Epitope Mapping of Antibodies Suggests the Novel Membrane Topology of B-Cell Receptor Associated Protein 31 on the Cell Surface of Embryonic Stem Cells: The Novel Membrane Topology of BAP31

When located in the endoplasmic reticulum (ER) membrane, B-cell receptor associated protein 31 (BAP31) is involved in the export of secreted proteins from the ER to the plasma membrane. In a previous study, we generated two monoclonal antibodies (mAbs), 297-D4 and 144-A8, that bound to surface molecules on human embryonic stem cells (hESCs), but not to surface molecules on mouse embryonic stem cells (mESCs). Subsequent studies revealed that the mAbs recognized BAP31 on the surface of hESCs. To investigate the membrane topology of BAP31 on the cell surface, we first examined the epitope specificity of 297-D4 and 144-A8, as well as a polyclonal anti-BAP31 antibody (alpha-BAP31). We generated a series of GST-fused BAP31 mutant proteins in which BAP31 was serially deleted at the C-terminus. GST-fused BAP31 mutant proteins were then screened to identify the epitopes targeted by the antibodies. Both 297-D4 and 144-A8 recognized C-terminal residues 208-217, while alpha-BAP31 recognized C-terminal residues 165-246, of BAP31 on hESCs, suggesting that the C-terminal domain of BAP31 is exposed on the cell surface. The polyclonal antibody alpha-BAP31 bound to mESCs, which confirmed that the C-terminal domain of BAP31 is also exposed on the surface of these cells. Our results show for the first time the novel membrane topology of cell surface-expressed BAP31 as the extracellular exposure of the BAP31 C-terminal domain was not predicted from previous studies.published_or_final_versio

Rabies Virus CVS-N2c(Delta G) Strain Enhances Retrograde Synaptic Transfer and Neuronal Viability

Summary Virally based transsynaptic tracing technologies are powerful experimental tools for neuronal circuit mapping. The glycoprotein-deletion variant of the SAD-B19 vaccine strain rabies virus (RABV) has been the reagent of choice in monosynaptic tracing, since it permits the mapping of synaptic inputs to genetically marked neurons. Since its introduction, new helper viruses and reagents that facilitate complementation have enhanced the efficiency of SAD-B19ΔG transsynaptic transfer, but there has been little focus on improvements to the core RABV strain. Here we generate a new deletion mutant strain, CVS-N2cΔG, and examine its neuronal toxicity and efficiency in directing retrograde transsynaptic transfer. We find that by comparison with SAD-B19ΔG, the CVS-N2cΔG strain exhibits a reduction in neuronal toxicity and a marked enhancement in transsynaptic neuronal transfer. We conclude that the CVS-N2cΔG strain provides a more effective means of mapping neuronal circuitry and of monitoring and manipulating neuronal activity in vivo in the mammalian CNS

Retinoid X receptor promotes hematopoietic stem cell fitness and quiescence and preserves hematopoietic homeostasis.

Hematopoietic stem cells (HSCs) balance self-renewal and differentiation to maintain hematopoietic fitness throughout life. In steady-state conditions, HSC exhaustion is prevented by the maintenance of most HSCs in a quiescent state, with cells entering the cell cycle only occasionally. HSC quiescence is regulated by retinoid and fatty-acid ligands of transcriptional factors of the nuclear retinoid X receptor (RXR) family. Here, we show that dual deficiency for hematopoietic RXRa and RXRb induces HSC exhaustion, myeloid cell/megakaryocyte differentiation, and myeloproliferative-like disease. RXRa and RXRb maintain HSC quiescence, survival, and chromatin compaction; moreover, transcriptome changes in RXRa;RXRb-deficient HSCs include premature acquisition of an aging-like HSC signature, MYC pathway upregulation, and RNA intron retention. Fitness loss and associated RNA transcriptome and splicing alterations in RXRa;RXRb-deficient HSCs are prevented by Myc haploinsufficiency. Our study reveals the critical importance of RXRs for the maintenance of HSC fitness and their protection from premature aging.We thank the members of the J.A.C. and M.R. laboratories for extensive discussions and critiques of the manuscript. We thank Daniel Metzger (Université de Strasbourg, France) for Rxrbf/f 418 mice, Juan Carlos Zúñiga-Pflücker (Sunnybrook Health Sciences Centre, Canada) for OP9-NL1 cells, Daniel Jiménez-Carretero (CNIC) for t-SNE analysis, the CRG (Barcelona, Spain) Genomics Unit for ATACseq sequencing, and S. Bartlett (CNIC) for editorial assistance. We also thank the staff of the CNIC Cellomics and Animal facilities for technical support. This study was supported by grants from the Spanish Ministerio de Ciencia e Innovación (MICIN) (SAF2017-90604-REDT-NurCaMein, RTI2018- 095928-B100, and PID2021-122552OB-I00), La Marató de TV3 Foundation (201605-32), and the Comunidad de Madrid (MOIR-B2017/BMD-3684) to M.R and from the Formación de Profesorado Universitario (FPU17/01731) program (MICIN) to J.P. The project also received funding from the US National Institutes of Health (R01 DK124115, P01 HL158688, R01 HL147536, R01 CA237016 and U54 DK126108 to J.A.C). The CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the Ministerio de Ciencia e Innovación (MCIN), and the Pro CNIC Foundation and is a Severo Ochoa Center of Excellence (grant CEX2020-001041-S funded by MICIN/AEI/10.13039/501100011033).S

Structural insight into SUMO chain recognition and manipulation by the ubiquitin ligase RNF4

The small ubiquitin-like modifier (SUMO) can form polymeric chains that are important signals in cellular processes such as meiosis, genome maintenance and stress response. The SUMO-targeted ubiquitin ligase RNF4 engages with SUMO chains on linked substrates and catalyses their ubiquitination, which targets substrates for proteasomal degradation. Here we use a segmental labelling approach combined with solution nuclear magnetic resonance (NMR) spectroscopy and biochemical characterization to reveal how RNF4 manipulates the conformation of the SUMO chain, thereby facilitating optimal delivery of the distal SUMO domain for ubiquitin transfer

Role of rapid urease test and histopathology in the diagnosis of Helicobacter pylori infection in a developing country

BACKGROUND: The aim of this study was to determine the effect of commonly self-prescribed proton pump inhibitors (PPI) on the results of rapid urease test and histology for the diagnosis of H. pylori infection. METHODS: One hundred-nine consecutive patients with dyspeptic symptoms attending the endoscopy suite were enrolled in this study. Antrum biopsy specimens were collected at endoscopy for the rapid urease test (Pronto Dry, Medical Instrument Corp, France) and histopathology. Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and like-hood ratio of a positive and negative of Pronto Dry test were compared against histology. The gold standard test for the diagnosis of H. pylori infection was histopathology. RESULTS: Sixty-one percent (66/109) patients were males with mean age of 43 ± 14.1 years and age range 17–80 years. Fifty-two percent (57/109) were not on any medications while 48% (52/109) used PPI before presentation to the outpatients. Pronto Dry was positive in 40% (44/109) and negative in 60% (65/109). Histopathology was positive for H. pylori in 57% (62/109) and negative in 43% (47/109). The sensitivity, specificity, PPV, NPV and like-hood ratio of a positive and negative Pronto Dry test with and without PPI were 43.3%, 86.4%, 81.3%, 3.18, 0.656 and 52.8% vs 71.9%, 80%, 82.1%, 69%, 3.59 and 0.35. CONCLUSION: This study shows that the sensitivity, specificity, NPV and PPV of rapid urease test was reduced in patients who are on PPI. The exclusive use of the rapid urease test for the diagnosis of Helicobacter pylori cannot be recommended in patients with prior PPI use

Abnormal ECG Findings in Athletes: Clinical Evaluation and Considerations.

PURPOSE OF REVIEW: Pre-participation cardiovascular evaluation with electrocardiography is normal practice for most sporting bodies. Awareness about sudden cardiac death in athletes and recognizing how screening can help identify vulnerable athletes have empowered different sporting disciplines to invest in the wellbeing of their athletes. RECENT FINDINGS: Discerning physiological electrical alterations due to athletic training from those representing cardiac pathology may be challenging. The mode of investigation of affected athletes is dependent on the electrical anomaly and the disease(s) in question. This review will highlight specific pathological ECG patterns that warrant assessment and surveillance, together with an in-depth review of the recommended algorithm for evaluation

Activity of a novel, dual PI3-kinase/mTor inhibitor NVP-BEZ235 against primary human pancreatic cancers grown as orthotopic xenografts

The phosphatidylinositol-3-kinase (PI3K)/Akt signalling pathway is frequently deregulated in pancreatic cancers, and is believed to be an important determinant of their biological aggression and drug resistance. NVP-BEZ235 is a novel, dual class I PI3K/mammalian target of rapamycin (mTor) inhibitor undergoing phase I human clinical trials. To simulate clinical testing, the effects of NVP-BEZ235 were studied in five early passage primary pancreatic cancer xenografts, grown orthotopically. These tumours showed activated PKB/Akt, and increased levels of at least one of the receptor tyrosine kinases that are commonly activated in pancreatic cancers. Pharmacodynamic effects were measured following acute single doses, and anticancer effects were determined in separate groups following chronic drug exposure. Acute oral dosing with NVP-BEZ235 strongly suppressed the phosphorylation of PKB/Akt, followed by recovery over 24 h. There was also inhibition of Ser235/236 S6 ribosomal protein and Thr37/46 4E-BP1, consistent with the effects of NVP-BEZ235 as a dual PI3K/mTor inhibitor. Chronic dosing with 45 mg kg−1 of NVP-BEZ235 was well tolerated, and produced significant tumour growth inhibition in three models. These results predict that agents targeting the PI3K/Akt/mTor pathway might have anticancer activity in pancreatic cancer patients, and support the testing of combination studies involving chemotherapy or other molecular targeted agents

Epistasis and genotype-by-environment interaction of grain protein content in durum wheat

Parental, F1 , F 2 , BC 1 and BC 2 generations of four crosses involving four cultivars of durum wheat (Triticum durum Desf.) were evaluated at two sites in Tunisia. A three-parameter model was found inadequate for all cases except crosses Chili x Cocorit 71 at site Sidi Thabet and Inrat 69 x Karim at both sites. In most cases a digenic epistatic model was sufficient to explain variation in generation means. Dominance effects (h) and additive x additive epistasis (i) (when significant) were more important than additive (d) effects and other epistatic components. Considering the genotype-by-environment interaction, the non-interactive model (m, d, h, e) was found adequate. Additive variance was higher than environmental variance in three crosses at both sites. The estimated values of narrow-sense heritability were dependent upon the cross and the sites and were 0%-85%. The results indicate that appropriate choice of environment and selection in later generations would increase grain protein content in durum wheat

Quality of life in patients with various Barrett's esophagus associated health states

BACKGROUND: The management of Barrett's esophagus (BE), particularly high grade dysplasia (HGD), is an area of much debate and controversy. Surgical esophagectomy, intensive endoscopic surveillance and mucosal ablative techniques, especially photodynamic therapy (PDT), have been proposed as possible management strategies. The purpose of this study was to determine the health related quality of life associated with Barrett's esophagus and many of the pivotal health states associated with Barrett's HGD management. METHODS: 20 patients with Barrett's esophagus were enrolled in a pilot survey study at a large urban hospital. The utility of Barrett's esophagus without dysplasia (current health state) as well as various health states associated with HGD management (hypothetical states as the subject did not have HGD) were measured using a validated health utility instrument (Paper Standard Gamble). These specific health states were chosen for the study because they are considered pivotal in Barrett's HGD decision making. Information regarding Barrett's HGD was presented to the subject in a standardized format that was designed to be easily comprehendible. RESULTS: The average utility scores (0–1 with 0 = death and 1 = perfect health) for the various Barrett's esophagus associated states were: BE without dysplasia-0.95; Post-esophagectomy for HGD with dysphagia-0.92; Post-PDT for HGD with recurrence uncertainty-0.93; Post-PDT for HGD with recurrence uncertainty and dysphagia-0.91; Intensive endoscopic surveillance for HGD-0.90. CONCLUSION: We present the scores for utilities associated with Barrett's esophagus as well as various states associated with the management of HGD. The results of our study may be useful in advising patients and providers regarding expected outcomes of the various HGD management strategies as well as providing utility scores for future cost-effectiveness analyses

Exploring Chromophore-Binding Pocket: High-Resolution Solid-State 1H–13C Interfacial Correlation NMR Spectra with Windowed PMLG Scheme

High-resolution two-dimensional (2D) 1H–13C heteronuclear correlation spectra are recorded for selective observation of interfacial 3–5.5 Å contacts of the uniformly 13C-labeled phycocyanobilin (PCB) chromophore with its unlabeled binding pocket. The experiment is based on a medium- and long-distance heteronuclear correlation (MELODI–HETCOR) method. For improving 1H spectral resolution, a windowed phase-modulated Lee–Goldburg (wPMLG) decoupling scheme is applied during the t1 evolution period. Our approach allows for identification of chromophore–protein interactions, in particular for elucidation of the hydrogen-bonding networks and charge distributions within the chromophore-binding pocket. The resulting pulse sequence is tested on the cyanobacterial (Cph1) phytochrome sensory module (residues 1–514, Cph1Δ2) containing uniformly 13C- and 15N-labeled PCB chromophore (u-[13C,15N]-PCB-Cph1Δ2) at 17.6 T