Food-specific serum IgG and symptom reduction with a personalized, unrestricted-calorie diet of six weeks in Irritable Bowel Syndrome (IBS).

BACKGROUND: Irritable Bowel Syndrome (IBS) is a widespread disease with variable symptoms that have an important impact on the quality of life. Despite the prevalence of IBS, its etiology and pathophysiology are still to be fully understood, but immune response is known to be involved. In this study, we investigated the variation of two specific cytokines, B-cell activating factor (BAFF) and platelet-activating factor (PAF), the levels of food-specific IgG and the symptom severity, using Irritable Bowel Syndrome-Symptom Severity Score (IBS-SSS), following a personalized and unrestricted-calorie diet. METHODS: We enrolled 30 subjects with diagnosis of IBS, according to Rome-IV criteria, whose inflammatory markers were measured at baseline and after 6 weeks of dietary intervention. The subjects were monitored in a general practice outpatient setting and nutritional advice was offered remotely via two telephone sessions with a nutritionist. RESULTS: BAFF and PAF values did not differ between baseline and end of study, both in compliant (C) and non-compliant (NC) subjects. IgG levels significantly decreased only in compliant subjects: 37.32 (23.24-93.67) IU/mL; 27.9 (7.56-93.96) IU/mL (p = 0.02) and in non-compliant went from 51.83 (13.17-113.1) IU/mL to 44.06 (4.96-255.4) IU/mL (p = 0.97, ns). IBS-SSS significantly decreased in both compliant subjects, from 245 (110-480) to 110 (0-140) (p < 0.0001), and non compliant subjects, from 250 (155-370) to 100 (7-220) (p < 0.0001). Comparing IBS-SSS between week 3 and week 6, only compliant subjects had a significant reduction, from 155 (50-355) to 110 (0-140) (p = 0.005), versus non-compliant, from 115 (35-315) to 100 (7-220) (p = 0.33, ns). CONCLUSION: These findings support the rapid efficacy and suitability of a personalized dietetic intervention with outside consultation in IBS. TRIAL REGISTRATION: ClinicalTrials.gov ID NCT04348760 Registered April 15, 2020 (retrospectively registered) https://clinicaltrials.gov/show/NCT04348760.

Methylglyoxal, Glycated Albumin, PAF, and TNF-α: Possible Inflammatory and Metabolic Biomarkers for Management of Gestational Diabetes.

BACKGROUND: In gestational diabetes mellitus (GDM), pancreatic ß-cell breakdown can result from a proinflammatory imbalance created by a sustained level of cytokines. In this study, we investigated the role of specific cytokines, such as B-cell activating factor (BAFF), tumor necrosis factor α (TNF-α), and platelet-activating factor (PAF), together with methylglyoxal (MGO) and glycated albumin (GA) in pregnant women affected by GDM. METHODS: We enrolled 30 women whose inflammation and metabolic markers were measured at recruitment and after 12 weeks of strict dietetic therapy. We compared these data to the data obtained from 53 randomly selected healthy nonpregnant subjects without diabetes, hyperglycemia, or any condition that can affect glycemic metabolism. RESULTS: In pregnant women affected by GDM, PAF levels increased from 26.3 (17.4-47.5) ng/mL to 40.1 (30.5-80.5) ng/mL (p < 0.001). Their TNF-α levels increased from 3.0 (2.8-3.5) pg/mL to 3.4 (3.1-5.8) pg/mL (p < 0.001). The levels of methylglyoxal were significantly higher in the women with GDM (p < 0.001), both at diagnosis and after 12 weeks (0.64 (0.46-0.90) µg/mL; 0.71 (0.47-0.93) µg/mL, respectively) compared to general population (0.25 (0.19-0.28) µg/mL). Levels of glycated albumin were significantly higher in women with GDM (p < 0.001) only after 12 weeks from diagnosis (1.51 (0.88-2.03) nmol/mL) compared to general population (0.95 (0.63-1.4) nmol/mL). CONCLUSION: These findings support the involvement of new inflammatory and metabolic biomarkers in the mechanisms related to GDM complications and prompt deeper exploration into the vicious cycle connecting inflammation, oxidative stress, and metabolic results.

An essential step of kinetochore formation controlled by the SNARE protein Snap29.

The kinetochore is an essential structure that mediates accurate chromosome segregation in mitosis and meiosis. While many of the kinetochore components have been identified, the mechanisms of kinetochore assembly remain elusive. Here, we identify a novel role for Snap29, an unconventional SNARE, in promoting kinetochore assembly during mitosis in Drosophila and human cells. Snap29 localizes to the outer kinetochore and prevents chromosome mis-segregation and the formation of cells with fragmented nuclei. Snap29 promotes accurate chromosome segregation by mediating the recruitment of Knl1 at the kinetochore and ensuring stable microtubule attachments. Correct Knl1 localization to kinetochore requires human or Drosophila Snap29, and is prevented by a Snap29 point mutant that blocks Snap29 release from SNARE fusion complexes. Such mutant causes ectopic Knl1 recruitment to trafficking compartments. We propose that part of the outer kinetochore is functionally similar to membrane fusion interfaces.

Control of lysosomal biogenesis and Notch-dependent tissue patterning by components of the TFEB-V-ATPase axis in Drosophila melanogaster.

In vertebrates, TFEB (transcription factor EB) and MITF (microphthalmia-associated transcription factor) family of basic Helix-Loop-Helix (bHLH) transcription factors regulates both lysosomal function and organ development. However, it is not clear whether these 2 processes are interconnected. Here, we show that Mitf, the single TFEB and MITF ortholog in Drosophila, controls expression of vacuolar-type H(+)-ATPase pump (V-ATPase) subunits. Remarkably, we also find that expression of Vha16-1 and Vha13, encoding 2 key components of V-ATPase, is patterned in the wing imaginal disc. In particular, Vha16-1 expression follows differentiation of proneural regions of the disc. These regions, which will form sensory organs in the adult, appear to possess a distinctive endolysosomal compartment and Notch (N) localization. Modulation of Mitf activity in the disc in vivo alters endolysosomal function and disrupts proneural patterning. Similar to our findings in Drosophila, in human breast epithelial cells we observe that impairment of the Vha16-1 human ortholog ATP6V0C changes the size and function of the endolysosomal compartment and that depletion of TFEB reduces ligand-independent N signaling activity. Our data suggest that lysosomal-associated functions regulated by the TFEB-V-ATPase axis might play a conserved role in shaping cell fate.

Immunohistochemical tools and techniques to visualize Notch in Drosophila melanogaster.

The ability to accurately visualize proteins in Drosophila tissues is critical for studying their abundance and localization relative to the morphology of cells during tissue development and homeostasis. Here we describe the procedure to visualize Notch localization in whole-mount preparations of several Drosophila organs using confocal microscopy. The use of monoclonal antibodies directed to distinct portions of Notch allows one to follow the fate of the receptor during constitutive and inductive processes. The protocol described here can be used to co-label with antibodies recognizing markers of subcellular compartments in wild-type as well as mutant tissues.

ESCRT-0 is not required for ectopic Notch activation and tumor suppression in Drosophila.

Multivesicular endosome (MVE) sorting depends on proteins of the Endosomal Sorting Complex Required for Transport (ESCRT) family. These are organized in four complexes (ESCRT-0, -I, -II, -III) that act in a sequential fashion to deliver ubiquitylated cargoes into the internal luminal vesicles (ILVs) of the MVE. Drosophila genes encoding ESCRT-I, -II, -III components function in sorting signaling receptors, including Notch and the JAK/STAT signaling receptor Domeless. Loss of ESCRT-I, -II, -III in Drosophila epithelia causes altered signaling and cell polarity, suggesting that ESCRTs genes are tumor suppressors. However, the nature of the tumor suppressive function of ESCRTs, and whether tumor suppression is linked to receptor sorting is unclear. Unexpectedly, a null mutant in Hrs, encoding one of the components of the ESCRT-0 complex, which acts upstream of ESCRT-I, -II, -III in MVE sorting is dispensable for tumor suppression. Here, we report that two Drosophila epithelia lacking activity of Stam, the other known components of the ESCRT-0 complex, or of both Hrs and Stam, accumulate the signaling receptors Notch and Dome in endosomes. However, mutant tissue surprisingly maintains normal apico-basal polarity and proliferation control and does not display ectopic Notch signaling activation, unlike cells that lack ESCRT-I, -II, -III activity. Overall, our in vivo data confirm previous evidence indicating that the ESCRT-0 complex plays no crucial role in regulation of tumor suppression, and suggest re-evaluation of the relationship of signaling modulation in endosomes and tumorigenesis.

EGF receptor exposed to oxidative stress acquires abnormal phosphorylation and aberrant activated conformation that impairs canonical dimerization.

Crystallographic studies have offered understanding of how receptor tyrosine kinases from the ErbB family are regulated by their growth factor ligands. A conformational change of the EGFR (ErbB1) was shown to occur upon ligand binding, where a solely ligand-mediated mode of dimerization/activation was documented. However, this dogma of dimerization/activation was revolutionized by the discovery of constitutively active ligand-independent EGFR mutants. In addition, other ligand-independent activation mechanisms may occur. We have shown that oxidative stress (ox-stress), induced by hydrogen peroxide or cigarette smoke, activates EGFR differently than its ligand, EGF, thereby inducing aberrant phosphorylation and impaired trafficking and degradation of EGFR. Here we demonstrate that ox-stress activation of EGFR is ligand-independent, does not induce "classical" receptor dimerization and is not inhibited by the tyrosine kinase inhibitor AG1478. Thus, an unprecedented, apparently activated, state is found for EGFR under ox-stress. Furthermore, this activation mechanism is temperature-dependent, suggesting the simultaneous involvement of membrane structure. We propose that ceramide increase under ox-stress disrupts cholesterol-enriched rafts leading to EGFR re-localization into the rigid, ceramide-enriched rafts. This increase in ceramide also supports EGFR aberrant trafficking to a peri-nuclear region. Therefore, the EGFR unprecedented and activated conformation could be sustained by simultaneous alterations in membrane structure under ox-stress.