Molecular association of glucose-6- phosphate isomerase and pyruvate kinase M2 with glyceraldehyde-3-phosphate dehydrogenase in cancer cells

Background: For a long time cancer cells are known for increased uptake of glucose and its metabolization through glycolysis. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a key regulatory enzyme of this pathway and can produce ATP through oxidative level of phosphorylation. Previously, we reported that GAPDH purified from a variety of malignant tissues, but not from normal tissues, was strongly inactivated by a normal metabolite, methylglyoxal (MG).Molecular mechanism behind MG mediated GAPDH inhibition in cancer cells is not well understood. Methods: GAPDH was purified from Ehrlich ascites carcinoma (EAC) cells based on its enzymatic activity. GAPDH associated proteins in EAC cells and 3-methylcholanthrene (3MC) induced mouse tumor tissue were detected by mass spectrometry analysis and immunoprecipitation (IP) experiment, respectively. Interacting domains of GAPDH and its associated proteins were assessed by in silico molecular docking analysis. Mechanism of MG mediated GAPDH inactivation in cancer cells was evaluated by measuring enzyme activity, Circular dichroism (CD) spectroscopy, IP and mass spectrometry analyses. Result: Here, we report that GAPDH is associated with glucose-6-phosphate isomerase (GPI) and pyruvate kinase M2 (PKM2) in Ehrlich ascites carcinoma (EAC) cells and also in 3-methylcholanthrene (3MC) induced mouse tumor tissue. Molecular docking analyses suggest C-terminal domain preference for the interaction between GAPDH and GPI. However, both C and N termini of PKM2 might be interacting with the C terminal domain of GAPDH. Expression of both PKM2 and GPI is increased in 3MC induced tumor compared with the normal tissue. In presence of 1 mM MG,association of GAPDH with PKM2 or GPI is not perturbed, but the enzymatic activity of GAPDH is reduced to 26.8 ± 5 % in 3MC induced tumor and 57.8 ± 2.3 % in EAC cells. Treatment of MG to purified GAPDH complex leads to glycation at R399 residue of PKM2 only, and changes the secondary structure of the protein complex. Conclusion: PKM2 may regulate the enzymatic activity of GAPDH. Increased enzymatic activity of GAPDH in tumor cells may be attributed to its association with PKM2 and GPI. Association of GAPDH with PKM2 and GPI could be a signature for cancer cells. Glycation at R399 of PKM2 and changes in the secondary structure of GAPDH complex could be one of the mechanisms by which GAPDH activity is inhibited in tumor cells by MG

Mortality and pulmonary complications in patients undergoing surgery with perioperative SARS-CoV-2 infection: an international cohort study

Background: The impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on postoperative recovery needs to be understood to inform clinical decision making during and after the COVID-19 pandemic. This study reports 30-day mortality and pulmonary complication rates in patients with perioperative SARS-CoV-2 infection. Methods: This international, multicentre, cohort study at 235 hospitals in 24 countries included all patients undergoing surgery who had SARS-CoV-2 infection confirmed within 7 days before or 30 days after surgery. The primary outcome measure was 30-day postoperative mortality and was assessed in all enrolled patients. The main secondary outcome measure was pulmonary complications, defined as pneumonia, acute respiratory distress syndrome, or unexpected postoperative ventilation. Findings: This analysis includes 1128 patients who had surgery between Jan 1 and March 31, 2020, of whom 835 (74·0%) had emergency surgery and 280 (24·8%) had elective surgery. SARS-CoV-2 infection was confirmed preoperatively in 294 (26·1%) patients. 30-day mortality was 23·8% (268 of 1128). Pulmonary complications occurred in 577 (51·2%) of 1128 patients; 30-day mortality in these patients was 38·0% (219 of 577), accounting for 81·7% (219 of 268) of all deaths. In adjusted analyses, 30-day mortality was associated with male sex (odds ratio 1·75 [95% CI 1·28–2·40], p\textless0·0001), age 70 years or older versus younger than 70 years (2·30 [1·65–3·22], p\textless0·0001), American Society of Anesthesiologists grades 3–5 versus grades 1–2 (2·35 [1·57–3·53], p\textless0·0001), malignant versus benign or obstetric diagnosis (1·55 [1·01–2·39], p=0·046), emergency versus elective surgery (1·67 [1·06–2·63], p=0·026), and major versus minor surgery (1·52 [1·01–2·31], p=0·047). Interpretation: Postoperative pulmonary complications occur in half of patients with perioperative SARS-CoV-2 infection and are associated with high mortality. Thresholds for surgery during the COVID-19 pandemic should be higher than during normal practice, particularly in men aged 70 years and older. Consideration should be given for postponing non-urgent procedures and promoting non-operative treatment to delay or avoid the need for surgery. Funding: National Institute for Health Research (NIHR), Association of Coloproctology of Great Britain and Ireland, Bowel and Cancer Research, Bowel Disease Research Foundation, Association of Upper Gastrointestinal Surgeons, British Association of Surgical Oncology, British Gynaecological Cancer Society, European Society of Coloproctology, NIHR Academy, Sarcoma UK, Vascular Society for Great Britain and Ireland, and Yorkshire Cancer Research

An internal segment (residues 58-119) of the hepatitis B virus X protein is sufficient to activate MAP kinase pathways in mouse liver

The human hepatitis B virus X protein (HBx) is known as a dual-specificity transactivator stimulating the transcriptional machinery in the nucleus and signal transduction pathways in the cytoplasm. HBx-induced activation of mitogen-activated protein kinase (MAPK) signaling cascades is considered to play an important role in hepatitis B virus-mediated hepatocarcinogenesis. Herein, we have identified the regions of HBx that are crucial for activating such signaling cascades in vivo. A truncated mutant incorporating regions C-E (amino acids 58-140) was as effective as the full-length HBx in activating MAPKs and enhancing activator protein-1 binding activity. While deletion of region C (amino acids 58-84) or D (amino acids 85-119) led to a drastic loss of function, region E (amino acids 120-140) was dispensable for the activation of signaling cascades. Overall, these findings provide the first evidence for the requirement of domain 58-119 of HBx in transmitting mitogenic signals to the nucleus in vivo

Hydrogen-Bonding-Induced Chain Folding and Vesicular Assembly of an Amphiphilic Polyurethane

We have reported synthesis and vesicular assembly of a novel amphiphilic polyurethane with hydrophobic backbone and hydrophilic pendant carboxylic acid groups which were periodically grafted to the backbone via a tertiary amine group. In aqueous medium the polymer chain adopted a folded conformation which was stabilized by intrachain H-bonding among the urethane groups. Such a model was supported by concentration and solvent-dependent FT-IR, powder XRD, and urea-mediated “denaturation” experiments. Folded polymer chains further formed vesicular assembly which was probed by dynamic light scattering, TEM, AFM, SEM, and fluorescence microscopic studies, and dye encapsulation experiments. pH-dependent DLS and fluorescence microscopic studies revealed stable polymersome in entire tested pH window of 3.5–11.0. Zeta potential measurements showed a negatively charged surface in basic pH while a charge-neutral surface in neutral and acidic pH. MTT assay with CHO cell line indicated good cell viability

An Alternatively Spliced Isoform of Non-muscle Myosin II-C Is Not Regulated by Myosin Light Chain Phosphorylation*

We report a novel isoform of non-muscle myosin II-C (NM II-C), NM II-C2, that is generated by alternative splicing of an exon, C2, encoding 41 amino acids in mice (33 in humans). The 41 amino acids are inserted into loop 2 of the NM II-C heavy chain within the actin binding region. Unlike most vertebrate non-muscle and smooth muscle myosin IIs, baculovirus-expressed mouse heavy meromyosin (HMM) II-C2 demonstrates no requirement for regulatory myosin light chain (MLC20) phosphorylation for maximum actin-activated MgATPase activity or maximum in vitro motility as measured by the sliding actin filament assay. In contrast, noninserted HMM II-C0 and another alternatively spliced isoform HMM II-C1, which contains 8 amino acids inserted into loop 1, are dependent on MLC20 phosphorylation for both actin-activated MgATPase activity and in vitro motility (Kim, K. Y., Kovacs, M., Kawamoto, S., Sellers, J. R., and Adelstein, R. S. (2005) J. Biol. Chem. 280, 22769-2277515845534). HMM II-C1C2, which contains both the C1 and C2 inserts, does not require MLC20 phosphorylation for full activity similar to HMM II-C2. These constitutively active C2-inserted isoforms of NM II-C are expressed only in neuronal tissue. This is in contrast to NM II-C1 and NM II-C0, both of which are ubiquitously expressed. Full-length NM II-C2-GFP expressed in COS-7 cells localizes to filaments in interphase cells and to the cytokinetic ring in dividing cells

Evaluation strategies for measuring pneumococcal conjugate vaccine impact in low-resource settings.

OBJECTIVES: Pneumococcal conjugate vaccines (PCVs) are effective in reducing pneumococcal disease. We measured 13-valent PCV (PCV13) effect on different pneumococcal outcomes using diverse studies in Lao People's Democratic Republic. METHODS: Studies included: pre-PCV13 population-based record review of hospitalized childhood pneumonia cases; acute respiratory infection (ARI) study post-PCV13 to demonstrate effectiveness (VE) against hypoxic pneumonia; invasive pneumococcal disease (IPD) surveillance in all ages (2004-2018); carriage studies in children hospitalized with ARI (2013-2019); community carriage surveys pre- and post-PCV13. RESULTS: Annual pneumonia incidence rate in children pre-PCV13 was 1,530 (95% confidence interval [CI] 1,477-1,584) per 100,000. Adjusted VE against hypoxic pneumonia was 37% (95% CI 6-57%). For IPD, 85% (11/13) of cases were due to vaccine-types pre-PCV13, and 43% (3/7) post-PCV13 in children aged <5 years; for ≥5 years, 61% (27/44) and 42% (17/40), respectively. For ARI cases, adjusted VE for vaccine-type carriage was 39% (95% CI 4-60) in <5 year olds; slightly higher than community surveys (23% [95% CI 4-39%] in 12-23 month olds). CONCLUSIONS: Despite limited baseline data, we found evidence of PCV13 impact on disease and carriage. Our approach could be used in similar settings to augment existing WHO PCV evaluation guidelines

Ablation of Nonmuscle Myosin II-B and II-C Reveals a Role for Nonmuscle Myosin II in Cardiac Myocyte Karyokinesis

Ablation of nonmuscle myosin (NM) II-B and II-C in mice results in a defect in cardiac myocyte karyokinesis. More than 90% of the double knockout cardiac myocytes demonstrate defects in chromatid segregation and mitotic spindle formation. The requirement for NM II in karyokinesis is demonstrated in both the intact heart and in HL-1 atrial myocytes in culture