rDNA-ITS phylogenetic analyses. A CODEHOP-PCR based strategy was followed to design degenerate primers for the screening of type III PKS genes from fungal endophytes. We had successfully amplified partial PKS genes from eight endophytes. The amplified PKS sequences showed 60–99% identity to already characterized/putative PKS genes. From the partial sequence of FiPKS from Fusarium incarnatum BMER1, a full-length gene was amplified, cloned and characterized. FiPKScDNA was cloned and expressed in E. coli Lemo21 (DE3) and the purified protein was shown to produce pyrones and resorcinols using acyl-CoA thioesters as substrates. FiPKS showed the highest catalytic efficiency of 7.6 × 104 s−1 M-1 with stearoyl CoA as a starter unit. This study reports the identification and characterization of type III PKS from endophytes of medicinal plants by CODEHOP PCR.
•The study reports the functional characterization of two fungal type III polyketide synthases, SmPKS and CtPKS with high catalytic efficiency.•Both the recombinant PKSs efficiently synthesize ...pyrones, resorcylic acids and resorcinols using various acyl- CoA (C4–C20) as starter units.•TrCtPKS produces pentaketide and hexaketide resorcinols from arachidoyl-CoA with a high catalytic efficiency of 6.2 × 104 s−1 M−1.•Enhanced binding interactions of CtPKS residues forming intermolecular contacts at the active site could be attributed to its high specificity.
Two putative type III polyketide synthase genes (PKS) were identified from Sordariomycetes fungi. These two type III PKS genes from Sordaria macrospora (SmPKS) and Chaetomium thermophilum (CtPKS), shared 59.8% sequence identity. Both, full-length and truncated versions of type III PKSs were successfully cloned and overexpressed in a bacterial host, Escherichia Coli BL21 (DE3) using a N-terminus hexa-histidine tag. The full-length and the truncated construct of PKSs showed similar activity profiles, suggesting that additional amino acid residues at the C-terminal of both SmPKS and CtPKS may not be involved in catalytic functions. We demonstrate that these two recombinant polyketide synthases could efficiently synthesize tri- and tetraketide pyrones, resorcinols and resorcylic acids using various acyl-CoAs (C4–C20) as starter units. The truncated S. macrospora polyketide synthases (TrSmPKS) showed a maximum of 7.0 × 104 s−1 M−1 catalytic efficiency towards stearoyl-CoA.Whereas, truncated C. thermophilum polyketide synthases (TrCtPKS) preferred the long-chain acyl-CoA starter arachidoyl-CoA, to produce pentaketide and hexaketide resorcinols with a high catalytic efficiency of 6.2 × 104 s−1 M−1. Homology model and substrate docking analyses suggest a shorter distance between sulfur of catalytic Cys152 and thioester carbonyl group of arachidoyl-CoA as well as stronger imidazolium–thiolate ion pair distance in TrCtPKS between catalytic Cys152-His309 compared to TrSmPKS- arachidoyl CoA complex. Enhanced binding interactions of CtPKS residues forming intermolecular contacts at the active site could be attributed to its high specificity towards arachidoyl-CoA. This study reports the functional characterization of two fungal type III polyketide synthases, SmPKS and CtPKS with high catalytic efficiency from S. macrospora and C. thermophilum respectively. Furthermore, the results suggested that the both SmPKS and CtPKS could be attractive targets for protein engineering to discern the unique substrate specificity and catalytic efficiency.
High-grade gliomas, such as glioblastomas (GBMs), are very aggressive, invasive brain tumors with low patient survival rates. The recent identification of distinct glioma tumor subtypes offers the ...potential for understanding disease pathogenesis, responses to treatment and identification of molecular targets for personalized cancer therapies. However, the key alterations that drive tumorigenesis within each subtype are still poorly understood. Although aberrant NF-kappaB activity has been implicated in glioma, the roles of specific members of this protein family in tumorigenesis and pathogenesis have not been elucidated. In this study, we show that the NF-kappaB protein RelB is expressed in a particularly aggressive mesenchymal subtype of glioma, and loss of RelB significantly attenuated glioma cell survival, motility and invasion. We find that RelB promotes the expression of mesenchymal genes including YKL-40, a marker of the MES glioma subtype. Additionally, RelB regulates expression of Olig2, a regulator of cancer stem cell proliferation and a candidate marker for the cell of origin in glioma. Furthermore, loss of RelB in glioma cells significantly diminished tumor growth in orthotopic mouse xenografts. The relevance of our studies for human disease was confirmed by analysis of a human GBM genome database, which revealed that high RelB expression strongly correlates with rapid tumor progression and poor patient survival rates. Thus, our findings demonstrate that RelB is an oncogenic driver of mesenchymal glioma tumor growth and invasion, highlighting the therapeutic potential of inhibiting the noncanonical NF-kappaB (RelB-mediated) pathway to treat these deadly tumors.
Postoperative pain management plays a vital role in the recovery after surgery. All these days opioids were used to accomplish this, but their use can be associated with adverse effects which can ...prolong the hospital stay. In the present era of opioid epidemic, it is our responsibility to look for other alternatives, other drugs and modalities which can be safely be given to opioid naive surgical patients. The concept of multi-modal analgesia came as a paradigm shift lately by employing loco regional analgesia as well as drugs acting on different receptors of nociception. One of the pharmaceutical agents is Lignocaine which when given intravenously as continuous infusion produced analgesia in concentrations similar to epidural route. We conducted a study on continuous intra operative infusion of lignocaine in 1mg/kg/hr after a bolus dose. Lignocaine produced better hemodynamic stability, reduced consumption of volatile anaesthetics, less postoperative nausea vomiting, sedation and facilitated early ambulation in patients after laparoscopic surgeries.
BACKGROUND Chronic obstructive pulmonary disease (COPD) is the third leading cause of death worldwide, and causes significant morbidity. Cardiovascular compromise is often associated with COPD, ...especially in acute exacerbations. The purpose of this study is to find out the incidence of elevation of troponin I levels in acute exacerbation of COPD patients and measuring the outcome in terms of need for ventilation (both invasive and non-invasive), length of hospital stay & mortality. METHODS This was a prospective analytical study done on 30 patients with acute exacerbation of COPD who were admitted in Government Mohan Kumaramangalam Medical College and Hospital, Salem, Tamil Nadu from December 2015 to June 2016. Troponin I levels were estimated for all patients on admission. A cut off value of more than 34.2 pg/ml was considered as elevation. A written informed consent was obtained. Clinical outcomes were studied by doing echocardiogram to measure the pulmonary artery systolic pressures (PASP), the need for mechanical ventilation (both invasive and non-invasive), length of stay in the hospital and mortality. RESULTS The pulmonary artery systolic pressures were 52 mm of Hg vs 40.3 mm Hg (P < 0.002), length of hospital stay was 9.67 vs 6.63 days (P < 0.027), patients who required ventilatory support were 13 out of the 30 and the mean duration of ventilation was higher in troponin I elevated patients 5.67 vs 3.57 days (P < 0.0015) and mortality was higher in patients with increased troponin I levels (2 deaths) when compared to patients with normal troponin I levels (1 death)and is statistically significant (P < 0.001). CONCLUSIONS There was a significant elevation of troponin I in acute exacerbation COPD patients. Our study concluded that the presence of elevated levels of troponin I in acute exacerbation of COPD is associated with increased morbidity in terms of increased need for mechanical ventilation, intensive care unit (ICU) stay and mortality. KEYWORDS Cardiac Biomarker, NIV, PASP, Troponin I
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![The NF-[kappa]B RelB Protei...](http://api.summon.serialssolutions.com/2.0.0/image/issn/XR4PS5YY4K/1932-6203/small "The NF-[kappa]B RelB Protein Is an Oncogenic Driver of Mesenchymal Glioma")
