Structural snapshots of R-loop formation by a type I-C CRISPR Cascade.

Type I CRISPR-Cas systems employ multi-subunit Cascade effector complexes to target foreign nucleic acids for destruction. Here, we present structures of D. vulgaris type I-C Cascade at various stages of double-stranded (ds)DNA target capture, revealing mechanisms that underpin PAM recognition and Cascade allosteric activation. We uncover an interesting mechanism of non-target strand (NTS) DNA stabilization via stacking interactions with the "belly" subunits, securing the NTS in place. This "molecular seatbelt" mechanism facilitates efficient R-loop formation and prevents dsDNA reannealing. Additionally, we provide structural insights into how two anti-CRISPR (Acr) proteins utilize distinct strategies to achieve a shared mechanism of type I-C Cascade inhibition by blocking PAM scanning. These observations form a structural basis for directional R-loop formation and reveal how different Acr proteins have converged upon common molecular mechanisms to efficiently shut down CRISPR immunity.

Transgenic ferret models define pulmonary ionocyte diversity and function.

Speciation leads to adaptive changes in organ cellular physiology and creates challenges for studying rare cell-type functions that diverge between humans and mice. Rare cystic fibrosis transmembrane conductance regulator (CFTR)-rich pulmonary ionocytes exist throughout the cartilaginous airways of humans1,2, but limited presence and divergent biology in the proximal trachea of mice has prevented the use of traditional transgenic models to elucidate ionocyte functions in the airway. Here we describe the creation and use of conditional genetic ferret models to dissect pulmonary ionocyte biology and function by enabling ionocyte lineage tracing (FOXI1-CreERT2::ROSA-TG), ionocyte ablation (FOXI1-KO) and ionocyte-specific deletion of CFTR (FOXI1-CreERT2::CFTRL/L). By comparing these models with cystic fibrosis ferrets3,4, we demonstrate that ionocytes control airway surface liquid absorption, secretion, pH and mucus viscosity-leading to reduced airway surface liquid volume and impaired mucociliary clearance in cystic fibrosis, FOXI1-KO and FOXI1-CreERT2::CFTRL/L ferrets. These processes are regulated by CFTR-dependent ionocyte transport of Cl- and HCO3-. Single-cell transcriptomics and in vivo lineage tracing revealed three subtypes of pulmonary ionocytes and a FOXI1-lineage common rare cell progenitor for ionocytes, tuft cells and neuroendocrine cells during airway development. Thus, rare pulmonary ionocytes perform critical CFTR-dependent functions in the proximal airway that are hallmark features of cystic fibrosis airway disease. These studies provide a road map for using conditional genetics in the first non-rodent mammal to address gene function, cell biology and disease processes that have greater evolutionary conservation between humans and ferrets.

Structural basis for mismatch surveillance by CRISPR-Cas9.

CRISPR-Cas9 as a programmable genome editing tool is hindered by off-target DNA cleavage1-4, and the underlying mechanisms by which Cas9 recognizes mismatches are poorly understood5-7. Although Cas9 variants with greater discrimination against mismatches have been designed8-10, these suffer from substantially reduced rates of on-target DNA cleavage5,11. Here we used kinetics-guided cryo-electron microscopy to determine the structure of Cas9 at different stages of mismatch cleavage. We observed a distinct, linear conformation of the guide RNA-DNA duplex formed in the presence of mismatches, which prevents Cas9 activation. Although the canonical kinked guide RNA-DNA duplex conformation facilitates DNA cleavage, we observe that substrates that contain mismatches distal to the protospacer adjacent motif are stabilized by reorganization of a loop in the RuvC domain. Mutagenesis of mismatch-stabilizing residues reduces off-target DNA cleavage but maintains rapid on-target DNA cleavage. By targeting regions that are exclusively involved in mismatch tolerance, we provide a proof of concept for the design of next-generation high-fidelity Cas9 variants.

Inflammatory Activity of Epithelial Stem Cell Variants from Cystic Fibrosis Lungs Is Not Resolved by CFTR Modulators.

Rationale: CFTR (cystic fibrosis transmembrane conductance regulator) modulator drugs restore function to mutant channels in patients with cystic fibrosis (CF) and lead to improvements in body mass index and lung function. Although it is anticipated that early childhood treatment with CFTR modulators will significantly delay or even prevent the onset of advanced lung disease, lung neutrophils and inflammatory cytokines remain high in patients with CF with established lung disease despite modulator therapy, underscoring the need to identify and ultimately target the sources of this inflammation in CF lungs. Objectives: To determine whether CF lungs, like chronic obstructive pulmonary disease (COPD) lungs, harbor potentially pathogenic stem cell "variants" distinct from the normal p63/Krt5 lung stem cells devoted to alveolar fates, to identify specific variants that might contribute to the inflammatory state of CF lungs, and to assess the impact of CFTR genetic complementation or CFTR modulators on the inflammatory variants identified herein. Methods: Stem cell cloning technology developed to resolve pathogenic stem cell heterogeneity in COPD and idiopathic pulmonary fibrosis lungs was applied to end-stage lungs of patients with CF (three homozygous CFTR:F508D, one CFTR F508D/L1254X; FEV1, 14-30%) undergoing therapeutic lung transplantation. Single-cell-derived clones corresponding to the six stem cell clusters resolved by single-cell RNA sequencing of these libraries were assessed by RNA sequencing and xenografting to monitor inflammation, fibrosis, and mucin secretion. The impact of CFTR activity on these variants after CFTR gene complementation or exposure to CFTR modulators was assessed by molecular and functional studies. Measurements and Main Results: End-stage CF lungs display a stem cell heterogeneity marked by five predominant variants in addition to the normal lung stem cell, of which three are proinflammatory both at the level of gene expression and their ability to drive neutrophilic inflammation in xenografts in immunodeficient mice. The proinflammatory functions of these three variants were unallayed by genetic or pharmacological restoration of CFTR activity. Conclusions: The emergence of three proinflammatory stem cell variants in CF lungs may contribute to the persistence of lung inflammation in patients with CF with advanced disease undergoing CFTR modulator therapy.

Distress tolerance is linked with substance use motivations and problems in young adults across four continents.

INTRODUCTION: People low in trait distress tolerance are at higher risk for harmful patterns of substance use. Some evidence suggests that maladaptive motives for substance use account for this correlation. However, the generality of these associations remains in doubt because virtually all available data come from North American samples. METHOD: Using data from 7 countries (total N = 5858; U.S., Argentina, Uruguay, Spain, South Africa, Canada, and England), we examined distress tolerance's association with alcohol- and cannabis-related problems in young adults. On an exploratory basis, we examined how distress tolerance related to different substance-use motivations. RESULTS: We found that distress tolerance was inversely related to problematic alcohol and cannabis use (rs = -0.14 and - 0.13). There was notable variation across countries in the magnitude of these effects, particularly for cannabis-related problems. Additionally, exploratory analyses revealed statistically significant (cross-sectional) indirect effects of distress tolerance on substance-related problems via substance-use motivations related to neutralizing negative emotions. CONCLUSIONS: Distress tolerance's role in substance-use problems appears to generalize beyond North America, although effect sizes were generally small and varied notably across geographical regions. Distress tolerance's connection with negative reinforcement processes (e.g., coping motives) warrants attention as a possible mediator of its association with problematic substance use.

Sonic Hedgehog Signaling Is Essential for Pulmonary Ionocyte Specification in Human and Ferret Airway Epithelia.

Pulmonary ionocytes express high levels of cystic fibrosis transmembrane conductance regulator (CFTR), an anion channel that is critical for hydration of the airways and mucociliary clearance. However, the cellular mechanisms that govern ionocyte specification and function remain unclear. We observed that increased abundance of ionocytes in cystic fibrosis (CF) airway epithelium was associated with enhanced expression of Sonic Hedgehog (SHH) effectors. In this study, we evaluated whether the SHH pathway directly impacts ionocyte differentiation and CFTR function in airway epithelia. Pharmacological HPI1-mediated inhibition of SHH signaling component GLI1 significantly impaired human basal cell specification of ionocytes and ciliated cells but significantly enhanced specification of secretory cells. By contrast, activation of the SHH pathway effector smoothened (SMO) with the chemical agonist SAG significantly enhanced ionocyte specification. The abundance of CFTR+ BSND+ ionocytes under these conditions had a direct relationship with CFTR-mediated currents in differentiated air-liquid interface (ALI) airway cultures. These findings were corroborated in ferret ALI airway cultures generated from basal cells in which the genes encoding the SHH receptor PTCH1 or its intracellular effector SMO were genetically ablated using CRISPR-Cas9, causing aberrant activation or suppression of SHH signaling, respectively. These findings demonstrate that SHH signaling is directly involved in airway basal cell specification of CFTR-expressing pulmonary ionocytes and is likely responsible for enhanced ionocyte abundance in the CF proximal airways. Pharmacologic approaches to enhance ionocyte and reduce secretory cell specification after CFTR gene editing of basal cells may have utility in the treatment of CF.

Brain Metastases from Biliary Tract Cancer: Case Series and Clinicogenomic Analysis.

BACKGROUND: Limited data from small series have suggested that brain metastases from biliary tract cancers (BrM-BTC) affect ≤2% of patients with BTC. We sought to review our experience with patients with BrM-BTC and to identify associations of tumor-related molecular alterations with outcomes. MATERIALS AND METHODS: A retrospective review of patients with BTC seen at a tertiary referral center from 2005 to 2021 was performed; patients with BrM-BTC were identified, and clinical and molecular data were collected. RESULTS: Twenty-one of 823 patients with BTC (2.6%) developed BrM. For patients with BrM-BTC, median follow-up time was 27.9 months after primary BTC diagnosis and 3.1 months after BrM diagnosis. Median time from primary diagnosis to diagnosis of BrM was 14.4 [range, 1.1-66.0] months. Median overall survival (OS) from primary diagnosis was 31.5 [2.9-99.8] months and median OS from BrM diagnosis was 4.2 [0.2-33.8] months. Patients who underwent BrM-directed therapy trended toward longer OS following BrM diagnosis than patients receiving supportive care only (median 6.5 vs 0.8 months, P = .060). The BrM-BTC cohort was enriched for BRAF (30%), PIK3CA (25%), and GNAS (20%) mutations. patients with BrM-BTC with BRAF mutations trended toward longer OS following BrM diagnosis (median 13.1 vs 4.2 months, P = .131). CONCLUSION: This is the largest series of patients with BrM-BTC to date and provides molecular characterization of this rare subgroup of patients with BTC. Patients with BrM-BTC may be more likely to have BRAF mutations. With advances in targeted therapy for patients with BTC with actionable mutations, continued examination of shifting patterns of failure, with emphasis on BrM, is warranted.

Bowel habits, faecal microbiota and faecal bile acid composition of healthy adults consuming fruit pomace fibres: two-arm, randomised, double-blinded, placebo-controlled trials.

Dietary fibre modulates gastrointestinal (GI) health and function, providing laxation, shifting microbiota, and altering bile acid (BA) metabolism. Fruit juice production removes the polyphenol- and fibre-rich pomace fraction. The effects of orange and apple pomaces on GI outcomes were investigated in healthy, free-living adults. Healthy adults were enrolled in two double-blinded, crossover trials, being randomised by baseline bowel movement (BM) frequency. In the first trial, subjects (n 91) received orange juice (OJ, 0 g fibre/d) or OJ + orange pomace (OJ + P, 10 g fibre/d) for 4 weeks, separated by a 3-week washout. Similarly, in the second trial, subjects (n 90) received apple juice (AJ, 0 g fibre/d) or AJ + apple pomace (AJ + P, 10 g fibre/d). Bowel habit diaries, GI tolerance surveys and 3-d diet records were collected throughout. Fresh faecal samples were collected from a participant subset for microbiota and BA analyses in each study. Neither pomace interventions influenced BM frequency. At Week 4, OJ + P tended to increase (P = 0·066) GI symptom occurrence compared with OJ, while AJ + P tended (P = 0·089) to increase flatulence compared with AJ. Faecalibacterium (P = 0·038) and Negativibacillus (P = 0·043) were differentially abundant between pre- and post-interventions in the apple trial but were no longer significant after false discovery rate (FDR) correction. Baseline fibre intake was independently associated with several microbial genera in both trials. Orange or apple pomace supplementation was insufficient to elicit changes in bowel habits, microbiota diversity or BA of free-living adults with healthy baseline BM. Future studies should consider baseline BM frequency and habitual fibre intake.

Use of Telehealth During the COVID-19 Pandemic Among Practicing Maternal-Fetal Medicine Clinicians.

Background: Limited knowledge exists about the drivers of telehealth use among obstetricians during COVID-19 in the United States. We investigated the use of live video visits by Maternal-Fetal Medicine (MFM) clinicians, the factors associated with use and interest in future use. Methods: We drew survey data from 373 clinicians on two outcomes: (1) use of any (vs. no) live video visits during COVID-19 and (2) among users, the extent of live video use. Bivariate and multivariate logistic regressions quantified the association between predisposing (demographic and practice setting characteristics) and enabling factors (prepandemic telehealth use, structural and perceived patient barriers) and each outcome. Results: During the pandemic, 88% reported any use, a jump from 29% prepandemic utilization. Users (vs. nonusers) were younger (p = 0.02); tended to provide comprehensive prenatal care (p = 0.01) and/or inpatient care (p = 0.02), practice in university settings (p = 0.01), engage in various telehealth modalities prepandemic (p ≤ 0.01), and to perceive challenges with technical (p < 0.01), reimbursement (p = 0.05), and patient barriers to internet or data plan access (p ≤ 0.001). After adjusting for covariates, only prepandemic communication through patient portal (adjusted odds ratio [aOR] = 3.85; 95% confidence interval [CI] = 1.33-11.12), perceived patient access barriers (aOR = 5.27; 95% CI = 1.95-14.23), and practice in multiple versus university settings (aOR = 0.18; 95% CI = 0.06-0.56) remained significantly associated with use. Approximately 44% were high users. Prepandemic ultrasound use (aOR = 1.92; 95% CI = 1.17-3.16), perceived patient access barriers (aOR = 1.85; 95% CI = 1.12-3.06) and Midwest versus North practice location (aOR = 0.46; 95% CI = 0.21-0.98) predicted high use. Among high users, 99% wanted to continue offering video visits. Conclusions: We found widespread use of live video obstetric care by MFM clinicians and continued interest in use postpandemic.

Genetic and Functional Modifications Associated with Ovarian Cancer Cell Aggregation and Limited Culture Conditions.

The aggregation of cancer cells provides a survival signal for disseminating cancer cells; however, the underlying molecular mechanisms have yet to be elucidated. Using qPCR gene arrays, this study investigated the changes in cancer-specific genes as well as genes regulating mitochondrial quality control, metabolism, and oxidative stress in response to aggregation and hypoxia in our progressive ovarian cancer models representing slow- and fast-developing ovarian cancer. Aggregation increased the expression of anti-apoptotic, stemness, epithelial-mesenchymal transition (EMT), angiogenic, mitophagic, and reactive oxygen species (ROS) scavenging genes and functions, and decreased proliferation, apoptosis, metabolism, and mitochondrial content genes and functions. The incorporation of stromal vascular cells (SVF) from obese mice into the spheroids increased DNA repair and telomere regulatory genes that may represent a link between obesity and ovarian cancer risk. While glucose had no effect, glutamine was essential for aggregation and supported proliferation of the spheroid. In contrast, low glucose and hypoxic culture conditions delayed adhesion and outgrowth capacity of the spheroids independent of their phenotype, decreased mitochondrial mass and polarity, and induced a shift of mitochondrial dynamics towards mitophagy. However, these conditions did not reduce the appearance of polarized mitochondria at adhesion sites, suggesting that adhesion signals that either reversed mitochondrial fragmentation or induced mitobiogenesis can override the impact of low glucose and oxygen levels. Thus, the plasticity of the spheroids' phenotype supports viability during dissemination, allows for the adaptation to changing conditions such as oxygen and nutrient availability. This may be critical for the development of an aggressive cancer phenotype and, therefore, could represent druggable targets for clinical interventions.

The interaction between the Dbf4 ortholog Chiffon and Gcn5 is conserved in Dipteran insect species.

Chiffon is the sole Drosophila ortholog of Dbf4, the regulatory subunit for the cell-cycle kinase Cdc7 that initiates DNA replication. In Drosophila, the chiffon gene encodes two polypeptides with independent activities. Chiffon-A contains the conserved Dbf4 motifs and interacts with Cdc7 to form the Dbf4-dependent Kinase (DDK) complex, which is essential for a specialized form of DNA replication. In contrast, Chiffon-B binds the histone acetyltransferase Gcn5 to form the Chiffon histone acetyltransferase (CHAT) complex, which is necessary for histone H3 acetylation and viability. Previous studies have shown that the Chiffon-B region is only present within insects. However, it was unclear how widely the interaction between Chiffon-B and Gcn5 was conserved among insect species. To examine this, we performed yeast two-hybrid assays using Chiffon-B and Gcn5 from a variety of insect species and found that Chiffon-B and Gcn5 interact in Diptera species such as Australian sheep blowfly and yellow fever mosquito. Protein domain analysis identified that Chiffon-B has features of acidic transcriptional activators such as Gal4 or VP16. We propose that the CHAT complex plays a critical role in a biological process that is unique to Dipterans and could therefore be a potential target for pest control strategies.

DHA Supplementation Attenuates Inflammation-Associated Gene Expression in the Mammary Gland of Lactating Mothers Who Deliver Preterm.

BACKGROUND: In a randomized trial of DHA supplementation to lactating mothers who delivered preterm, there were significant increases in DHA status in the mother and her infant. OBJECTIVES: Our objective here was to characterize the mammary gland transcriptomes from the above study. We hypothesized that proinflammatory gene expression would be attenuated in the increased DHA group compared with the standard DHA group. METHODS: In the original trial, mothers delivering at <29 wk gestation at the University of Cincinnati Medical Center and intending to express their milk were randomly assigned to supplementation with 200 mg/d DHA (standard group: STD) or 1000 mg/d DHA (experimental group: EXP) within 7 d of delivery. Here, we conducted RNA-seq transcriptome analysis of n = 5 EXP and n = 4 STD extracellular mammary mRNA samples extracted from the fat layer of milk samples obtained 4 wk postenrollment. Transcripts were assessed for differential expression (false discovery rate adjusted P value <0.05) and clustering between EXP compared with STD groups. Ontological analysis of all differentially expressed genes (DEGs) was performed with Toppcluster. RESULTS: There were 409 DEGs. We observed 5 main groups of biological processes that were upregulated, including those associated with improved immune regulation and management of oxidative stress; and 3 main groups of biological processes that were downregulated, including 1 associated with immune dysregulation. For example, we observed upregulation of inflammation-inhibiting genes including NFKB inhibitor alpha (NFKBIA; fold-change (FC), adjusted P value: FC = 1.70, P = 0.007) and interleukin-18 binding protein (IL18BP: FC = 2.2, adjusted P = 0.02); and downregulation of proinflammatory genes including interleukin 7 receptor (IL7R: FC = -1.9, adjusted P = 0.02) and interleukin 1 receptor like 1 (IL1RL1: FC = -13.0, adjusted P = 0.02). CONCLUSIONS: Increased DHA supplementation during lactation can modulate the expression of inflammation-related genes within the mammary gland. This might translate to milk composition with a more optimal inflammasome profile. Future research with a larger clinical trial and greater interrogation of clinical outcomes is warranted.

Ideational factors associated with net care behaviour: a multi-country analysis.

BACKGROUND: Malaria is endemic to sub-Saharan African countries. Mass and routine distribution, promotion, and use of ITNs are critical components of malaria prevention programmes. Correct and consistent use of insecticide-treated mosquito nets (ITN) is an effective strategy for malaria prevention. To extend bed-net lifespan, the World Health Organization recommends folding or tying up ITNs when they are not in use. This study analyses factors associated with net care practices in three African countries. METHODS: Researchers collected household data nationwide in Côte d'Ivoire, from the North and Far North regions of Cameroon, and from Port Loko and Bo districts in Sierra Leone, between 2018 and 2019. The dependent variable was respondents reporting that they fold or tie up their nets. The study adjusted for selected sociodemographic, ideational (psychosocial), and household variables using multilevel models. The analysis was limited to women of reproductive age and their male spouses/partners from households with at least one ITN: 2,940 respondents in Cameroon, 6,105 in Côte d'Ivoire, and 2,730 in Sierra Leone. RESULTS: Among respondents, 50.2% in Cameroon, 52.0% in Côte d'Ivoire and 75.6% in Sierra Leone reported folding or tying up their net when it was not in use. In all three countries, the data showed significant clustering at both household and community levels, indicating the influence of factors operating at these levels on net-care behaviour. The odds of reporting the behaviour varied significantly by geographic unit in each country. Consistent use of nets was strongly correlated with net-care behaviour. Furthermore, five ideational variables were positively associated with the outcome behaviour in all three countries: positive attitude towards net care, perceived susceptibility for malaria, response-efficacy of ITNs, perceived self-efficacy for net use, and the perception that net use was a community norm. Additional significant ideational variables included positive attitudes towards net use (Cameroon and Côte d'Ivoire), perceived severity of malaria (Côte d'Ivoire), and interpersonal communication about malaria (Côte d'Ivoire). CONCLUSIONS: The study identified ideational variables associated with recommended net-care practice. Programme efforts designed to promote net-care practices and extend average lifespan of ITNs may be more effective if they emphasize positive attitudes towards net care, perceived susceptibility of malaria infection, response-efficacy of ITNs, perceived self-efficacy for net use, and promote net-care behaviour as a positive community norm.

Ideational factors associated with consistent use of insecticide-treated nets: a multi-country, multilevel analysis.

BACKGROUND: Malaria remains a major cause of morbidity and mortality in sub-Saharan Africa. Using insecticide-treated nets (ITNs) every night, year-round is critical to maximize protection against malaria. This study describes sociodemographic, psychosocial, and household factors associated with consistent ITN use in Cameroon, Côte d'Ivoire and Sierra Leone. METHODS: Cross-sectional household surveys employed similar sampling procedures, data collection tools, and methods in three countries. The survey sample was nationally representative in Côte d'Ivoire, representative of the North and Far North regions in Cameroon, and representative of Bo and Port Loko districts in Sierra Leone. Analysis used multilevel logistic regression and sociodemographic, ideational, and household independent variables among households with at least one ITN to identify correlates of consistent ITN use, defined as sleeping under an ITN every night the preceding week. FINDINGS: Consistent ITN use in Côte d'Ivoire was 65.4%, 72.6% in Cameroon, and 77.1% in Sierra Leone. While several sociodemographic and ideational variables were correlated with consistent ITN use, these varied across countries. Multilevel logistic regression results showed perceived self-efficacy to use ITNs and positive attitudes towards ITN use were variables associated with consistent use in all three countries. The perception of ITN use as a community norm was positively linked with consistent use in Cameroon and Côte d'Ivoire but was not significant in Sierra Leone. Perceived vulnerability to malaria was positively linked with consistent use in Cameroon and Sierra Leone but negatively correlated with the outcome in Côte d'Ivoire. Household net sufficiency was strongly and positively associated with consistent use in all three countries. Finally, the findings revealed strong clustering at the household and enumeration area (EA) levels, suggesting similarities in net use among respondents of the same EA and in the same household. CONCLUSIONS: There are similarities and differences in the variables associated with consistent ITN use across the three countries and several ideational variables are significant. The findings suggest that a social and behaviour change strategy based on the ideation model is relevant for increasing consistent ITN use and can inform specific strategies for each context. Finally, ensuring household net sufficiency is essential.

Evaluating capacity strengthening for social and behavior change communication: a systematic review.

International social and behavior change communication (SBCC) programs often include capacity strengthening (CS). Quality evaluations of CS can help justify investing in these activities and guide the design of future CS activities. To inform and improve future CS efforts, a comprehensive examination of ways in which activities aimed at strengthening capacity for improved SBCC are assessed is needed. Unfortunately, systematic literature reviews about the assessment of CS activities in SBCC programs are rare. This systematic review helped fill this gap and explored ways in which CS interventions for improved SBCC in low- and middle-income countries (LMICs) evaluated their success. A search of electronic research databases yielded a total of 1033 potentially eligible publications. Reviewers identified 19 eligible publications that assessed the effects of activities for improved SBCC capacity. Reviewers identified seven findings, including the fact that evaluating CS for improved SBCC is rare, with only three publications having focused exclusively on evaluating SBCC capacity. This current review also identified several shortcomings around the quality of writing as well as sufficient detail to support certain claims and conclusions, especially around issues of sustainability. Until quality evaluations of CS activities are better documented, future CS activities for SBCC will find it difficult to identify effective CS approaches and demonstrate their contribution to improved SBCC in LMICs. The review discusses several implications and offers practical recommendations regarding ways to improve the evaluation of CS activities in SBCC.

Minimal-contact physical interventions for pregnant women with musculoskeletal disorders: a systematic review of randomised and non-randomised clinical trials.

This review summarised minimal-contact physical interventions and their effects on pain, disability and quality of life in pregnant women with musculoskeletal disorders. Twelve bibliographic databases were systematically searched until December 31 2020. PEDro Scale was used for quality assessments. Narrative synthesis of 10 eligible studies was conducted. Education and multimodal home exercises plus handbooks/multimodal individual/group exercises; and self-management programmes improved pain intensity, sick leave and disability in pregnant women with lumbopelvic pain. Individual home-based progressive muscle relaxation exercises; unsupervised water exercises plus information using handbooks/videos/music; group multimodal exercises plus home exercises and information/education; and partner massage plus information using booklets/photographs reduced pain intensity in pregnant women with low back pain. Non-rigid/customised lumbopelvic belts plus information reduced pain intensity more significantly than rigid belts or stabilisation exercises plus information among pregnant women with pelvic girdle pain. Minimal contact interventions are effective and may be utilised during infectious disease pandemics.

Biology, pathotype, and virulence of Globodera rostochiensis populations from Kenya.

The potato cyst nematodes (PCN), Globodera rostochiensis (Woll.) and G. pallida (Stone), are important pests of potato globally. Due to their extensive damage potential and the challenge of managing them, these nematodes are under strict regulations in many countries; however, despite these regulations, PCN continue to spread into new areas and countries. In Kenya, G. rostochiensis was first reported in 2015 and G. pallida was reported three years later, both in Nyandarua County. Research was conducted to characterize the biology, pathotype, and virulence of G. rostochiensis populations from Kenya in glasshouse and laboratory studies. The development of G. rostochiensis was assessed in roots of susceptible potato 'Désirée' and resistant 'Laura' carrying the H1 resistance gene. The 'HAR1' population from Kenya and 'Ecosse' from Germany were not able to produce females in the roots of the resistant potato 'Laura'. The rate of root penetration by G. rostochiensis juveniles did not differ (p > 0.05) between populations and cultivars. However, in the resistant cultivar, juveniles developed into males only. A total of 736 cumulative degree-days at 6°C base temperature (DD6) were required by 'HAR1' to complete the life cycle on 'Désirée', whereas 'Ecosse' completed the life cycle within 645 DD6. The Kenyan populations lacked obligatory diapause and high numbers of juveniles hatched immediately after maturity. Consequently, the Kenyan populations had the potential to complete up to three reproduction cycles in less than a year. On selected potato cultivars, the populations from Kenya failed to reproduce on 10 out of 13 commercial cultivars tested. The 10 cultivars carried the H1 resistance gene, which suggests that the G. rostochiensis populations tested belong to the Ro1/4 pathotype group. The virulence of the G. rostochiensis populations from Kenya did not differ from that of the standard reference population 'Ecosse' and therefore can be effectively managed with the commercially available potato cultivars carrying the H1 resistance gene.

Bioenergetics underlying single-cell migration on aligned nanofiber scaffolds.

Cell migration is centrally involved in a myriad of physiological processes, including morphogenesis, wound healing, tissue repair, and metastatic growth. The bioenergetics that underlie migratory behavior are not fully understood, in part because of variations in cell culture media and utilization of experimental cell culture systems that do not model physiological connective extracellular fibrous networks. In this study, we evaluated the bioenergetics of C2C12 myoblast migration and force production on fibronectin-coated nanofiber scaffolds of controlled diameter and alignment, fabricated using a nonelectrospinning spinneret-based tunable engineered parameters (STEP) platform. The contribution of various metabolic pathways to cellular migration was determined using inhibitors of cellular respiration, ATP synthesis, glycolysis, or glucose uptake. Despite immediate effects on oxygen consumption, mitochondrial inhibition only modestly reduced cell migration velocity, whereas inhibitors of glycolysis and cellular glucose uptake led to striking decreases in migration. The migratory metabolic sensitivity was modifiable based on the substrates present in cell culture media. Cells cultured in galactose (instead of glucose) showed substantial migratory sensitivity to mitochondrial inhibition. We used nanonet force microscopy to determine the bioenergetic factors responsible for single-cell force production and observed that neither mitochondrial nor glycolytic inhibition altered single-cell force production. These data suggest that myoblast migration is heavily reliant on glycolysis in cells grown in conventional media. These studies have wide-ranging implications for the causes, consequences, and putative therapeutic treatments aimed at cellular migration.