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Abstract Epiphytic microbes are those that live for some or all of their life cycle on the surface of plant leaves. Leaf surfaces are a topologically complex, physicochemically heterogeneous habitat that is home to extensive, mixed communities of resident and transient inhabitants from all three domains of life. In this review, we discuss the origins of leaf surface microbes and how different biotic and abiotic factors shape their communities. We discuss the leaf surface as a habitat and microbial adaptations which allow some species to thrive there, with particular emphasis on microbes that occupy the continuum between epiphytic specialists and phytopathogens, groups which have considerable overlap in terms of adapting to the leaf surface and between which a single virulence determinant can move a microbial strain. Finally, we discuss the recent findings that the wheat pathogenic fungus Zymoseptoria tritici spends a considerable amount of time on the leaf surface, and ask what insights other epiphytic organisms might provide into this pathogen, as well as how Z. tritici might serve as a model system for investigating plant–microbe-microbe interactions on the leaf surface.

Objective Lifestyle intervention can produce clinically significant weight loss and reduced disease risk/severity for many individuals with overweight/obesity. Dietary lapses, instances of non-adherence to the recommended dietary goal(s) in lifestyle intervention, are associated with less weight loss and higher energy intake. There are distinct “types” of dietary lapse (e.g., eating an off-plan food, eating a larger portion), and behavioral, psychosocial, and contextual mechanisms may differ across dietary lapse types. Some lapse types also appear to impact weight more than others. Elucidating clear lapse types thus has potential for understanding and improving adherence to lifestyle intervention. Methods This 18-month observational cohort study will use real-time digital assessment tools within a multi-level factor analysis framework to uncover “lapse phenotypes” and understand their impact on clinical outcomes. Adults with overweight/obesity ( n = 150) will participate in a 12-month online lifestyle intervention and 6-month weight loss maintenance period. Participants will complete 14-day lapse phenotyping assessment periods at baseline, 3, 6, 12, and 18 months in which smartphone surveys, wearable devices, and geolocation will assess dietary lapses and relevant phenotyping characteristics. Energy intake (via 24-h dietary recall) and weight will be collected at each assessment period. Results This trial is ongoing; data collection began on 31 October 2022 and is scheduled to complete by February 2027. Conclusion Results will inform novel precision tools to improve dietary adherence in lifestyle intervention, and support updated theoretical models of adherence behavior. Additionally, these phenotyping methods can likely be leveraged to better understand non-adherence to other health behavior interventions. Trial Registration This study was prospectively registered https://clinicaltrials.gov/study/NCT05562427

How molecules interact governs how they move. Single-molecule tracking (SMT) thus provides a unique window into the dynamic interactions of biomolecules within live cells. Using transcription regulation as a case study, we describe how SMT works, what it can tell us about molecular biology, and how it has changed our perspective on the inner workings of the nucleus. We also describe what SMT cannot yet tell us and how new technical advances seek to overcome its limitations. This ongoing progress will be imperative to address outstanding questions about how dynamic molecular machines function in live cells.

ObjectivesTo identify incident SARS-CoV-2 infections and inform effective mitigation strategies in university settings, we piloted an integrated symptom and exposure monitoring and testing system among a cohort of university students and employees.DesignProspective cohort study.SettingA public university in California from June to August 2020.Participants2180 university students and 738 university employees.Primary outcome measuresAt baseline and endline, we tested participants for active SARS-CoV-2 infection via quantitative PCR (qPCR) test and collected blood samples for antibody testing. Participants received notifications to complete additional qPCR tests throughout the study if they reported symptoms or exposures in daily surveys or were selected for surveillance testing. Viral whole genome sequencing was performed on positive qPCR samples, and phylogenetic trees were constructed with these genomes and external genomes.ResultsOver the study period, 57 students (2.6%) and 3 employees (0.4%) were diagnosed with SARS-CoV-2 infection via qPCR test. Phylogenetic analyses revealed that a super-spreader event among undergraduates in congregate housing accounted for at least 48% of cases among study participants but did not spread beyond campus. Test positivity was higher among participants who self-reported symptoms (incidence rate ratio (IRR) 12.7; 95% CI 7.4 to 21.8) or had household exposures (IRR 10.3; 95% CI 4.8 to 22.0) that triggered notifications to test. Most (91%) participants with newly identified antibodies at endline had been diagnosed with incident infection via qPCR test during the study.ConclusionsOur findings suggest that integrated monitoring systems can successfully identify and link at-risk students to SARS-CoV-2 testing. As the study took place before the evolution of highly transmissible variants and widespread availability of vaccines and rapid antigen tests, further research is necessary to adapt and evaluate similar systems in the present context.

SARS coronavirus 2 (SARS-CoV-2) has caused an ongoing global pandemic with significant mortality and morbidity. At this time, the only FDA-approved therapeutic for COVID-19 is remdesivir, a broad-spectrum antiviral nucleoside analog. Efficacy is only moderate, and improved treatment strategies are urgently needed. To accomplish this goal, we devised a strategy to identify compounds that act synergistically with remdesivir in preventing SARS-CoV-2 replication. We conducted combinatorial high-throughput screening in the presence of submaximal remdesivir concentrations, using a human lung epithelial cell line infected with a clinical isolate of SARS-CoV-2. This identified 20 approved drugs that act synergistically with remdesivir, many with favorable pharmacokinetic and safety profiles. Strongest effects were observed with established antivirals, Hepatitis C virus nonstructural protein 5A (HCV NS5A) inhibitors velpatasvir and elbasvir. Combination with their partner drugs sofosbuvir and grazoprevir further increased efficacy, increasing remdesivir's apparent potency > 25-fold. We report that HCV NS5A inhibitors act on the SARS-CoV-2 exonuclease proofreader, providing a possible explanation for the synergy observed with nucleoside analog remdesivir. FDA-approved Hepatitis C therapeutics Epclusa® (velpatasvir/sofosbuvir) and Zepatier® (elbasvir/grazoprevir) could be further optimized to achieve potency and pharmacokinetic properties that support clinical evaluation in combination with remdesivir.

Abstract Objective Greater perceived social support (PSS) is associated with more favorable changes in weight loss, activity behaviors, and eating regulation after metabolic and bariatric surgery (MBS). However, studies have relied on generic, retrospective PSS measures, and stability of PSS levels and relations with weight loss and weight‐related behaviors over time is unknown. Using smartphone‐based Ecological Momentary Assessment, this study evaluated pre‐to 1‐year post‐MBS changes in daily weight management‐focused PSS and associations with weight loss, device‐measured activity behaviors, and eating regulation before and during the initial year after MBS. Method Adult MBS patients (n = 71) received (1) an accelerometer to measure daily moderate‐to‐vigorous intensity physical activity (MVPA) and sedentary time (ST) minutes/day, and (2) a smartphone to complete morning weight‐focused PSS ratings and eating regulation (dietary restraint/disinhibition) ratings at four semi‐random times daily for 10 days at pre‐ and 3, 6, and 12‐month postoperative. Generalized linear mixed models analyzed the associations of PSS with total weight loss (%TWL) and activity/eating outcomes. Results Participants on average reported relatively stable moderate‐to‐high PSS (3.98 on one to five scale) across assessments. Perceived social support was not related to %TWL, MVPA, or ST. Participants with higher PSS reported lower disinhibition and higher restraint than those with lower PSS (ps

Single-molecule imaging provides a powerful way to study biochemical processes in live cells, yet it remains challenging to track single molecules while simultaneously detecting their interactions. Here, we describe a novel property of rhodamine dyes, proximity-assisted photoactivation (PAPA), in which one fluorophore (the 'sender') can reactivate a second fluorophore (the 'receiver') from a dark state. PAPA requires proximity between the two fluorophores, yet it operates at a longer average intermolecular distance than Förster resonance energy transfer (FRET). We show that PAPA can be used in live cells both to detect protein-protein interactions and to highlight a subpopulation of labeled protein complexes in which two different labels are in proximity. In proof-of-concept experiments, PAPA detected the expected correlation between androgen receptor self-association and chromatin binding at the single-cell level. These results establish a new way in which a photophysical property of fluorophores can be harnessed to study molecular interactions in single-molecule imaging of live cells.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), has emerged as a major global health threat. The COVID-19 pandemic has resulted in over 168 million cases and 3.4 million deaths to date, while the number of cases continues to rise. With limited therapeutic options, the identification of safe and effective therapeutics is urgently needed. The repurposing of known clinical compounds holds the potential for rapid identification of drugs effective against SARS-CoV-2. Here, we utilized a library of FDA-approved and well-studied preclinical and clinical compounds to screen for antivirals against SARS-CoV-2 in human pulmonary epithelial cells. We identified 13 compounds that exhibit potent antiviral activity across multiple orthogonal assays. Hits include known antivirals, compounds with anti-inflammatory activity, and compounds targeting host pathways such as kinases and proteases critical for SARS-CoV-2 replication. We identified seven compounds not previously reported to have activity against SARS-CoV-2, including B02, a human RAD51 inhibitor. We further demonstrated that B02 exhibits synergy with remdesivir, the only antiviral approved by the FDA to treat COVID-19, highlighting the potential for combination therapy. Taken together, our comparative compound screening strategy highlights the potential of drug repurposing screens to identify novel starting points for development of effective antiviral mono- or combination therapies to treat COVID-19.

The most common method for RNA detection involves reverse transcription followed by quantitative polymerase chain reaction (RT-qPCR) analysis. Commercial one-step master mixes-which include both a reverse transcriptase and a thermostable polymerase and thus allow performing both the RT and qPCR steps consecutively in a sealed well-are key reagents for SARS-CoV-2 diagnostic testing; yet, these are typically expensive and have been affected by supply shortages in periods of high demand. As an alternative, we describe here how to express and purify Taq polymerase and M-MLV reverse transcriptase and assemble a homemade one-step RT-qPCR master mix. This mix can be easily assembled from scratch in any laboratory equipped for protein purification. We also describe two simple alternative methods to prepare clinical swab samples for SARS-CoV-2 RNA detection by RT-qPCR: heat-inactivation for direct addition, and concentration of RNA by isopropanol precipitation. Finally, we describe how to perform RT-qPCR using the homemade master mix, how to prepare in vitro-transcribed RNA standards, and how to use a fluorescence imager for endpoint detection of RT-PCR amplification in the absence of a qPCR machine In addition to being useful for diagnostics, these versatile protocols may be adapted for nucleic acid quantification in basic research. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Preparation of a one-step RT-qPCR master mix using homemade enzymes Basic Protocol 2: Preparation of swab samples for direct RT-PCR Alternate Protocol 1: Concentration of RNA from swab samples by isopropanol precipitation Basic Protocol 3: One-step RT-qPCR of RNA samples using a real-time thermocycler Support Protocol: Preparation of RNA concentration standards by in vitro transcription Alternate Protocol 2: One-step RT-PCR using endpoint fluorescence detection.

Re-opening of communities in the midst of the ongoing COVID-19 pandemic has ignited new waves of infections in many places around the world. Mitigating the risk of reopening will require widespread SARS-CoV-2 testing, which would be greatly facilitated by simple, rapid, and inexpensive testing methods. This study evaluates several protocols for RNA extraction and RT-qPCR that are simpler and less expensive than prevailing methods. First, isopropanol precipitation is shown to provide an effective means of RNA extraction from nasopharyngeal (NP) swab samples. Second, direct addition of NP swab samples to RT-qPCRs is evaluated without an RNA extraction step. A simple, inexpensive swab collection solution suitable for direct addition is validated using contrived swab samples. Third, an open-source master mix for RT-qPCR is described that permits detection of viral RNA in NP swab samples with a limit of detection of approximately 50 RNA copies per reaction. Quantification cycle (Cq) values for purified RNA from 30 known positive clinical samples showed a strong correlation (r2 = 0.98) between this homemade master mix and commercial TaqPath master mix. Lastly, end-point fluorescence imaging is found to provide an accurate diagnostic readout without requiring a qPCR thermocycler. Adoption of these simple, open-source methods has the potential to reduce the time and expense of COVID-19 testing.

Re-opening of communities in the midst of the ongoing COVID-19 pandemic has ignited a second wave of infections in many places around the world. Mitigating the risk of reopening will require widespread SARS-CoV-2 testing, which would be greatly facilitated by simple, rapid, and inexpensive testing methods. To this end, we evaluated several protocols for RNA extraction and RT-qPCR that are simpler and less expensive than prevailing methods. First, we show that isopropanol precipitation provides an effective means of RNA extraction from nasopharyngeal (NP) swab samples. Second, we evaluate direct addition of NP swab samples to RT-qPCR reactions without an RNA extraction step. We describe a simple, inexpensive swab collection solution suitable for direct addition, which we validate using contrived swab samples. Third, we describe an open-source master mix for RT-qPCR and show that it permits detection of viral RNA in NP swab samples. Lastly, we show that an end-point fluorescence measurement provides an accurate diagnostic readout without requiring a qPCR thermocycler. Adoption of these simple, inexpensive methods has the potential to significantly reduce the time and expense of COVID-19 testing.

Introduction: Tobacco smoking is a priority public health concern, and a leading cause of deathand disability globally. While the daily smoking prevalence in Canada is approximately 9.7%,the proportion of smokers amongst emergency department (ED) patients has been found to besignificantly higher. The purpose of this survey study was to determine the smoking prevalence ofadult ED patients presenting to three urban Canadian hospitals, and to determine whether there wasan increased prevalence compared to the general public.Methods: A verbal questionnaire was administered to adult patients aged 18 years and olderpresenting to Royal University Hospital, St. Paul’s Hospital, and Saskatoon City Hospital inSaskatoon, Saskatchewan. We compared patients’ smoking habits to Fagerström tobaccodependence scores, readiness to quit smoking, chief complaints, Canadian Triage Acuity Scalescores, and willingness to partake in ED-specific cessation interventions.Results: A total of 1190 eligible patients were approached, and 1078 completed the questionnaire.Adult Saskatoon ED patients demonstrated a cigarette smoking prevalence of 19.6%, which issignificantly higher than the adult Saskatchewan public at 14.65% (P

Abstract Objective Lapses from the dietary prescription in lifestyle modification interventions for overweight/obesity are common and impact weight loss outcomes. While it is expected that lapses influence weight via increased consumption, there are no studies that have evaluated how dietary lapses affect dietary intake during treatment. This study examined the association between daily lapses and daily energy and macronutrient intake during a lifestyle modification intervention. Methods This study used an intensive longitudinal design to observe participants throughout a 6‐month lifestyle modification intervention. Participants (n = 32) were adults with overweight/obesity (body mass index 25–50 kg/m2) and a diagnosed cardiovascular disease risk factor (e.g., hypertension) with a desire to lose weight. Participants underwent a gold‐standard individual in‐person lifestyle modification protocol consisting of 3 months of weekly sessions with 3 months of monthly sessions. Each participant's dietary prescription included a calorie target range that was based on their starting weight. Participants completed ecological momentary assessment (EMA; repeated daily smartphone surveys) every other week to self‐report on dietary lapses and telephone‐based 24‐h dietary recalls every 6 weeks. Results On days with EMA and recalled intake (n = 210 days), linear mixed models demonstrated significant associations between daily lapse and higher total daily caloric intake (B = 139.20, p

IntroductionThe aim of this feasibility and proof-of-concept study was to examine the use of a novel wearable device for automatic food intake detection to capture the full range of free-living eating environments of adults with overweight and obesity. In this paper, we document eating environments of individuals that have not been thoroughly described previously in nutrition software as current practices rely on participant self-report and methods with limited eating environment options.MethodsData from 25 participants and 116 total days (7 men, 18 women, Mage = 44 ± 12 years, BMI 34.3 ± 5.2 kg/mm2), who wore the passive capture device for at least 7 consecutive days (≥12h waking hours/d) were analyzed. Data were analyzed at the participant level and stratified amongst meal type into breakfast, lunch, dinner, and snack categories. Out of 116 days, 68.1% included breakfast, 71.5% included lunch, 82.8% included dinner, and 86.2% included at least one snack.ResultsThe most prevalent eating environment among all eating occasions was at home and with one or more screens in use (breakfast: 48.1%, lunch: 42.2%, dinner: 50%, and snacks: 55%), eating alone (breakfast: 75.9%, lunch: 89.2%, dinner: 74.3%, snacks: 74.3%), in the dining room (breakfast: 36.7%, lunch: 30.1%, dinner: 45.8%) or living room (snacks: 28.0%), and in multiple locations (breakfast: 44.3%, lunch: 28.8%, dinner: 44.8%, snacks: 41.3%).DiscussionResults suggest a passive capture device can provide accurate detection of food intake in multiple eating environments. To our knowledge, this is the first study to classify eating occasions in multiple eating environments and may be a useful tool for future behavioral research studies to accurately codify eating environments.

Purpose . Identifying factors that influence moderate-to-vigorous intensity physical activity (MVPA) and sedentary time in metabolic and bariatric surgery (MBS) patients is necessary to inform the development of interventions. Weather/environmental factors may be especially important considering rapid climate change and the vulnerability of people with obesity to heat and pollution. Our study aimed to examine the associations of weather (maximal, average and Wet Bulb Globe Temperatures), and air pollution indices (air quality index [AQI]) with daily physical activity (PA) of both light (LPA) and MVPA and sedentary time before and after MBS. Materials and methods . Participants (n = 77) wore an accelerometer at pre- and 3, 6, and 12-months post-MBS to assess LPA/MVPA/ sedentary time (min/d). These data were combined with participants’ local (Boston, MA or Providence, RI, USA) daily weather and AQI data (extracted from federal weather and environmental websites). Results . Multilevel generalized additive models showed inverted U-shaped associations between weather indices and MVPA, with a marked reduction in MVPA for daily maximal temperatures ≥20 °C. Sensitivity analysis showed a less marked decrease of MVPA (min/d) during higher temperatures after versus before MBS. Both MVPA before and after MBS and sedentary time before MBS were negatively impacted by higher AQI levels. Conclusion . This study is the first to show that weather and air pollution indices, even in locations with good AQI and moderate temperatures, are related to variability in activity behaviors, particularly MVPA, during pre- and post-MBS. Weather/environmental conditions should be considered in MVPA prescription/strategies for adults who have undergone MBS.

BackgroundChildren who have Autism Spectrum Disorder (ASD) show preferences for processed foods, such as salty and sugary snacks (SSS) and sugar-sweetened beverages (SSB), while healthier foods, such as fruits and vegetables (FV), are consumed less. Innovative tools are needed that can efficiently disseminate evidence-based interventions and engage autistic children to improve their diet.AimThe aim of this 3-month randomized trial was to test the initial efficacy of a mobile health (mHealth) nutrition intervention on changing consumption of targeted healthy (FV) and less healthy foods/beverages (SSS, SSB) in children who have ASD, ages 6–10, who were picky eaters.MethodsThirty-eight parent-child dyads were randomly assigned to either an intervention (technology) group or a wait list control (education) group. The intervention included behavioral skills training, a high level of personalization for dietary goals, and involved parents as “agents of change.” Parents in the education group received general nutrition education and the dietary goals but did not receive skills training. Children's intake was assessed at baseline and at 3 months using 24-hour dietary recalls.ResultsWhile there were no significant group-by-time interactions (P > 0.25) for any of the primary outcomes, we found a significant main effect of time for FV intake (P = 0.04) indicating that both groups consumed more FV at 3 months (2.58 ± 0.30 servings/day) than at baseline (2.17 ± 0.28 servings/day; P = 0.03). Children in the intervention group who consumed few FV at baseline and showed high engagement with the technology increased their FV intake by 1.5 servings/day (P

Abstract Background Bariatric surgery produces weight loss in part by impacting appetite and eating behavior. Research suggests physical activity (PA) assists with regulation of appetite and eating during non‐surgical weight loss, although whether PA carries similar benefits in the context of bariatric surgery is unknown. Objective Evaluate associations of moderate‐to‐vigorous intensity PA (MVPA) and sedentary time (ST) with appetite sensations (hunger [homeostatic/hedonic], satiety) and eating regulation behaviors (restraint, disinhibition) before and during the initial year following bariatric surgery. Method Adult bariatric patients received an accelerometer to measure MVPA/ST and a smartphone to complete appetite/eating ratings at four semi‐random times daily for 10 days at pre‐ and 3‐, 6‐, and 12‐months post‐surgery. Data were analyzed using generalized linear mixed models. Results Higher MVPA levels related to more satiety across time (p = 0.045) and more restraint at 3‐months post‐surgery (p