Novel Components of the Stress Assembly Sec Body Identified by Proximity Labeling

Sec bodies are membraneless stress-induced assemblies that form by the coalescence of endoplasmic reticulum exit sites (ERES). Through APEX2 tagging of Sec24AB, we biotinylated and identified the full complement of Sec body proteins. In the presence of biotin-phenol and H2O2 (APEX on), APEX2 facilitates the transfer of a biotin moiety to nearby interactors of chimeric Sec24AB. Using this unbiased approach comparing APEX on and off (−H2O2) conditions, we identified 52 proteins specifically enriched in Sec bodies. These include a large proportion of ER and Golgi proteins, packaged without defined stoichiometry, which we could selectively verify by imaging. Interestingly, Sec body components are neither transcriptionally nor translationally regulated under the conditions that induce Sec body formation, suggesting that incorporation of these proteins into granules may be driven instead by the aggregation of nucleating proteins with a high content of intrinsically disordered regions. This reinforces the notion that Sec bodies may act as storage for ERES, ER and Golgi components during stress

Tailoring limb length based on total small bowel length in one anastomosis gastric bypass surgery (TAILOR study):study protocol for a randomized controlled trial

Background: The one anastomosis gastric bypass (OAGB) is being performed by an increasing number of bariatric centers over the world. However, the optimal length of the biliopancreatic (BP) limb remains a topic of discussion. Retrospective studies suggest the benefit of tailoring BP-limb length; however, randomized trials are lacking. The aim of this study is to investigate whether tailoring the length of the BP-limb based on total small bowel length (TSBL) leads to better results in terms of weight loss, vitamin deficiencies, and bowel movements compared to a fixed BP-limb length. Methods: The TAILOR study is a double-blind single-center randomized controlled trial. Patients scheduled for primary OAGB surgery will be randomly allocated either to a standard BP-limb of 150 cm or to a BP-limb length based on their TSBL: TSBL 700 cm, BP-limb 210 cm. The primary outcome is to compare the percent total weight loss (%TWL) at 5 years between the two groups. Secondary outcomes include nutritional deficiencies, remission of comorbidities, symptoms of dumping, quality of life, and daily bowel movements. The study includes a total of 212 patients and is designed to detect a 5% difference in the primary endpoint. Discussion: The TAILOR study will provide new insights into the effect of different BP-limb lengths and the role of the TSBL in the OAGB. The study is designed to provide guidance for bariatric surgeons to determine the optimal BP-limb length in the OAGB

Genotype-dependent N-glycosylation and newly exposed O-glycosylation affect plasmin-induced cleavage of histidine-rich glycoprotein (HRG)

Histidine-rich glycoprotein (HRG) is an abundant plasma protein harboring at least three N-glycosylation sites. HRG integrates many biological processes, such as coagulation, antiangiogenic activity, and pathogen clearance. Importantly, HRG is known to exhibit five genetic variants with minor allele frequencies of more than 10%. Among them, Pro204Ser can induce a fourth N-glycosylation site (Asn202). Considerable efforts have been made to reveal the biological function of HRG, whereas data on HRG glycosylation are scarcer. To close this knowledge gap, we used C18-based LC–MS/MS to study the glycosylation characteristics of six HRG samples from different sources. We used endogenous HRG purified from human plasma and compared its glycosylation to that of the recombinant HRG produced in Chinese hamster ovary cells or human embryonic kidney 293 cells, targeting distinct genotypic isoforms. In endogenous plasma HRG, every N-glycosylation site was occupied predominantly with a sialylated diantennary complex–type glycan. In contrast, in the recombinant HRGs, all glycans showed different antennarities, sialylation, and core fucosylation, as well as the presence of oligomannose glycans, LacdiNAcs, and antennary fucosylation. Furthermore, we observed two previously unreported O-glycosylation sites in HRG on residues Thr273 and Thr274. These sites together showed more than 90% glycan occupancy in all HRG samples studied. To investigate the potential relevance of HRG glycosylation, we assessed the plasmin-induced cleavage of HRG under various conditions. These analyses revealed that the sialylation of the N- and O-glycans as well as the genotype-dependent N-glycosylation significantly influenced the kinetics and specificity of plasmin-induced cleavage of HRG

Glycoproteomics-Compatible MS/MS-Based Quantification of Glycopeptide Isomers

Glycosylation is an essential protein modification occurring on the majority of extracellular human proteins, with mass spectrometry (MS) being an indispensable tool for its analysis, that not only determines glycan compositions, but also the position of the glycan at specific sites via glycoproteomics. However, glycans are complex branching structures with monosaccharides interconnected in a variety of biologically relevant linkages, isomeric properties that are invisible when the readout is mass alone. Here, we developed an LC-MS/MS-based workflow for determining glycopeptide isomer ratios. Making use of isomerically defined glyco(peptide) standards, we observed marked differences in fragmentation behavior between isomer pairs when subjected to collision energy gradients, specifically in terms of the galactosylation/sialylation branching and linkage. These behaviors were developed into component variables that allowed for relative quantification of isomerism within mixtures. Importantly, at least for small peptides, the isomer quantification appeared to be largely independent from the peptide portion of the conjugate, allowing a broad application of the method

Laparoscopic Small Bowel Length Measurement in Bariatric Surgery Using a Hand-Over-Hand Technique with Marked Graspers:an Ex Vivo Experiment

Introduction Tailoring limb length in bariatric surgery is a subject of many studies. To acquire the optimal limb length, accurate measurement of the small bowel length is essential. Objective To assess the intra- and inter-individual variability of laparoscopic bowel length measurement using a hand-over-hand technique with marked graspers. Method Four bariatric surgeons and four surgical residents performed measurements on cadaver porcine intestine in a laparoscopic box using marked graspers. Each participant performed 10 times a measurement of three different lengths: 150, 180, and 210 cm. Acceptable percentage deviation from the goal lengths was defined as less than 10%, while unacceptable deviations were defined as more than 15%. Results The bariatric surgeons measured the 150-, 180-, and 210-cm tasks with 4% (CI 0.4, 9), - 6% (CI - 11, - 0.8), and 1% (CI - 4, 6) deviation, respectively. In total, the bariatric surgeons estimated 58 out of 119 times (49%) between the margins of 10% deviation and 36 times (30%) outside the 15% margin. Considerable inter-individual differences were found between the surgeons. The surgical residents underestimated the tasks with 12% (CI - 18, - 6), 16% (CI - 19, - 13), and 18% (CI - 22, - 13), respectively. Conclusion Bariatric surgeons estimated bowel length with on average less than 10% deviation. However, this still resulted in 30% of the measurements with more than 15% deviation. There were considerable inter-individual differences between the surgeons and residents structurally underestimated the bowel length. Ascertainment of measurement accuracy and adequate training is essential for bariatric procedures in which limb length is of importance

Applying an Anti-reflux Suture in the One Anastomosis Gastric Bypass to Prevent Biliary Reflux:a Long-Term Observational Study

INTRODUCTION: The one anastomosis gastric bypass (OAGB) is an effective treatment to induce sustained weight loss in morbidly obese patients. Concerns remain regarding the development of reflux. The aim of this study was to investigate the effect of an "anti-reflux suture" as anti-reflux modification to prevent reflux. METHOD: This is a single-center retrospective cohort study of patients who underwent a primary OAGB at the Center Obesity North-Netherlands (CON) between January 2015 and December 2016. Reflux was defined as symptoms of acid/bilious regurgitation or pyrosis. This was consequently asked and reported at each follow-up visit. Outcomes of patients with an anti-reflux suture were compared to those without. RESULTS: In 414 (59%) of the 703 included patients, an anti-reflux suture was applied. Follow-up at 3 years was 74%. The incidence of reflux did not differ between patients with or without an anti-reflux suture (57 versus 56%, respectively; P = 0.9). The presence of an anti-reflux suture was significantly associated with a lower incidence of conversion to Roux-en-Y gastric bypass (RYGB) for reflux (OR 0.56, 95%CI 0.34-0.91). Patients preoperatively diagnosed with gastroesophageal reflux disease (GERD) were 5.2 times more likely to need a conversion to RYGB for reflux (95%CI 2.7-10.1). CONCLUSION: The presence of preoperative GERD should be weighted heavily in the decision to perform an OAGB as this is a major risk factor for conversion surgery due to reflux. The anti-reflux suture might be a valuable addition to the procedure of the OAGB because it results in fewer conversion surgeries for reflux

Contemporary human H3N2 influenza a viruses require a low threshold of suitable glycan receptors for efficient infection

Recent human H3N2 influenza A viruses (IAV) have evolved to employ elongated glycans terminating in α2,6-linked sialic acid as their receptors. These glycans are displayed in low abundancies by (humanized) Madin-Darby Canine Kidney cells (MDCK and hCK) which are commonly employed to propagate IAV, resulting in low or no viral propagation. Here, we examined whether the overexpression of the glycosyltransferases B3GNT2 and B4GALT1, which are responsible for the elongation of poly-N-acetyllactosamines (LacNAc), would result in improved A/H3N2 propagation. Stable overexpression of B3GNT2 and B4GALT1 in MDCK and hCK cells was achieved by lentiviral integration and subsequent antibiotic selection and confirmed by qPCR and protein mass spectrometry experiments. Flow cytometry and glycan mass spectrometry experiments using the B3GNT2 and/or B4GALT1 knock-in cells demonstrated increased binding of viral hemagglutinins and the presence of a larger number of LacNAc repeating units, especially on hCK-B3GNT2 cells. An increase in the number of glycan receptors did, however, not result in a greater infection efficiency of recent human H3N2 viruses. Based on these results, we propose that H3N2 IAVs require a low number of suitable glycan receptors to infect cells and that an increase in the glycan receptor display above this threshold does not result in improved infection efficiency

Коло Марусі Чурай

In this article Marusya Churay*s (a character famous in story and song) life history is researched. On the basis of real events and historical facts the author tells about people who were related to the life of this personality

The pleiotropic phenotype of FlbA of Aspergillus niger is explained in part by the activity of seven of its downstream-regulated transcription factors

Inactivation of flbA in Aspergillus niger results in thinner cell walls, increased cell lysis, abolished sporulation, and an increased secretome complexity. A total of 36 transcription factor (TF) genes are differentially expressed in ΔflbA. Here, seven of these genes (abaA, aslA, aslB, azf1, htfA, nosA, and srbA) were inactivated. Inactivation of each of these genes affected sporulation and, with the exception of abaA, cell wall integrity and protein secretion. The impact on secretion was strongest in the case of ΔaslA and ΔaslB that showed increased pepsin, cellulase, and amylase activity. Biomass was reduced of agar cultures of ΔabaA, ΔaslA, ΔnosA, and ΔsrbA, while biomass was higher in liquid shaken cultures of ΔaslA and ΔaslB. The ΔaslA and ΔhtfA strains showed increased resistance to H2O2, while ΔaslB was more sensitive to this reactive oxygen species. Together, inactivation of the seven TF genes impacted biomass formation, sporulation, protein secretion, and stress resistance, and thereby these genes explain at least part of the pleiotropic phenotype of ΔflbA of A. niger

A case series exploring the human milk polyclonal IgA1 response to repeated SARS-CoV-2 vaccinations by LC-MS based fab profiling

INTRODUCTION: Upon vaccination against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) humans will start to produce antibodies targeting virus specific antigens that will end up in circulation. In lactating women such antibodies will also end up in breastmilk, primarily in the form of secretory immunoglobulin A1 (SIgA1), the most abundant immunoglobulin (Ig) in human milk. Here we set out to investigate the SIgA1 clonal repertoire response to repeated SARS-CoV-2 vaccination, using a LC-MS fragment antigen-binding (Fab) clonal profiling approach. METHODS: We analyzed the breastmilk of six donors from a larger cohort of 109 lactating mothers who received one of three commonly used SARS-CoV-2 vaccines. We quantitatively monitored the SIgA1 Fab clonal profile over 16 timepoints, from just prior to the first vaccination until 15  days after the second vaccination. RESULTS: In all donors, we detected a population of 89-191 vaccine induced clones. These populations were unique to each donor and heterogeneous with respect to individual clonal concentrations, total clonal titer, and population size. The vaccine induced clones were dominated by persistent clones (68%) which came up after the first vaccination and were retained or reoccurred after the second vaccination. However, we also observe transient SIgA1 clones (16%) which dissipated before the second vaccination, and vaccine induced clones which uniquely emerged only after the second vaccination (16%). These distinct populations were observed in all analyzed donors, regardless of the administered vaccine. DISCUSSION: Our findings suggest that while individual donors have highly unique human milk SIgA1 clonal profiles and a highly personalized SIgA1 response to SARS-CoV-2 vaccination, there are also commonalities in vaccine induced responses