The Role of CBL Family of E3 Ubiquitin Ligases in Intestinal Epithelial Homeostasis

All adult organs are endowed with a small pool of resident stem cells that must be maintained throughout life to provide for cell turnover during homeostasis and tissue repair following any injury. The unique ability to self-renew as well as to differentiate into functional cells of organs in which they reside makes stem cells essential for the maintenance of organ systems. It is now becoming increasingly evident that aberrant activity of stem cells contributes to diseases such as cancer or tissue/organ atrophy. Thus, it is critical to better understand the mechanisms that regulate adult stem cells. Ligand-activated receptor tyrosine kinases (RTKs) represent an important means to regulate adult stem cells. We and others have established the CBL-family ubiquitin ligases as key negative regulators of RTKs. However, physiological roles of CBL proteins in intestinal epithelial homeostasis are unknown. Recently, we have shown that CBL and CBL-B function as redundant but essential regulators of hematopoietic stem cell (HSC) quiescence and their combined deletion in HSCs of mice leads to a rapidly-fatal myeloproliferative disorder, mimicking a similar leukemic disease associated with mutations of CBL in humans. CBL and CBL-B have also been shown to help maintain asymmetric neural stem cell division. Any role of CBL proteins in the regulation of epithelial stem cells has not been reported. To address such a role, we generated a novel mouse model (Cbl-flox/flox; Cbl-b-null; Lgr5-EGFP-IRES-CreERT2; Rosa26-LacZ) to confer concurrent tamoxifen-inducible loss of CBL and CBL-B in the Lgr5-expressing intestinal epithelial stem cells (IESCs). Tamoxifen injection in this inducible CBL/CBL-B double knockout (iDKO) mouse model resulted in a rapid and significant reduction in the Lgr5-High IESC pool with a concomitant increase in the Lgr5-Lo transit amplifying cells. Lineage tracing using LacZ-staining revealed an increase in the number of blue progeny in the iDKOs, suggesting an increased IESC commitment to differentiation. Of the progeny, iDKO animals showed a propensity towards enterocyte and goblet cell fate at the expense of Paneth cells. Loss of IESCs in iDKO mice led to slower recovery from intestinal epithelial injury due to X-ray radiation of the abdomen. In vitro deletion of CBL proteins in the crypt culture recapitulated the loss of self renewal phenotype and was associated with hyperactivation of MAPK pathway and downregulation of Wnt pathway effector TCF4. These results demonstrate a novel requirement of CBL/CBL-B in the maintenance of a well-studied epithelial stem cell compartment, the IESC, and suggest that CBL proteins by regulating MAPK pathway protect IESCs from exhaustion

Experience of managing first fifty COVID-19 positive peripartum women in a tertiary care centre in North India

Background: SARS-CoV-2 has caused significant morbidity and mortality worldwide. Analysis of the clinical profile of COVID-19 positive pregnant women is important to understand the pathophysiology, transmission and outcome of the disease in Indian population.Methods: It is a retrospective observational study of first fifty pregnant patients tested positive for COVID-19 by qRT PCR admitted for delivery in our hospital.Results: In this audit, first fifty COVID-19 pregnant women were studied and the mean age of the patients in this study was 26 years. 98% of these women were admitted for obstetric indications. Seventy two percent of these women were admitted with spontaneous onset of labour. Based on disease severity, 49 (98%) were either asymptomatic or exhibited mild disease and only 1 (2%) had severe disease who succumbed to her illness. Forty six percent patients delivered vaginally and 54% required cesarean delivery. The most common indication for LSCS was fetal distress (43%). Eighteen percent had preterm delivery. Among the newborn babies, one died due to severe birth asphyxia. Sixteen percent babies required NICU stay. Five babies tested positive for COVID-19 of which one baby was positive on day one of life.Conclusions: As per our results, majority of the COVID-19 positive pregnant women had mild disease. There has been increase in cesarean section rate as compared to the previous hospital figures. Only one baby tested positive within 24 hours of delivery so the possibility vertical transmission can not be commented upon as of today

Role of visual inspection of cervix with acetic acid and high risk human papilloma virus DNA testing in screening for cervical cancer

Background: To evaluate the role of VIA alone and in combination with high risk Human Papilloma virus DNA testing as a screening test for cervical dysplasia and cancer.Methods: 400 symptomatic patients from the gynecology outpatient department were screened using Pap smear and VIA. HPV DNA testing was done for 62 VIA positive and 100 VIA negative women. Colposcopy was done for all women. Those found positive on any or all of the screening tests were subjected to cervical biopsy. The results were analysed for PAP, VIA, HPV and a combined test using VIA and HPV both.Results: VIA had the highest sensitivity (91%) to detect any grade of dysplasia. The sensitivity of the combination test (VIA + HPV) was 80.6% which was lower than that of VIA (91%) and also lower than that of HR HPV DNA detection (86%). The specificity of the combination test (VIA + HPV) was 68.3 % which was significantly higher than that of VIA alone (39%) (p = 0.000) and also higher than that for HPV DNA detection when used alone (56%). Pap smear had the highest specificity (95.12 %) but sensitivity was much lower at 52.7 %.Conclusions: VIA is a highly sensitive screening test. The main disadvantage is its low specificity. However the combination test of VIA + HR HPV testing overcomes this and at the same time maintains a high sensitivity. Thus a test which combines VIA plus HR HPV testing is better screening method than either of the three tests (VIA, HPV, PAP) done alone

Loss of Cbl and Cbl-b ubiquitin ligases abrogates hematopoietic stem cell quiescence and sensitizes leukemic disease to chemotherapy.

Cbl and Cbl-b are tyrosine kinase-directed RING finger type ubiquitin ligases (E3s) that negatively regulate cellular activation pathways. E3 activity-disrupting human Cbl mutations are associated with myeloproliferative disorders (MPD) that are reproduced in mice with Cbl RING finger mutant knock-in or hematopoietic Cbl and Cbl-b double knockout. However, the role of Cbl proteins in hematopoietic stem cell (HSC) homeostasis, especially in the context of MPD is unclear. Here we demonstrate that HSC expansion and MPD development upon combined Cbl and Cbl-b deletion are dependent on HSCs. Cell cycle analysis demonstrated that DKO HSCs exhibit reduced quiescence associated with compromised reconstitution ability and propensity to undergo exhaustion. We show that sustained c-Kit and FLT3 signaling in DKO HSCs promotes loss of colony-forming potential, and c-Kit or FLT3 inhibition in vitro protects HSCs from exhaustion. In vivo, treatment with 5-fluorouracil hastens DKO HSC exhaustion and protects mice from death due to MPD. Our data reveal a novel and leukemia therapy-relevant role of Cbl and Cbl-b in the maintenance of HSC quiescence and protection against exhaustion, through negative regulation of tyrosine kinase-coupled receptor signaling

Cbl and Cbl-b ubiquitin ligases are essential for intestinal epithelial stem cell maintenance

Summary: Receptor tyrosine kinases (RTKs) control stem cell maintenance vs. differentiation decisions. Casitas B-lineage lymphoma (CBL) family ubiquitin ligases are negative regulators of RTKs, but their stem cell regulatory roles remain unclear. Here, we show that Lgr5+ intestinal stem cell (ISC)-specific inducible Cbl-knockout (KO) on a Cblb null mouse background (iDKO) induced rapid loss of the Lgr5Hi ISCs with transient expansion of the Lgr5Lo transit-amplifying population. LacZ-based lineage tracing revealed increased ISC commitment toward enterocyte and goblet cell fate at the expense of Paneth cells. Functionally, Cbl/Cblb iDKO impaired the recovery from radiation-induced intestinal epithelial injury. In vitro, Cbl/Cblb iDKO led to inability to maintain intestinal organoids. Single-cell RNA sequencing in organoids identified Akt-mTOR (mammalian target of rapamycin) pathway hyperactivation upon iDKO, and pharmacological Akt-mTOR axis inhibition rescued the iDKO defects. Our results demonstrate a requirement for Cbl/Cblb in the maintenance of ISCs by fine-tuning the Akt-mTOR axis to balance stem cell maintenance vs. commitment to differentiation

Deep Underground Neutrino Experiment (DUNE), Far Detector Technical Design Report, Volume I Introduction to DUNE

International audienceThe preponderance of matter over antimatter in the early universe, the dynamics of the supernovae that produced the heavy elements necessary for life, and whether protons eventually decay—these mysteries at the forefront of particle physics and astrophysics are key to understanding the early evolution of our universe, its current state, and its eventual fate. The Deep Underground Neutrino Experiment (DUNE) is an international world-class experiment dedicated to addressing these questions as it searches for leptonic charge-parity symmetry violation, stands ready to capture supernova neutrino bursts, and seeks to observe nucleon decay as a signature of a grand unified theory underlying the standard model. The DUNE far detector technical design report (TDR) describes the DUNE physics program and the technical designs of the single- and dual-phase DUNE liquid argon TPC far detector modules. This TDR is intended to justify the technical choices for the far detector that flow down from the high-level physics goals through requirements at all levels of the Project. Volume I contains an executive summary that introduces the DUNE science program, the far detector and the strategy for its modular designs, and the organization and management of the Project. The remainder of Volume I provides more detail on the science program that drives the choice of detector technologies and on the technologies themselves. It also introduces the designs for the DUNE near detector and the DUNE computing model, for which DUNE is planning design reports. Volume II of this TDR describes DUNE's physics program in detail. Volume III describes the technical coordination required for the far detector design, construction, installation, and integration, and its organizational structure. Volume IV describes the single-phase far detector technology. A planned Volume V will describe the dual-phase technology