Rapid determination of substrate specificity of Clostridium histolyticum beta-collagenase using an immobilized peptide library.

The molecular basis of the substrate specificity of Clostridium histolyticum beta-collagenase was investigated using a combinatorial method. An immobilized positional peptide library, which contains 24,000 sequences, was constructed with a 7-hydroxycoumarin-4-propanoyl (Cop) fluorescent group attached at the N terminus of each sequence. This immobilized peptide library was incubated with C. histolyticum beta-collagenase, releasing fluorogenic fragments in the solution phase. The relative substrate specificity (k(cat)/K(m)) for each member of the library was determined by measuring fluorescence intensity in the solution phase. Edman sequencing was used to assign structure to subsites of active substrate mixtures. Collectively, the substrate preference for subsites (P(3)-P(4)') of C. histolyticum beta-collagenase was determined. The last position on the C-terminal side in which the identity of the amino acids affects the activity of the enzyme is P(4)', and an aromatic side chain is preferred in this position. The optimal P(1)'-P(3)' extended substrate sequence is P(1)'-Gly/Ala, P(2)'-Pro/Xaa, and P(3)'-Lys/Arg/Pro/Thr/Ser. The Cop group in either the P(2) or P(3) position is required for a high substrate activity with C. histolyticum beta-collagenase. S(2) and S(3) sites of the protease play a dominant role in fixing the substrate specificity. The immobilized peptide library proved to be a powerful approach for assessing the substrate specificity of C. histolyticum beta-collagenase, so it may be applied to the study of other proteases of interest.     

Multiomic Metabolic Enrichment Network Analysis Reveals Metabolite–Protein Physical Interaction Subnetworks Altered in Cancer

Metabolism is recognized as an important driver of cancer progression and other complex diseases, but global metabolite profiling remains a challenge. Protein expression profiling is often a poor proxy since existing pathway enrichment models provide an incomplete mapping between the proteome and metabolism. To overcome these gaps, we introduce multiomic metabolic enrichment network analysis (MOMENTA), an integrative multiomic data analysis framework for more accurately deducing metabolic pathway changes from proteomics data alone in a gene set analysis context by leveraging protein interaction networks to extend annotated metabolic models. We apply MOMENTA to proteomic data from diverse cancer cell lines and human tumors to demonstrate its utility at revealing variation in metabolic pathway activity across cancer types, which we verify using independent metabolomics measurements. The novel metabolic networks we uncover in breast cancer and other tumors are linked to clinical outcomes, underscoring the pathophysiological relevance of the findings.     

Cloning and characterization of a SnRK1-encoding gene from <Emphasis Type="Italic">Malus hupehensis</Emphasis> Rehd. and heterologous expression in tomato

Sucrose non-fermenting-1-related protein kinase-1 (SnRK1) plays an important role in metabolic regulation in plant. To understand the molecular mechanism of amino acids and carbohydrate metabolism in Malus hupehensis Rehd. var. pinyiensis Jiang (Pingyi Tiancha, PYTC), a full-length cDNA clone encoding homologue of SnRK1 was isolated from PYTC by Rapid Amplification of cDNA Ends (RACE). The clone, designated as MhSnRK1, contains 2063 nucleotides with an open reading frame of 1548 nucleotides. The deduced 515 amino acids showed high identities with other plant SnRK1 genes. Quantitative real-time PCR analysis revealed this gene was expressed in roots, stems and leaves. Exposing seedlings to nitrate caused and initial decrease in expression of the MhSnRK1 gene in roots, leaves and stems in short term. Ectopic expression of MhSnRK1 in tomato mainly resulted in higher starch content in leaf and red-ripening fruit than wild-type plants. This result supports the hypothesis that overexpression of SnRK1 causes the accumulation of starch in plant cells. All the results suggest that MhSnRK1 may play important roles in carbohydrate and amino acid metabolisms.     

High Rates of Obesity and Chronic Disease Among United Methodist Clergy

We used self‐reported data from United Methodist clergy to assess the prevalence of obesity and having ever been told certain chronic disease diagnoses. Of all actively serving United Methodist clergy in North Carolina (NC) 95% (n = 1726) completed self‐report height and weight items and diagnosis questions from the Behavioral Risk Factor Surveillance Survey (BRFSS). We calculated BMI categories and diagnosis prevalence rates for the clergy and compared them to the NC population using BRFSS data. The obesity rate among clergy aged 35–64 years was 39.7%, 10.3% (95% CI = 8.5%, 12.1%) higher than their NC counterparts. Clergy also reported significantly higher rates of having ever been given diagnoses of diabetes, arthritis, high blood pressure, angina, and asthma compared to their NC peers. Health interventions that address obesity and chronic disease among clergy are urgently needed.     

恶性疟原虫抗原表位的亚单位疫苗与DNA疫苗的联合免疫

构建了含有恶性疟原虫抗原基因 ( AWTE)的真核表达质粒 p CMV- AWTE,以及能在大肠杆菌中得到分泌性表达的原核表达质粒 p MC0 5 ,表达的蛋白 AWTE保持了疟原虫抗原的抗原性。将 p CMV- AWTE以及 AWTE两者混合各 1 0μg鼻腔免疫小鼠 ,一次后诱导机体产生了较高水平的体液免疫及细胞免疫     

Genome Sequence of Staphylococcus epidermidis Strain AU12-03, Isolated from an Intravascular Catheter

In recent years, Staphylococcus epidermidis has become a major nosocomial pathogen and the most common cause of intravascular catheter-related bacteremia, which can increase morbidity and mortality and significantly affect patient recovery. We report a draft genome sequence of Staphylococcus epidermidis AU12-03, isolated from an intravascular catheter tip.     

Sphingosine kinase 1 regulates HMGB1 translocation by directly interacting with calcium/calmodulin protein kinase II-δ in sepsis-associated liver injury

Previously, we confirmed that sphingosine kinase 1 (SphK1) inhibition improves sepsis-associated liver injury. High-mobility group box 1 (HMGB1) translocation participates in the development of acute liver failure. However, little information is available on the association between SphK1 and HMGB1 translocation during sepsis-associated liver injury. In the present study, we aimed to explore the effect of SphK1 inhibition on HMGB1 translocation and the underlying mechanism during sepsis-associated liver injury. Primary Kupffer cells and hepatocytes were isolated from SD rats. The rat model of sepsis-associated liver damage was induced by intraperitoneal injection with lipopolysaccharide (LPS). We confirmed that Kupffer cells were the cells primarily secreting HMGB1 in the liver after LPS stimulation. LPS-mediated HMGB1 expression, intracellular translocation, and acetylation were dramatically decreased by SphK1 inhibition. Nuclear histone deacetyltransferase 4 (HDAC4) translocation and E1A-associated protein p300 (p300) expression regulating the acetylation of HMGB1 were also suppressed by SphK1 inhibition. HDAC4 intracellular translocation has been reported to be controlled by the phosphorylation of HDAC4. The phosphorylation of HDAC4 is modulated by CaMKII-δ. However, these changes were completely blocked by SphK1 inhibition. Additionally, by performing coimmunoprecipitation and pull-down assays, we revealed that SphK1 can directly interact with CaMKII-δ. The colocalization of SphK1 and CaMKII-δ was verified in human liver tissues with sepsis-associated liver injury. In conclusion, SphK1 inhibition diminishes HMGB1 intracellular translocation in sepsis-associated liver injury. The mechanism is associated with the direct interaction of SphK1 and CaMKII-δ.Subject terms: Hepatotoxicity, Sepsis     

Evolution of green lacewings (Neuroptera: Chrysopidae): a molecular supermatrix approach

We present a time‐calibrated phylogeny of the charismatic green lacewings (Neuroptera: Chrysopidae). Previous phylogenetic studies on the family using DNA sequences have suffered from sparse taxon sampling and/or limited amounts of data. Here we combine all available previously published DNA sequence data and add to it new DNA sequences generated for this study. We analysed these data in a supermatrix using Bayesian and maximum likelihood methods and provide a phylogenetic hypothesis for the family that recovers strong support for the monophyly of all subfamilies and resolves relationships among a large proportion of chrysopine genera. Chrysopinae tribes Leucochrysini and Belonopterygini were recovered as monophyletic sister clades, while the species‐rich tribe Chrysopini was rendered paraphyletic by Ankylopterygini. Relationships among the subfamilies were resolved, although with relatively low statistical support, and the topology varied based on the method of analysis. Greatest support was found for Apochrysinae as sister to Nothochrysinae and Chrysopinae, which is in contrast to traditional concepts that place Nothochrysinae as sister to the rest of the family. Divergence estimates suggest that the stem groups to the various subfamilies diverged during the Triassic‐Jurassic, and that stem groups of the chrysopine tribes diverged during the Cretaceous.     

A fast dynamic mode of the EF‐G‐bound ribosome

A key intermediate in translocation is an ‘unlocked state’ of the pre‐translocation ribosome in which the P‐site tRNA adopts the P/E hybrid state, the L1 stalk domain closes and ribosomal subunits adopt a ratcheted configuration. Here, through two‐ and three‐colour smFRET imaging from multiple structural perspectives, EF‐G is shown to accelerate structural and kinetic pathways in the ribosome, leading to this transition. The EF‐G‐bound ribosome remains highly dynamic in nature, wherein, the unlocked state is transiently and reversibly formed. The P/E hybrid state is energetically favoured, but exchange with the classical P/P configuration persists; the L1 stalk adopts a fast dynamic mode characterized by rapid cycles of closure and opening. These data support a model in which P/E hybrid state formation, L1 stalk closure and subunit ratcheting are loosely coupled, independent processes that must converge to achieve the unlocked state. The highly dynamic nature of these motions, and their sensitivity to conformational and compositional changes in the ribosome, suggests that regulating the formation of this intermediate may present an effective avenue for translational control.