Crystal structure of the H256A mutant of rat testis fructose-6- phosphate,2-kinase/fructose-2,6-bisphosphatase: Fructose 6-phosphate in the active site leads to mechanisms for both mutant and wild type bisphosphatase activities

Fructose-6-phosphate,2-kinase/fructose-2,6-bisphosphatase (Fru-6-P,2- kinase/Fru-2,6-Pase) is a bifunctional enzyme, catalyzing the interconversion of β-D-fructose-6-phosphate (Fru-6-P) and fructose-2,6-bisphosphate (Fru- 2,6-P2) at distinct active sites. A mutant rat testis isozyme with an alanine replacement for the catalytic histidine (H256A) in the Fru-2,6-Pase domain retains 17% of the wild type activity (Mizuguchi, H., Cook, P. F., Tai, C-H., Hasemann, C. A., and Uyeda, K. (1998) J. Biol. Chem. 274, 2166- 2175). We have solved the crystal structure of H256A to a resolution of 2.4 Å by molecular replacement. Clear electron density for Fru-6-P is found at the Fru-2,6-Pase active site, revealing the important interactions in substrate/product binding. A superposition of the H256A structure with the RT2K-Wo structure reveals no significant reorganization of the active site resulting from the binding of Fru-6-P or the H256A mutation. Using this superposition, we have built a view of the Fru-2,6-P2-bound enzyme and identify the residues responsible for catalysis. This analysis yields distinct catalytic mechanisms for the wild type and mutant proteins. The wild type mechanism would lead to an inefficient transfer of a proton to the leaving group Fru-6-P, which is consistent with a view of this event being rate-limiting, explaining the extremely slow turnover (0.032 s-1) of the Fru-2,6-Pase in all Fru-6-P,2-kinase/Fru-2,6-Pase isozymes

Protein profiling in hepatocellular carcinoma by label-free quantitative proteomics in two west african populations.

Background Hepatocellular Carcinoma is the third most common cause of cancer related death worldwide, often diagnosed by measuring serum AFP; a poor performance stand-alone biomarker. With the aim of improving on this, our study focuses on plasma proteins identified by Mass Spectrometry in order to investigate and validate differences seen in the respective proteomes of controls and subjects with LC and HCC. Methods Mass Spectrometry analysis using liquid chromatography electro spray ionization quadrupole time-of-flight was conducted on 339 subjects using a pooled expression profiling approach. ELISA assays were performed on four significantly differentially expressed proteins to validate their expression profiles in subjects from the Gambia and a pilot group from Nigeria. Results from this were collated for statistical multiplexing using logistic regression analysis. Results Twenty-six proteins were identified as differentially expressed between the three subject groups. Direct measurements of four; hemopexin, alpha-1-antitrypsin, apolipoprotein A1 and complement component 3 confirmed their change in abundance in LC and HCC versus control patients. These trends were independently replicated in the pilot validation subjects from Nigeria. The statistical multiplexing of these proteins demonstrated performance comparable to or greater than ALT in identifying liver cirrhosis or carcinogenesis. This exercise also proposed preliminary cut offs with achievable sensitivity, specificity and AUC statistics greater than reported AFP averages. Conclusions The validated changes of expression in these proteins have the potential for development into high-performance tests usable in the diagnosis and or monitoring of HCC and LC patients. The identification of sustained expression trends strengthens the suggestion of these four proteins as worthy candidates for further investigation in the context of liver disease. The statistical combinations also provide a novel inroad of analyses able to propose definitive cut-offs and combinations for evaluation of performance

First-in-class humanized FSH blocking antibody targets bone and fat

Blocking the action of FSH genetically or pharmacologically in mice reduces body fat, lowers serum cholesterol, and increases bone mass, making an anti-FSH agent a potential therapeutic for three global epidemics: obesity, osteoporosis, and hypercholesterolemia. Here, we report the generation, structure, and function of a first-in-class, fully humanized, epitope-specific FSH blocking antibody with a KD of 7 nM. Protein thermal shift, molecular dynamics, and fine mapping of the FSH-FSH receptor interface confirm stable binding of the Fab domain to two of five receptor-interacting residues of the FSHβ subunit, which is sufficient to block its interaction with the FSH receptor. In doing so, the humanized antibody profoundly inhibited FSH action in cell-based assays, a prelude to further preclinical and clinical testing

TSPYL2 Is Important for G1 Checkpoint Maintenance upon DNA Damage

Nucleosome assembly proteins play important roles in chromatin remodeling, which determines gene expression, cell proliferation and terminal differentiation. Testis specific protein, Y-encoded-like 2 (TSPYL2) is a nucleosome assembly protein expressed in neuronal precursors and mature neurons. Previous studies have shown that TSPYL2 binds cyclin B and inhibits cell proliferation in cultured cells suggesting a role in cell cycle regulation. To investigate the physiological significance of TSPYL2 in the control of cell cycle, we generated mice with targeted disruption of Tspyl2. These mutant mice appear grossly normal, have normal life span and do not exhibit increased tumor incidence. To define the role of TSPYL2 in DNA repair, checkpoint arrest and apoptosis, primary embryonic fibroblasts and thymocytes from Tspyl2 deficient mice were isolated and examined under unperturbed and stressed conditions. We show that mutant fibroblasts are impaired in G1 arrest under the situation of DNA damage induced by gamma irradiation. This is mainly attributed to the defective activation of p21 transcription despite proper p53 protein accumulation, suggesting that TSPYL2 is additionally required for p21 induction. TSPYL2 serves a biological role in maintaining the G1 checkpoint under stress condition

Pseudomonas aeruginosa Eliminates Natural Killer Cells via Phagocytosis-Induced Apoptosis

Pseudomonas aeruginosa (PA) is an opportunistic pathogen that causes the relapse of illness in immunocompromised patients, leading to prolonged hospitalization, increased medical expense, and death. In this report, we show that PA invades natural killer (NK) cells and induces phagocytosis-induced cell death (PICD) of lymphocytes. In vivo tumor metastasis was augmented by PA infection, with a significant reduction in NK cell number. Adoptive transfer of NK cells mitigated PA-induced metastasis. Internalization of PA into NK cells was observed by transmission electron microscopy. In addition, PA invaded NK cells via phosphoinositide 3-kinase (PI3K) activation, and the phagocytic event led to caspase 9-dependent apoptosis of NK cells. PA-mediated NK cell apoptosis was dependent on activation of mitogen-activated protein (MAP) kinase and the generation of reactive oxygen species (ROS). These data suggest that the phagocytosis of PA by NK cells is a critical event that affects the relapse of diseases in immunocompromised patients, such as those with cancer, and provides important insights into the interactions between PA and NK cells

Influenza A H5N1 Immigration Is Filtered Out at Some International Borders

Geographic spread of highly pathogenic influenza A H5N1, the bird flu strain, appears a necessary condition for accelerating the evolution of a related human-to-human infection. As H5N1 spreads the virus diversifies in response to the variety of socioecological environments encountered, increasing the chance a human infection emerges. Genetic phylogenies have for the most part provided only qualitative evidence that localities differ in H5N1 diversity. For the first time H5N1 variation is quantified across geographic space.We constructed a statistical phylogeography of 481 H5N1 hemagglutinin genetic sequences from samples collected across 28 Eurasian and African localities through 2006. The MigraPhyla protocol showed southern China was a source of multiple H5N1 strains. Nested clade analysis indicated H5N1 was widely dispersed across southern China by both limited dispersal and long distance colonization. The UniFrac metric, a measure of shared phylogenetic history, grouped H5N1 from Indonesia, Japan, Thailand and Vietnam with those from southeastern Chinese provinces engaged in intensive international trade. Finally, H5N1's accumulative phylogenetic diversity was greatest in southern China and declined beyond. The gradient was interrupted by areas of greater and lesser phylogenetic dispersion, indicating H5N1 migration was restricted at some geopolitical borders. Thailand and Vietnam, just south of China, showed significant phylogenetic clustering, suggesting newly invasive H5N1 strains have been repeatedly filtered out at their northern borders even as both countries suffered recurring outbreaks of endemic strains. In contrast, Japan, while successful in controlling outbreaks, has been subjected to multiple introductions of the virus.The analysis demonstrates phylogenies can provide local health officials with more than hypotheses about relatedness. Pathogen dispersal, the functional relationships among disease ecologies across localities, and the efficacy of control efforts can also be inferred, all from viral genetic sequences alone

The effects of β-glucan on human immune and cancer cells

Non-prescriptional use of medicinal herbs among cancer patients is common around the world. The alleged anti-cancer effects of most herbal extracts are mainly based on studies derived from in vitro or in vivo animal experiments. The current information suggests that these herbal extracts exert their biological effect either through cytotoxic or immunomodulatory mechanisms. One of the active compounds responsible for the immune effects of herbal products is in the form of complex polysaccharides known as β-glucans. β-glucans are ubiquitously found in both bacterial or fungal cell walls and have been implicated in the initiation of anti-microbial immune response. Based on in vitro studies, β-glucans act on several immune receptors including Dectin-1, complement receptor (CR3) and TLR-2/6 and trigger a group of immune cells including macrophages, neutrophils, monocytes, natural killer cells and dendritic cells. As a consequence, both innate and adaptive response can be modulated by β-glucans and they can also enhance opsonic and non-opsonic phagocytosis. In animal studies, after oral administration, the specific backbone 1→3 linear β-glycosidic chain of β-glucans cannot be digested. Most β-glucans enter the proximal small intestine and some are captured by the macrophages. They are internalized and fragmented within the cells, then transported by the macrophages to the marrow and endothelial reticular system. The small β-glucans fragments are eventually released by the macrophages and taken up by other immune cells leading to various immune responses. However, β-glucans of different sizes and branching patterns may have significantly variable immune potency. Careful selection of appropriate β-glucans is essential if we wish to investigate the effects of β-glucans clinically. So far, no good quality clinical trial data is available on assessing the effectiveness of purified β-glucans among cancer patients. Future effort should direct at performing well-designed clinical trials to verify the actual clinical efficacy of β-glucans or β-glucans containing compounds

Real-world Effectiveness and Tolerability of Interferon-free Direct-acting Antiviral for 15,849 Patients with Chronic Hepatitis C: A Multinational Cohort Study

BACKGROUND AND AIMS: As practice patterns and hepatitis C virus (HCV) genotypes (GT) vary geographically, a global real-world study from both East and West covering all GTs can help inform practice policy toward the 2030 HCV elimination goal. This study aimed to assess the effectiveness and tolerability of DAA treatment in routine clinical practice in a multinational cohort for patients infected with all HCV GTs, focusing on GT3 and GT6. METHODS: We analyzed the sustained virological response (SVR12) of 15,849 chronic hepatitis C patients from 39 Real-World Evidence from the Asia Liver Consortium for HCV clinical sites in Asia Pacific, North America, and Europe between 07/01/2014-07/01/2021. RESULTS: The mean age was 62±13 years, with 49.6% male. The demographic breakdown was 91.1% Asian (52.9% Japanese, 25.7% Chinese/Taiwanese, 5.4% Korean, 3.3% Malaysian, and 2.9% Vietnamese), 6.4% White, 1.3% Hispanic/Latino, and 1% Black/African-American. Additionally, 34.8% had cirrhosis, 8.6% had hepatocellular carcinoma (HCC), and 24.9% were treatment-experienced (20.7% with interferon, 4.3% with direct-acting antivirals). The largest group was GT1 (10,246 [64.6%]), followed by GT2 (3,686 [23.2%]), GT3 (1,151 [7.2%]), GT6 (457 [2.8%]), GT4 (47 [0.3%]), GT5 (1 [0.006%]), and untyped GTs (261 [1.6%]). The overall SVR12 was 96.9%, with rates over 95% for GT1/2/3/6 but 91.5% for GT4. SVR12 for GT3 was 95.1% overall, 98.2% for GT3a, and 94.0% for GT3b. SVR12 was 98.3% overall for GT6, lower for patients with cirrhosis and treatment-experienced (TE) (93.8%) but ≥97.5% for treatment-naive patients regardless of cirrhosis status. On multivariable analysis, advanced age, prior treatment failure, cirrhosis, active HCC, and GT3/4 were independent predictors of lower SVR12, while being Asian was a significant predictor of achieving SVR12. CONCLUSIONS: In this diverse multinational real-world cohort of patients with various GTs, the overall cure rate was 96.9%, despite large numbers of patients with cirrhosis, HCC, TE, and GT3/6. SVR12 for GT3/6 with cirrhosis and TE was lower but still excellent (\u3e91%)

Search for light resonances decaying to boosted quark pairs and produced in association with a photon or a jet in proton–proton collisions at √s=13 TeV with the ATLAS detector

This Letter presents a search for new light resonances decaying to pairs of quarks and produced in association with a high-pT photon or jet. The dataset consists of proton–proton collisions with an integrated luminosity of 36.1 fb−1at a centre-of-mass energy of √s=13TeV recorded by the ATLAS detector at the Large Hadron Collider. Resonance candidates are identified as massive large-radius jets with substructure consistent with a particle decaying into a quark pair. The mass spectrum of the candidates is examined for local excesses above background. No evidence of a new resonance is observed in the data, which are used to exclude the production of a lepto-phobic axial-vector Z boson

Correlated long-range mixed-harmonic fluctuations measured in pp, p+Pb and low-multiplicity Pb+Pb collisions with the ATLAS detector

For abstract see published article