The effects of phloretin a phytoalexin from apple, was tested on Pectobacterium brasiliense (Pb1692), an emerging soft-rot pathogen of potato. Exposure of Pb1692 to 0.2 mM phloretin a concentration that does not affect growth, or to 0.4 mM a 50% growth inhibiting concentration (50% MIC), reduced motility, biofilm formation, secretion of plant cell wall-degrading enzymes, production of acyl-homoserine lactone (AHL) signaling molecules and infection, phenotypes that are associated with bacterial population density-dependent system known as quorum sensing (QS). To analyze the effect of growth inhibition on QS, the activity of ciprofloxacin, an antibiotic that impairs cell division, was compared to that of phloretin at 50% MIC. Unlike phloretin, the antibiotic hardly affected the tested phenotypes. The use of DH5 alpha, a QS-negative Escherichia coli strain, transformed with an AHL synthase (ExpI) from Pb1692, allowed to validate direct inhibition of AHL production by phloretin, as demonstrated by two biosensor strains, Chromobacterium violaceaum (CV026) and E. coli (pSB401). Expression analysis of virulence-related genes revealed downregulation of QS-regulated genes (expI, expR, luxS, rsmB), plant cell wall degrading enzymes genes (pel, peh and prt) and motility genes (motA, fim, fliA, flhC and flhD) following exposure to both phloretin concentrations. The results support the inhibition of ExpI activity by phloretin. Docking simulations were used to predict the molecular associations between phloretin and the active site of ExpI, to suggest a likely mode of action for the compound's inhibition of virulence.

Bacteria show distinct and characteristic fatty acid (FA) patterns which can be modified by environmental conditions. In this study, we cultivated six plant-pathogenic bacteria of agricultural concern and performed a detailed analysis of the fatty acid composition. The study covered four strains of the gram-negative Xanthomonas campestris pathovar (pv) campestris (Xcc), Xanthomonas perforans (Xp), Acidovorax citrulli (Ac) and Pseudomonas syringae pv. tomato (Pst), and two strains of the gram-positive Clavibacter michiganensis subsp. michiganensis (Cmm) and Streptomyces scabies (Ssc). After cultivation, freeze-dried bacteria samples were transesterified and analysed by gas chromatography with mass spectrometry in full scan and selected ion monitoring (SIM) modes. Altogether, 44 different FAs were detected in the six strains with individual contributions of 0.01-43.8% to the total FAs. The variety in the six strains ranged between 12 and 31 individual FAs. The FA composition of Xcc, Xp, Cmm and Ssc were dominated by iso- and anteiso-fatty acids (especially i15:0, a15:0, i16:0), which is typical for most bacteria. In contrast to this, Ac and Pst showed only saturated and monounsaturated FAs. Four of the six bacteria showed similar FA patterns as reported before in the literature. Differences were observed in the case of Cmm where many more FAs were detected in the present study. In addition, to the best of our knowledge, the FA pattern of Xp was presented for the first time.

Objective: Broccoli is a ``functional food'' that contains bioactive compounds and phytochemicals that have beneficial health-promoting effects. This study aimed at investigating the effects of broccoli consumption on lipid and glucose metabolism and gut microbiota. Methods: Male C57BL/6J mice (7-8 wk old) were fed ad libitum with a normal diet supplemented with or without 10% (w/w) broccoli florets or broccoli stalks. Oral glucose tolerance tests were performed at week 15. After 17 wk, blood and tissues were collected. Serum parameters, histology, gene and protein expression, and intestinal microbiota composition were evaluated. Results: Stalk supplementation led to reductions in fasting glucose levels, serum insulin, and the homeostasis model assessment-insulin resistance (HOMA-IR) index. Liver enzymes improved in both experimental groups, and broccoli florets decreased total triacylglycerols. The stalks group had elevated fatty acid oxidation-related genes and proteins (AMPK, PPARa, and CPT1). Diverse microbiota populations were observed in both broccoli groups. Broccoli stalks were found to be richer in Akkermansia muciniphila, while broccoli florets reduced Mucispirillum schaedleri abundance and increased bacterial richness. Conclusions: Long-term whole broccoli supplementation decreased inflammation, improved lipid parameters and insulin sensitivity, and altered the gut microbiome in mice. Our data provide new information regarding the potential benefits of broccoli stalks in metabolic parameters. (C) 2021 Elsevier Inc. All rights reserved.

REV-ERB alpha is a nuclear receptor that inhibits Bmal1 transcription as part of the circadian clock molecular mechanism. Mammalian target of rapamycin (mTOR) complex 1 (mTORC1) is a master regulator of cell and whole-body energy homeostasis, that serves as an important link between metabolism and circadian clock, in part, by regulating BMAL1 activity. While the connection of REV-ERB alpha to the circadian clock molecular mechanism is well characterized, the interaction between mTORC1, REV-ERB alpha and the circadian clock machinery is not very clear. We used leucine and rapamycin to modulate mTORC1 activation and evaluate this effect on circadian rhythms. In the liver, mTORC1 was inhibited by leucine. REV-ERB alpha overexpression activated the mTORC1 signaling pathway via transcription inhibition of mTORC1 inhibitor, Tsc1, antagonizing the effect of leucine, while its silencing downregulated mTORC1 signaling. Activation of mTORC1 led to increased BMAL1 phosphorylation. Activation as well as inhibition of mTORC1 led to altered circadian rhythms in mouse muscle. Inhibition of liver mTORC1 by leucine or rapamycin led to low-amplitude circadian rhythms. In summary, our study shows that leucine inhibits liver mTORC1 pathway leading to dampened circadian rhythms. REV-ERB alpha activates the mTORC1 pathway, leading to phosphorylation of the clock protein BMAL1.

Background: Nonalcoholic fatty-liver disease (NAFLD) is a global health problem associated with gut dys-biosis and intestinal permeability. Broccoli is a natural source of bioactive phytochemicals, characterized by health-promoting properties. Objective: This study evaluated the effect of broccoli florets and stalks on liver fat accumulation, inflamma-tion, gut microbiome, and intestinal barrier integrity. Design: Male C57BL/6J mice (n = 32, 8-week-old) were fed with a high-fat high-cholesterol diet (HFCD) with/without 15% broccoli (florets or stalks) for 7 weeks. Liver damage was evaluated by changes in glucose response and histological and biochemical parameters. Protein and gene expressions related to liver inflam-mation were examined. The effect of broccoli on microbiota population together with genes related to barrier integrity in the gut was investigated. Results: Dietary broccoli improved the glycemic response assessed by oral glucose tolerance test (OGTT). Histological evaluation showed no change in hepatic steatosis. Broccoli consumption also attenuated inflam-mation as revealed by lower inducible nitric oxide synthase (iNOS) and serum amyloid A1 (SAA1) expression levels in broccoli-supplemented groups. Gut microbiota analysis demonstrated elevated Acidifaciens and re-duced Mucispirillum schaedleri abundance in the stalks group, whereas Proteobacteria strains abundance was increased in the florets group. Gut integrity remained unchanged. Conclusion: Broccoli supplementation improves glucose tolerance, attenuates liver inflammation, and alters microbial composition, but does not affect gut integrity. This research provides new evidence on the effects of dietary broccoli under HFCD.

Aims The Mediterranean fruit fly (the medfly) causes major losses of agricultural fruits. Its microbiome is mainly composed of various Enterobacteriaceae that contribute to nutrient acquisition and are associated with the fly's development. Moreover, the performance of males produced by the sterile insect technique is improved by providing mass-reared insects with specific gut bacteria. Bdellovibrio and like organisms (BALOs) are obligate predators of Gram-negative bacteria that efficiently preys upon diverse Enterobacteriaceae, making it a potential disruptor of the fly's microbiome. We hypothesized that the fly's microbiome can be targeted to control the insect. Methods and Results Inoculation of B. bacteriovorus as free-swimming or encapsulated cells into gut extracts significantly reduced gut bacterial abundance, sustaining predator survival. Similar treatments applied to adult flies showed that the predators also survived in the gut environment. While addition of the predators did not affect total gut bacterial abundance and end-point fly mortality, a shift in the gut community structure, measured by high-throughput community sequencing was observed. Conclusions The bacterial predator of bacteria B. bacteriovorus can prey and survive in vivo in the medfly gut. Significance and Impact of the Study This study establishes the potential of BALOs to affect the microbiome of insect hosts.

Nonalcoholic fatty liver disease (NAFLD) is linked to obesity, type 2 diabetes, hyperlipidemia, and gut dysbiosis. Gut microbiota profoundly affects the host energy homeostasis, which, in turn, is affected by a high-fat diet (HFD) through the liver-gut axis, among others. Broccoli contains beneficial bioactive compounds and may protect against several diseases. This study aimed to determine the effects of broccoli supplementation to an HFD on metabolic parameters and gut microbiome in mice. Male (7-8 weeks old) C57BL/J6 mice were divided into four groups: normal diet (ND), high-fat diet (HFD), high-fat diet+10% broccoli florets (HFD + F), and high-fat diet + 10% broccoli stalks (HFD + S). Liver histology and serum biochemical factors were evaluated. Alterations in protein and gene expression of the key players in lipid and carbohydrate metabolism as well as in gut microbiota alterations were also investigated. Broccoli florets addition to the HFD significantly reduced serum insulin levels, HOMA-IR index, and upregulated adiponectin receptor expression. Conversely, no significant difference was found in the group supplemented with broccoli stalks. Both broccoli stalks and florets did not affect fat accumulation, carbohydrate, or lipid metabolism-related parameters. Modifications in diversity and in microbial structure of proteobacteria strains, Akermansia muciniphila and Mucispirillum schaedleri were observed in the broccoli-supplemented HFD-fed mice. The present study suggests that dietary broccoli alters parameters related to insulin sensitivity and modulates the intestinal environment. More studies are needed to confirm the results of this study and to investigate the mechanisms underlying these beneficial effects.

Hydrophobic carriers were examined for geosmin and 2-methylisoborneol removal from water derived from an aquaculture system. A combination of adsorption and biodegradation was found to underlie the removal of the off-flavor compounds. Adsorption of these compounds by the carriers was unaffected by the presence of organic matter in the water to be treated. A model based on adsorption/desorption and first-order degradation kinetics provided an accurate prediction for experimentally determined 2-methylisoborneol removal rates. Steady removal of geosmin and 2-methylisoborneol as well as nitrate reduction were observed during long-term operation of the plug-flow reactors with water derived from an aquaculture facility. Metagenomic analysis of the microbial community on the carriers during long-term operation of the reactors revealed a predominance of denitrifying bacteria. It was found that geosmin and 2-methylisoborneol led to statistically significant changes in the abundances of 21 contigs that contained genes involved in terpene degradation. This study shows that at low ambient concentrations of geosmin and 2-methylisoborneol in nitrate and organic-rich water, such as found in aquaculture systems, their biodegradation can be accomplished by terpene-degrading denitrifiers that develop on hydrophobic carriers used for filtration of the contaminated water.

Nitrites and nitrates are traditional food additives used as curing agents in the food industry. They inhibit the growth of microorganisms and give a typical pink color to meat. Besides the positive effects of nitrite in foods, if present at high levels in the body, may induce hypoxia and contribute to the production of pro-carcinogenic secondary N-nitrosamines. This study investigated the whole-body metabolic effects of hemin and nitrite added to a high fat diet as red and processed red meat nutritional models. Mice were fed for 11 weeks with five different diets-(1) control diet (ND), (2) high fat diet (HFD) with 60% fat, (3) HFD with hemin (HFD + H, red meat model), (4) HFD with hemin and nitrite (HFD + HN, processed meat model), and (5) HFD with hemin, nitrite, and secondary amine (HFD + HNN, N-nitrosamine generating model)-and several metabolic parameters were determined and respiratory measurements were performed. Mice fed with the HFD + H or HFD + HNN diet had a lower epididymal white adipose tissue (eWAT) : body ratio and lower fasting glucose level than those fed the HFD alone. In addition, our results demonstrated a relief in hepatosteatosis grade among the HFD + H and HFD + HNN diet fed mice. Nitrite added to the HFD impaired the ability to use fat for energy, opposite to the effect of hemin. This study shows that nitrite in addition to pro-carcinogenesis and hypoxia can impact metabolic disease progression when added to meat.

Naturally occurring sugars usually have d-chirality. While a change in chirality typically affects ligand–receptor interaction, non-caloric l-glucose was reported as sweet for humans. Here we show that l- and d-glucose have similar sensory detection thresholds (0.041 ± 0.006 M for d-glucose, and 0.032 ± 0.007 M for l-glucose) and similar sweetness intensities at suprathreshold concentrations. We demonstrate that l-glucose acts via the sweet taste receptor TAS1R2/TAS1R3, eliciting a dose-dependent activation in cell-based functional assays. Computational docking of glucose to the VFT domain of TAS1R2 suggests two sub-pockets, each compatible with each of the enantiomers. While some polar residues (Y103, D142, N143, S144, Y215) are unique for sub-pocket A and others (D307, T326, E382, R383) for sub-pocket B, no interaction is unique for only one enantiomer. The many options for creating hydrogen bonds with the hydroxyl moieties of glucose explain how both enantiomers can fit each one of the sub-pockets.

Cryopreservation of adherent cells is crucial for commercial cell therapy technology, including effective distribution and storage. Fast thawing has been shown to increase cell recovery in vitrified samples. Previously, radiofrequency (RF) has been investigated as a heating source on large samples, either with or without magnetic particles. Also, laser heating with the aid of dye or nanoparticles has been utilized on sub–millimeter samples successfully. For slow freezing cryopreservation methods, the influence of rate of thawing on viability is less clear. Cryopreservation of surface adhered cells result in many cases in detachment from the surface. We illustrate how intense infrared radiation from a focused halogen illuminator accelerates thawing. We show that two epithelial cell lines, retinal pigment epithelium cells and heterogeneous human epithelial colorectal adenocarcinoma cells, can be effectively cryopreserved and recovered using a combination of slow freezing and fast thawing under infrared illumination. We were able to successfully thaw samples, of 2–4 mm thick, including the media, on the order of a second, providing a heating rate of thousands of Kelvin per minute. Under optimal conditions, we observed higher post–thawing cell viability rates and higher cell adhesion with infrared thawing than with water bath thawing. We suggest that bulk warming with infrared radiation has an advantage over surface warming of surface–attached cells, as it alleviates cell stress during the process of thawing. These findings will pave the way for novel approaches to treating substrate–adhered cells and 3D scaffolds with cells and organoids. This technology may serve as a crucial component in lab–on–chip systems for medical testing and therapeutic use.

Ultra-processed foods have known negative implications for health; however, their effect on skeletal development has never been explored. Here, we show that young rats fed ultra-processed food rich in fat and sugar suffer from growth retardation due to lesions in their tibial growth plates. The bone mineral density decreases significantly, and the structural parameters of the bone deteriorate, presenting a sieve-like appearance in the cortices and poor trabecular parameters in long bones and vertebrae. This results in inferior mechanical performance of the entire bone with a high fracture risk. RNA sequence analysis of the growth plates demonstrated an imbalance in extracellular matrix formation and degradation and impairment of proliferation, differentiation and mineralization processes. Our findings highlight, for the first time, the severe impact of consuming ultra-processed foods on the growing skeleton. This pathology extends far beyond that explained by the known metabolic effects, highlighting bone as a new target for studies of modern diets.

ObjectiveBroccoli is a “functional food” that contains bioactive compounds and phytochemicals that have beneficial health-promoting effects. This study aimed at investigating the effects of broccoli consumption on lipid and glucose metabolism and gut microbiota. Methods Male C57BL/6J mice (7–8 wk old) were fed ad libitum with a normal diet supplemented with or without 10% (w/w) broccoli florets or broccoli stalks. Oral glucose tolerance tests were performed at week 15. After 17 wk, blood and tissues were collected. Serum parameters, histology, gene and protein expression, and intestinal microbiota composition were evaluated. Results Stalk supplementation led to reductions in fasting glucose levels, serum insulin, and the homeostasis model assessment–insulin resistance (HOMA-IR) index. Liver enzymes improved in both experimental groups, and broccoli florets decreased total triacylglycerols. The stalks group had elevated fatty acid oxidation–related genes and proteins (AMPK, PPARα, and CPT1). Diverse microbiota populations were observed in both broccoli groups. Broccoli stalks were found to be richer in Akkermansia muciniphila, while broccoli florets reduced Mucispirillum schaedleri abundance and increased bacterial richness. Conclusions Long-term whole broccoli supplementation decreased inflammation, improved lipid parameters and insulin sensitivity, and altered the gut microbiome in mice. Our data provide new information regarding the potential benefits of broccoli stalks in metabolic parameters.

Drug development is a long, expensive and multistage process geared to achieving safe drugs with high efficacy. A crucial prerequisite for completing the medication regimen for oral drugs, particularly for pediatric and geriatric populations, is achieving taste that does not hinder compliance. Currently, the aversive taste of drugs is tested in late stages of clinical trials. This can result in the need to reformulate, potentially resulting in the use of more animals for additional toxicity trials, increased financial costs and a delay in release to the market. Here we present BitterIntense, a machine learning tool that classifies molecules into “very bitter” or “not very bitter”, based on their chemical structure. The model, trained on chemically diverse compounds, has above 80% accuracy on several test sets. Our results suggest that about 25% of drugs are predicted to be very bitter, with even higher prevalence (~40%) in COVID19 drug candidates and in microbial natural products. Only ~10% of toxic molecules are predicted to be intensely bitter, and it is also suggested that intense bitterness does not correlate with hepatotoxicity of drugs. However, very bitter compounds may be more cardiotoxic than not very bitter compounds, possessing significantly lower QPlogHERG values. BitterIntense allows quick and easy prediction of strong bitterness of compounds of interest for food, pharma and biotechnology industries. We estimate that implementation of BitterIntense or similar tools early in drug discovery process may lead to reduction in delays, in animal use and in overall financial burden.

Smell loss is important for coronavirus disease-2019 (COVID-19) screening and diagnosis. Particular attention should be paid to individuals with pre-COVID-19 chronic hyposmia or anosmia. We report a case of reversible taste impairment in a COVID-19 patient with chronically impaired sense of smell. This case emphasizes the importance of COVID-19-related taste assessment.

ObjectivesTo characterize longitudinal symptoms of mild coronavirus disease 2019 (COVID-19) patients for a period of 6 months, to potentially aid in disease management. Methods Phone interviews were conducted with 103 patients with mild COVID-19 in Israel over a 6-month period (April 2020 to October 2020). Patients were recruited via social media and word to mouth and were interviewed up to 4 times, depending on reports of their unresolved symptoms. Inclusion criteria required participants to be residents of Israel aged 18 years or older, with positive COVID-19 real-time PCR results and nonsevere symptoms. The onset, duration, severity and resolution of symptoms were analysed. Results A total of 44% (45/103), 41% (42/103), 39% (40/103) and 38% (39/103) of patients experienced headache, fever, muscle ache and dry cough as the first symptom respectively. Smell and taste changes were experienced at 3.9 ± 5.4 and 4.6 ± 5.7 days (mean ± standard deviation (SD)) after disease onset respectively. Among prevalent symptoms, fever had the shortest duration (5.8 ± 8.6 days), and taste and smell changes were the longest-lasting symptoms (17.2 ± 17.6 and 18.9 ± 19.7 days; durations censored at 60 days). Longer recovery of the sense of smell correlated with the extent of smell change. At the 6-month follow-up, 46% (47/103) of the patients had at least one unresolved symptom, most commonly fatigue (22%, 23/103), smell and taste changes (15%, 15/103 and 8%, 8/103 respectively) and breathing difficulties (8%, 8/103). Conclusions Long-lasting effects of mild COVID-19 manifested in almost half of the participants reporting at least one unresolved symptom after 6 months.

Obesity is a global epidemic causing morbidity and impaired quality of life. The melanocortin receptor 4 (MC4R) is at the crux of appetite, energy homeostasis, and body-weight control in the central nervous system and is a prime target for anti-obesity drugs. Here, we present the cryo-EM structure of the human MC4R-Gs signaling complex bound to the agonist setmelanotide, a cyclic peptide recently approved for the treatment of obesity. The work reveals the mechanism of MC4R activation, highlighting a molecular switch that initiates satiation signaling. In addition, our findings indicate that Ca2+ is required for agonist but not antagonist efficacy. These results fill a gap in understanding MC4R activation and could guide the design of future weight management drugs.

Hydrogen to deuterium isotopic substitution has only a minor effect on physical and chemical properties of water and, as such, is not supposed to influence its neutral taste. Here we conclusively demonstrate that humans are, nevertheless, able to distinguish D2O from H2O by taste. Indeed, highly purified heavy water has a distinctly sweeter taste than same-purity normal water and can add to perceived sweetness of sweeteners. In contrast, mice do not prefer D2O over H2O, indicating that they are not likely to perceive heavy water as sweet. HEK 293T cells transfected with the TAS1R2/TAS1R3 heterodimer and chimeric G-proteins are activated by D2O but not by H2O. Lactisole, which is a known sweetness inhibitor acting via the TAS1R3 monomer of the TAS1R2/TAS1R3, suppresses the sweetness of D2O in human sensory tests, as well as the calcium release elicited by D2O in sweet taste receptor-expressing cells. The present multifaceted experimental study, complemented by homology modelling and molecular dynamics simulations, resolves a long-standing controversy about the taste of heavy water, shows that its sweet taste is mediated by the human TAS1R2/TAS1R3 taste receptor, and opens way to future studies of the detailed mechanism of action.

Directional solidification occurs in industrial and natural processes, such as freeze-casting, metal processing, biological cryopreservation and freezing of soils. Translational temperature gradient stage allows to control the process of directional solidification and to visualize it with optical microscope. In this stage freezing velocity and temperature gradient are decoupled and are independently controlled. Here we study the dynamics of the phase transition interface in thin water samples using translational temperature gradient stage. We follow position of the ice–water interface with optical microscopy and compare it to solution of one dimensional Stefan problem in the low velocity limit. We find an agreement between experimental observations and theoretical predictions for constant velocity and during acceleration of the ice front. This work presents a practical framework for analysis and design of experiments on a translational temperature gradient stage.