Olfactory, Taste, and Photo Sensory Receptors in Non-sensory Organs: It Just Makes Sense

Sensory receptors that detect and respond to light, taste, and smell primarily belong to the G-protein-coupled receptor (GPCR) superfamily. In addition to their established roles in the nose, tongue, and eyes, these sensory GPCRs have been found in many ‘non-sensory' organs where they respond to different physicochemical stimuli, initiating signaling cascades in these extrasensory systems. For example, taste receptors in the airway, and photoreceptors in vascular smooth muscle cells, both cause smooth muscle relaxation when activated. In addition, olfactory receptors are present within the vascular system, where they play roles in angiogenesis as well as in modulating vascular tone. By better understanding the physiological and pathophysiological roles of sensory receptors in non-sensory organs, novel therapeutic agents can be developed targeting these receptors, ultimately leading to treatments for pathological conditions and potential cures for various disease states

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field

Cabbage and fermented vegetables : From death rate heterogeneity in countries to candidates for mitigation strategies of severe COVID-19

Large differences in COVID-19 death rates exist between countries and between regions of the same country. Some very low death rate countries such as Eastern Asia, Central Europe, or the Balkans have a common feature of eating large quantities of fermented foods. Although biases exist when examining ecological studies, fermented vegetables or cabbage have been associated with low death rates in European countries. SARS-CoV-2 binds to its receptor, the angiotensin-converting enzyme 2 (ACE2). As a result of SARS-CoV-2 binding, ACE2 downregulation enhances the angiotensin II receptor type 1 (AT(1)R) axis associated with oxidative stress. This leads to insulin resistance as well as lung and endothelial damage, two severe outcomes of COVID-19. The nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is the most potent antioxidant in humans and can block in particular the AT(1)R axis. Cabbage contains precursors of sulforaphane, the most active natural activator of Nrf2. Fermented vegetables contain many lactobacilli, which are also potent Nrf2 activators. Three examples are: kimchi in Korea, westernized foods, and the slum paradox. It is proposed that fermented cabbage is a proof-of-concept of dietary manipulations that may enhance Nrf2-associated antioxidant effects, helpful in mitigating COVID-19 severity.Peer reviewe

Nrf2-interacting nutrients and COVID-19 : time for research to develop adaptation strategies

There are large between- and within-country variations in COVID-19 death rates. Some very low death rate settings such as Eastern Asia, Central Europe, the Balkans and Africa have a common feature of eating large quantities of fermented foods whose intake is associated with the activation of the Nrf2 (Nuclear factor (erythroid-derived 2)-like 2) anti-oxidant transcription factor. There are many Nrf2-interacting nutrients (berberine, curcumin, epigallocatechin gallate, genistein, quercetin, resveratrol, sulforaphane) that all act similarly to reduce insulin resistance, endothelial damage, lung injury and cytokine storm. They also act on the same mechanisms (mTOR: Mammalian target of rapamycin, PPAR gamma:Peroxisome proliferator-activated receptor, NF kappa B: Nuclear factor kappa B, ERK: Extracellular signal-regulated kinases and eIF2 alpha:Elongation initiation factor 2 alpha). They may as a result be important in mitigating the severity of COVID-19, acting through the endoplasmic reticulum stress or ACE-Angiotensin-II-AT(1)R axis (AT(1)R) pathway. Many Nrf2-interacting nutrients are also interacting with TRPA1 and/or TRPV1. Interestingly, geographical areas with very low COVID-19 mortality are those with the lowest prevalence of obesity (Sub-Saharan Africa and Asia). It is tempting to propose that Nrf2-interacting foods and nutrients can re-balance insulin resistance and have a significant effect on COVID-19 severity. It is therefore possible that the intake of these foods may restore an optimal natural balance for the Nrf2 pathway and may be of interest in the mitigation of COVID-19 severity

Mortality from gastrointestinal congenital anomalies at 264 hospitals in 74 low-income, middle-income, and high-income countries: a multicentre, international, prospective cohort study

Summary Background Congenital anomalies are the fifth leading cause of mortality in children younger than 5 years globally. Many gastrointestinal congenital anomalies are fatal without timely access to neonatal surgical care, but few studies have been done on these conditions in low-income and middle-income countries (LMICs). We compared outcomes of the seven most common gastrointestinal congenital anomalies in low-income, middle-income, and high-income countries globally, and identified factors associated with mortality. Methods We did a multicentre, international prospective cohort study of patients younger than 16 years, presenting to hospital for the first time with oesophageal atresia, congenital diaphragmatic hernia, intestinal atresia, gastroschisis, exomphalos, anorectal malformation, and Hirschsprung’s disease. Recruitment was of consecutive patients for a minimum of 1 month between October, 2018, and April, 2019. We collected data on patient demographics, clinical status, interventions, and outcomes using the REDCap platform. Patients were followed up for 30 days after primary intervention, or 30 days after admission if they did not receive an intervention. The primary outcome was all-cause, in-hospital mortality for all conditions combined and each condition individually, stratified by country income status. We did a complete case analysis. Findings We included 3849 patients with 3975 study conditions (560 with oesophageal atresia, 448 with congenital diaphragmatic hernia, 681 with intestinal atresia, 453 with gastroschisis, 325 with exomphalos, 991 with anorectal malformation, and 517 with Hirschsprung’s disease) from 264 hospitals (89 in high-income countries, 166 in middleincome countries, and nine in low-income countries) in 74 countries. Of the 3849 patients, 2231 (58·0%) were male. Median gestational age at birth was 38 weeks (IQR 36–39) and median bodyweight at presentation was 2·8 kg (2·3–3·3). Mortality among all patients was 37 (39·8%) of 93 in low-income countries, 583 (20·4%) of 2860 in middle-income countries, and 50 (5·6%) of 896 in high-income countries (p<0·0001 between all country income groups). Gastroschisis had the greatest difference in mortality between country income strata (nine [90·0%] of ten in lowincome countries, 97 [31·9%] of 304 in middle-income countries, and two [1·4%] of 139 in high-income countries; p≤0·0001 between all country income groups). Factors significantly associated with higher mortality for all patients combined included country income status (low-income vs high-income countries, risk ratio 2·78 [95% CI 1·88–4·11], p<0·0001; middle-income vs high-income countries, 2·11 [1·59–2·79], p<0·0001), sepsis at presentation (1·20 [1·04–1·40], p=0·016), higher American Society of Anesthesiologists (ASA) score at primary intervention (ASA 4–5 vs ASA 1–2, 1·82 [1·40–2·35], p<0·0001; ASA 3 vs ASA 1–2, 1·58, [1·30–1·92], p<0·0001]), surgical safety checklist not used (1·39 [1·02–1·90], p=0·035), and ventilation or parenteral nutrition unavailable when needed (ventilation 1·96, [1·41–2·71], p=0·0001; parenteral nutrition 1·35, [1·05–1·74], p=0·018). Administration of parenteral nutrition (0·61, [0·47–0·79], p=0·0002) and use of a peripherally inserted central catheter (0·65 [0·50–0·86], p=0·0024) or percutaneous central line (0·69 [0·48–1·00], p=0·049) were associated with lower mortality. Interpretation Unacceptable differences in mortality exist for gastrointestinal congenital anomalies between lowincome, middle-income, and high-income countries. Improving access to quality neonatal surgical care in LMICs will be vital to achieve Sustainable Development Goal 3.2 of ending preventable deaths in neonates and children younger than 5 years by 2030

A novel in vitro model for microvasculature reveals regulation of circumferential ECM organization by curvature.

In microvascular vessels, endothelial cells are aligned longitudinally whereas several components of the extracellular matrix (ECM) are organized circumferentially. While current three-dimensional (3D) in vitro models for microvasculature have allowed the study of ECM-regulated tubulogenesis, they have limited control over topographical cues presented by the ECM and impart a barrier for the high-resolution and dynamic study of multicellular and extracellular organization. Here we exploit a 3D fibrin microfiber scaffold to develop a novel in vitro model of the microvasculature that recapitulates endothelial alignment and ECM deposition in a setting that also allows the sequential co-culture of mural cells. We show that the microfibers' nanotopography induces longitudinal adhesion and alignment of endothelial colony-forming cells (ECFCs), and that these deposit circumferentially organized ECM. We found that ECM wrapping on the microfibers is independent of ECFCs' actin and microtubule organization, but it is dependent on the curvature of the microfiber. Microfibers with smaller diameters (100-400 µm) guided circumferential ECM deposition, whereas microfibers with larger diameters (450 µm) failed to support wrapping ECM. Finally, we demonstrate that vascular smooth muscle cells attached on ECFC-seeded microfibers, depositing collagen I and elastin. Collectively, we establish a novel in vitro model for the sequential control and study of microvasculature development and reveal the unprecedented role of the endothelium in organized ECM deposition regulated by the microfiber curvature

ECFCs attached and aligned on fibrin hydrogel microfibers.

<p>(<b>A</b>) Schematic of experimental procedure including electrospinning, ECFC seeding, and tumbling. Drawing not to scale. SEM of critical-point dried fibrin microfiber showing aligned topography on the microfiber surface. Scale bar is 10 µm. (<b>B–D</b>) Confocal z-stack image reconstructions of ECFCs seeded on fibrin hydrogel microfibers horizontally aligned after 5 days in culture. F-actin (phalloidin staining) is shown in green, EC-specific markers (VECad, CD31, or vWF) in red, and nuclei in blue. Yellow arrows indicate the direction of stretching and nanotopography on microfiber surface. Scale bars are 50 µm. n≥2 per stain with quadruplicates.</p

Co-cultured vSMCs deposit new ECM.

<p>Confocal z-stack image reconstructions of fibrin microfibers seeded with ECFCs followed by (<b>A</b>) co-culture of vSMCs for 2 days. n = 2 with quadruplicates. Scale bars are 200 µm. Co-cultured vSMCs for 3 days showing (<b>B</b>) wrapping and (<b>C</b>) aligned arrangement. Scale bars are 100 µm. (<b>D</b>) Collagen I deposited by co-cultured vSMCs after 3 days in co-culture. Scale bars are 100 µm. (<b>E</b>) Cross-sectional projection of confocal z-stack images of vSMCs after 5 days in co-culture. Arrowheads indicate SM22⁻ cells. Scale bars are 50 µm. B–E n≥3 with quadruplicates. (<b>F</b>) Confocal z-stack image reconstruction and (<b>G</b>) cross-sectional projection of co-cultured vSMCs after 5 days in co-culture. n = 2 with quadruplicates. Scale bars are 50 µm. SM22 is shown in green, CD31 in red, collagen I and elastin in red, and nuclei in blue.</p

ECFCs deposit ECM circumferentially on fibrin hydrogel microfibers.

<p>Confocal z-stack image reconstructions of ECFCs on fibrin microfibers after (<b>A</b>) 1 and (<b>B</b>) 5 days in culture. Scale bars are 200 µm. (<b>C</b>) High magnification confocal images of laminin, fibronectin and collagen IV wrapping around the fibrin microfibers. (<b>D</b>) TEM images of cross-sectional slices of a cell-fibrin microfiber construct after 5 days in culture (i–ii) with cells and (iii) without cells. (ii) is a higher magnification image for the boxed area in (i). F  =  Fibrin; E  =  ECM; C  =  Cells. (<b>E</b>) Cross-sectional projections of confocal z-stack images of ECFCs on fibrin microfibers after 5 days. F-actin (phalloidin) is shown in green, ECM proteins (collagen IV, laminin, fibronectin) in red or magenta, and nuclei in blue. Yellow arrows indicate the direction of nanotopography on microfiber surface. A n≥2, B–E n≥5 per stain with quadruplicates.</p