Proximity Mapping of Desmosomes Reveals a Striking Shift in Their Molecular Neighborhood Associated With Maturation.

Desmosomes are multiprotein adhesion complexes that link intermediate filaments to the plasma membrane, ensuring the mechanical integrity of cells across tissues, but how they participate in the wider signaling network to exert their full function is unclear. To investigate this, we carried out protein proximity mapping using biotinylation (BioID). The combined interactomes of the essential desmosomal proteins desmocollin 2a, plakoglobin, and plakophilin 2a (Pkp2a) in Madin-Darby canine kidney epithelial cells were mapped and their differences and commonalities characterized as desmosome matured from Ca2+ dependence to the mature, Ca2+-independent, hyper-adhesive state, which predominates in tissues. Results suggest that individual desmosomal proteins have distinct roles in connecting to cellular signaling pathways and that these roles alter substantially when cells change their adhesion state. The data provide further support for a dualistic concept of desmosomes in which the properties of Pkp2a differ from those of the other, more stable proteins. This body of data provides an invaluable resource for the analysis of desmosome function.

Bovine Herpes Virus Type 1 (BoHV-1) seroprevalence, risk factor and Bovine Viral Diarrhoea (BVD) co-infection analysis from Ireland.

Surveillance of endemic pathogens is essential for disease control, providing an evidence base for policy and advice. Bovine Herpes Virus Type 1 (BoHV-1), the causative agent of Infectious Bovine Rhinotracheitis (IBR), has been found to have high seroprevalence within the Irish cattle population. The aim of the present study was to establish seroprevalence levels for culled cattle in Ireland aged < 30 months and to establish whether BVD exposure and other factors was associated with BoHV-1 exposure. We employed random effects logit models coupled with repeated bootstrap sampling to provide robust estimates. The final dataset contained results for 5273 animals tested over two study years, 2018 and 2020. The animal-level seroprevalence of BoHV-1 was 21.43% (1130/5273; 95%CI: 20.32-22.53%). Univariable analysis suggested that BoHV-1 seropositivity risk was associated with BVDV serodiagnosis status, age, sex, year sampled, herd type, herd-size, and metrics of movement into the herd. Final random-effects multivariable models suggested increased risk associated with increasing herd size of the last herd, movements made by animals during the previous year, and the year the animal was sampled. Despite BVDV status and sex being retained in the final model, repeated bootstrap sampling of the regression model to estimate biased-corrected 95%CI suggested that these associations were not robust. The overall apparent prevalence of BoHV-1 exposure for culled cattle in Ireland declined in 2020 relative to 2018 (from 23.32 to 17.61%). Herd-size and the movement of animals were found to be important factors associated with animal-level risk, but there was less statistical support for sex-based or BVDV status associations.

Pachyonychia Congenita: A Research Agenda Leading to New Therapeutic Approaches.

Pachyonychia congenita (PC) is a dominantly inherited genetic disorder of cornification. PC stands out among other genodermatoses because despite its rarity, it has been the focus of a very large number of pioneering translational research efforts over the past 2 decades, mostly driven by a patient support organization, the Pachyonychia Congenita Project. These efforts have laid the ground for innovative strategies that may broadly impact approaches to the management of other inherited cutaneous and noncutaneous diseases. This article outlines current avenues of research in PC, expected outcomes, and potential hurdles.

Paeoniflorin shows chondroprotective effects under IL-1ß stress by regulating circ-PREX1/miR-140-3p/WNT5B axis.

BACKGROUND: Osteoarthritis (OA) is a chronic and degenerative bone and joint disease, and paeoniflorin shows anti-arthritis role in OA. This study planned to investigate the mechanism related to chondroprotective role of paeoniflorin in OA. METHODS: Real-time quantitative PCR and western blotting were performed to measure expression levels of circ-PREX1, microRNA (miR)-140-3p, Wingless-type MMTV integration site family, member 5B (WNT5B), B cell lymphoma (Bcl)-2, and Bcl-2 Associated X Protein (Bax). MTT assay, EdU assay, flow cytometry and enzyme-linked immunosorbent assay evaluated cell viability, proliferation, apoptosis and inflammatory response, respectively. Dual-luciferase reporter assay and RNA immunoprecipitation assay identified the relationship among circ-PREX1, miR-140-3p, and WNT5B. RESULTS: IL-1ß highly induced apoptosis rate, Bax expression and TNF-α product, accompanied with decreased cell viability, cell proliferation and IL-10 secretion, whereas these effects were partially reversed after paeoniflorin pretreatment. Expression of circ-PREX1 was upregulated and miR-140-3p was downregulated in cartilage tissues of patients with knee OA (KOA) and IL-1ß-induced human chondrocytes (C28/I2). Circ-PREX1 overexpression and miR-140-3p silencing attenuated the suppressive effect of paeoniflorin in IL-1ß-induced C28/I2 cells. Furthermore, miR-140-3p was negatively regulated by circ-PREX1. WNT5B was a downstream target of miR-140-3p and could be modulated by the circ-PREX1/miR-140-3p pathway in IL-1ß-induced C28/I2 cells. CONCLUSION: Paeoniflorin might protect human chondrocytes from IL-1ß-induced inflammatory injury via circ-PREX1-miR-140-3p-WNT5B pathway, suggesting a potential preventative agent and a novel target for the treatment of KOA.

Global expression analysis of endometrial cancer cells in response to progesterone identifies new therapeutic targets.

Progesterone prevents development of endometrial cancers through its receptor (PR) although the molecular mechanisms have yet to be fully characterized. In this study, we performed a global analysis of gene regulation by progesterone using human endometrial cancer cells that expressed PR endogenously or exogenously. We found progesterone strongly inhibits multiple components of the platelet derived growth factor receptor (PDGFR), Janus kinase (JAK), signal transducer and activator of transcription (STAT) pathway through PR. The PDGFR/JAK/STAT pathway signals to control numerous downstream targets including AP-1 transcription factors Fos and Jun. Treatment with inhibitors of the PDGFR/JAK/STAT pathway significantly blocked proliferation in multiple novel patient-derived organoid models of endometrial cancer, and activation of this pathway was found to be a poor prognostic signal for the survival of patients with endometrial cancer from The Cancer Genome Atlas. Our study identifies this pathway as central to the growth-limiting effects of progesterone in endometrial cancer and suggests that inhibitors of PDGFR/JAK/STAT should be considered for future therapeutic interventions.

Somatic mutations in facial skin from countries of contrasting skin cancer risk.

The incidence of keratinocyte cancer (basal cell and squamous cell carcinomas of the skin) is 17-fold lower in Singapore than the UK1-3, despite Singapore receiving 2-3 times more ultraviolet (UV) radiation4,5. Aging skin contains somatic mutant clones from which such cancers develop6,7. We hypothesized that differences in keratinocyte cancer incidence may be reflected in the normal skin mutational landscape. Here we show that, compared to Singapore, aging facial skin from populations in the UK has a fourfold greater mutational burden, a predominant UV mutational signature, increased copy number aberrations and increased mutant TP53 selection. These features are shared by keratinocyte cancers from high-incidence and low-incidence populations8-13. In Singaporean skin, most mutations result from cell-intrinsic processes; mutant NOTCH1 and NOTCH2 are more strongly selected than in the UK. Aging skin in a high-incidence country has multiple features convergent with cancer that are not found in a low-risk country. These differences may reflect germline variation in UV-protective genes.

Using the theoretical domains framework to determine the barriers and facilitators to medication adherence in Parkinson's disease.

Background: Patient medication adherence in Parkinson's Disease (PD) is often suboptimal. This may lead to poor symptom management, greater disease burden, decreased quality of life and increased healthcare costs. Use of psychological theory such as the Theoretical Domains Framework (TDF) has effectively captured barriers and facilitators to medication adherence in other long-term conditions. Applying this framework to medication adherence in PD could provide a better understanding of the challenges to inform the development of effective interventions. Objectives: The aim of the study was to apply the TDF to determine the barriers and facilitators to medication adherence in people with PD. Methodology: This qualitative study employed online interviews to explore medication adherence in a small group of people with PD recruited via Parkinson's UK and social media. A semi-structured interview schedule was designed informed by the 14 TDF domains. All interviews were audio-recorded, transcribed verbatim and mapped to the TDF using Framework Analysis. Results: Twelve participants diagnosed with PD were interviewed, 11 of whom were currently taking prescribed medication plus another self-medicating with Vitamin B1. All TDF domains were evident in the data. Predominant facilitators were Domains 1 - Knowledge, 6 - Social Influence, and 12 - Beliefs about Consequences and barriers were 7 - Reinforcement, 10 - Memory, Attention and Decision Processes, and 11 - Environmental Context and Resources. Other themes were not related to medication adherence. Conclusion: In this small group, all data relating to the barriers and facilitators for medication adherence in PD were successfully mapped onto the TDF. This indicates the utility of the framework for determining and structuring the factors to consider when providing medication support for this patient population in an accessible and coherent way. Further quantitative studies are needed to determine the extent to which these factors can be generalised to other PD patients.

Parenting in the early years and self-harm in adolescence: The role of control and reward systems in childhood.

BACKGROUND: Research suggests that early parenting may contribute to the development of self-harm but this has not been examined longitudinally. In this study, we explored the relationship between early parenting and self-harm in adolescence and considered whether (1) emotion regulation and (2) decision-making in childhood mediate the relationship between early parenting and self-harm. METHOD: Using longitudinal data from the Millennium Cohort Study (MCS), we tested mediation models exploring the relationship between early parenting and self-harm in adolescence via emotion regulation and decision-making. Parenting was assessed at age 3 with measures of conflict, closeness and discipline. The trajectories of independence & self-regulation and emotional dysregulation were modelled from ages 3 to 7 years through latent growth curve analysis, with individual predicted slope and intercept values used in the mediation models. Decision-making (deliberation time, total time, delay aversion, quality of decision making, risk adjustment, risk-taking) was assessed using the Cambridge Gambling Task (CGT) at age 11. RESULTS: In our sample (n = 11,145), we found no evidence of a direct association between early parenting and self-harm in adolescence. However, there were indirect effects of parenting (conflict and closeness) on self-harm via the slope of emotional dysregulation. Furthermore, delay aversion was positively associated with self-harm in adolescence. LIMITATIONS: It must be acknowledged that we cannot determine causality and that self-report measures of parenting are vulnerable to several biases. CONCLUSION: The findings support early identification and interventions for children exhibiting chronic emotional dysregulation and decision-making characterised by a bias for smaller, immediate over larger, delayed rewards.

Patient preferences for key drivers and facilitators of adoption of mHealth technology to manage depression: A discrete choice experiment.

BACKGROUND: In time, we may be able to detect the early onset of symptoms of depression and even predict relapse using behavioural data gathered through mobile technologies. However, barriers to adoption exist and understanding the importance of these factors to users is vital to ensure maximum adoption. METHOD: In a discrete choice experiment, people with a history of depression (N = 171) were asked to select their preferred technology from a series of vignettes containing four characteristics: privacy, clinical support, established benefit and device accuracy (i.e., ability to detect symptoms), with different levels. Mixed logit models were used to establish what was most likely to affect adoption. Sub-group analyses explored effects of age, gender, education, technology acceptance and familiarity, and nationality. RESULTS: Higher level of privacy, greater clinical support, increased perceived benefit and better device accuracy were important. Accuracy was the most important, with only modest compromises willing to be made to increase other factors such as privacy. Established benefit was the least valued of the attributes with participants happy with technology that had possible but unknown benefits. Preferences were moderated by technology acceptance, age, nationality, and educational background. CONCLUSION: For people with a history of depression, adoption of technology may be driven by the desire for accurate detection of symptoms. However, people with lower technology acceptance and educational attainment, those who were younger, and specific nationalities may be willing to compromise on some accuracy for more privacy and clinical support. These preferences should help shape design of mHealth tools.

Time trade-off study to establish utility decrements in individuals with a spinal cord injury who perform intermittent catheterization.

AIMS: To generate utility decrements for three attributes associated with catheterization for individuals with a spinal cord injury (SCI): the process of catheterization, the physical impact of urinary tract infections (UTIs) and worry associated with hospitalization. MATERIALS AND METHODS: Health state vignettes comprising various levels of the three attributes were developed. Two cohorts of respondents, corresponding to people with SCIs and a sample broadly representative of the UK population, were presented with nine vignettes (three vignettes for the mild, moderate and severe health states in addition to a random set of six vignettes). It was assumed no or a nominal decrement was associated with the mild health state. Utility decrements were derived from analysing the data obtained from the online time trade-off (TTO). A proportion of the SCI cohort (n = 57) also completed the EQ-5D-5L questionnaire. RESULTS: Utility decrements were generated using statistical models for the general population (n = 358), the SCI population (n = 48) and the two populations combined (merged model, n = 406). Results from the two cohorts showed minimal differences. For the merged model, SCI status was not statistically significant. All interaction terms, excluding SCI and the severe level of the physical attribute, were not statistically significant. Compared to the mild level, the greatest utility decrement calculated was the severe level of the emotional (worry) attribute (0.09, p < .001) for the SCI population. A significant decrement of 0.02 (p < .001) was calculated for the moderate level of the emotional attribute for all models. The mean utility score for those with SCI having completed the EQ-5D-5L was 0.371. LIMITATIONS: Modest sample size of respondents from the SCI population (n = 48). CONCLUSIONS: Worry associated with hospitalization had the greatest impact on patients' health-related quality of life (HRQoL). The catheterization process, such as the lubrication and repositioning of the catheter, also impacted on patients' HRQoL.

Rapid, sensitive detection of PFOA with smartphone-based flow rate analysis utilizing competitive molecular interactions during capillary action.

Perfluorinated-alkyl substances (PFAS) pose an unmet threat to the public because they are not strictly monitored and regulated. Perfluorinated-carbon alkyl chains (PFOA), a type of PFAS, at 70 fg/µL is the current health and safety recommendation. Current testing methods for PFOA and PFAS chemicals include HPLC-MS/MS and molecularly imprinted polymers, which are expensive, time-consuming, and require training. In this work, PFOA and PFOS detection was performed on a paper microfluidic chip using competitive interactions between PFOA/PFOS, cellulose fibers, and various reagents (L-lysine, casein, and albumin). Such interactions altered the surface tension at the wetting front and, subsequently, the capillary flow rate. A smartphone captured the videos of this capillary action. The samples flowed through the channel in less than 2 min. Albumin worked the best in detecting PFOA, followed by casein. The detection limit was 10 ag/µL in DI water and 1 fg/µL in effluent (processed) wastewater. Specificity to other non-fluorocarbon surfactants was also tested, using anionic sodium dodecyl sulfate (SDS), non-ionic Tween 20, and cationic cetrimonium bromide (CTAB). A combination of the reagents successfully distinguished PFOA from all three surfactants at 100% accuracy. This low-cost, handheld assay can be an accessible alternative for rapid in situ estimation of PFOA concentration.

Xeno-free self-assembling peptide scaffolds for building 3D organotypic skin cultures.

Organotypic skin cultures represent in vitro models of skin which can be used for disease modeling, tissue engineering, and screening applications. Non-human collagen is currently the gold standard material used for the construction of the supporting matrix, however, its clinical applications are limited due to its xenogeneic origin. We have developed a novel peptide hydrogel-based skin construct that shows a pluristratified epidermis, basement membrane, and dermal compartment after 3 weeks of in vitro culture. Peptide-based constructs were compared to collagen-based constructs and stratification marker expression was histologically higher in peptide constructs than in collagen constructs. Transepithelial electrical resistance also showed mature barrier function in peptide constructs. This study presents a novel application of the self-assembling peptide hydrogel in a defined xeno-free in vitro system.

Investigating a psychological model of mental conditions and coping during the COVID-19 pandemic driven by participatory methods.

BACKGROUND: There is evidence of increased mental health problems during the early stages of the COVID-19 pandemic. We aimed to identify the factors that put certain groups of people at greater risk of mental health problems. METHODS: We took a participatory approach, involving people with lived experience of mental health problems and/or carers, to generate a set of risk factors and potential moderators of the effects of COVID on mental health. An online cross-sectional survey was completed by 1464 United Kingdom residents between 24th April and 27th June 2020. The survey had questions on whether respondents were existing mental health service users and or carers, level of depression (PHQ9) and anxiety (GAD7), demographics, threat and coping appraisals, perceived resilience (BRS), and specific coping behaviours (validated as part of this study). The relationship between responses and coping strategies was measured using tetrachoric correlations. Structural equation modelling was used to test the model. RESULTS: A model significantly fit our data (rel χ2 = 2.05, RMSEA = 0.029 95%, CI (0.016, 0.042), CFI = 0.99, TLI = 0.98, SRMR = 0.014). Age and coping appraisal predicted anxiety and depression. Whereas, threat appraisal and ethnicity only predicted anxiety, and resilience only predicted depression. Additionally, specific coping behaviours predicted anxiety and depression, with overlap on distraction. CONCLUSIONS: Some, but not all, risk factors significantly predict anxiety and depression. While there is a relationship between anxiety and depression, different factors may put people at greater risk of one or the other during the pandemic.

Sensitive, smartphone-based SARS-CoV-2 detection from clinical saline gargle samples.

Saliva specimens have drawn interest for diagnosing respiratory viral infections due to their ease of collection and decreased risk to healthcare providers. However, rapid and sensitive immunoassays have not yet been satisfactorily demonstrated for such specimens due to their viscosity and low viral loads. Using paper microfluidic chips and a smartphone-based fluorescence microscope, we developed a highly sensitive, low-cost immunofluorescence particulometric SARS-CoV-2 assay from clinical saline gargle samples. We demonstrated the limit of detection of 10 ag/µL. With easy-to-collect saline gargle samples, our clinical sensitivity, specificity, and accuracy were 100%, 86%, and 93%, respectively, for n = 27 human subjects with n = 13 RT-qPCR positives.

Mogroside V Improves Follicular Development and Ovulation in Young-Adult PCOS Rats Induced by Letrozole and High-Fat Diet Through Promoting Glycolysis.

Polycystic ovary syndrome (PCOS) is a heterogeneous endocrine disorder characterized by hyperandrogenism, ovulatory dysfunction, and polycystic ovaries. In this study, we induced a young-adult PCOS rat model by oral administration of letrozole combined with a high-fat diet and then treated with mogroside V (MV) to evaluate the protective effects of MV on endocrine and follicle development in young-adult PCOS rats. MV (600 mg/kg/day) administration not only significantly reduced the body weight and ovary weight, but also attenuated the disrupted estrous cycle and decreased the level of testosterone. MV restored the follicular development, especially by increasing the number of corpus luteum and the thickness of the granular layer in young-adult POCS rats. Moreover, metabolomics showed that MV markedly increased the levels of D-Glucose 6-phosphate, lactate and GTP, while decreased the level of pyruvate. Bioinformatic analysis revealed that MV recovered multiple metabolism-related processes including gluconeogenesis, glycolysis and glucose metabolic process. Further real-time quantitative PCR analysis showed that MV upregulated the expression of lactate dehydrogenase A (Ldha), hexokinase 2 (Hk2) and pyruvate kinase M2 (Pkm2). Western blotting and immunohistochemistry analysis showed that MV restored the expression of lactate dehydrogenase A (Ldha), hexokinase 2 (Hk2) and pyruvate kinase M2 (Pkm2). Collectively, these findings indicated that MV could effectively improve the ovarian microenvironment by upregulating the expression of LDHA, HK2 and PKM2 in granulosa cells and enhancing lactate and energy production, which may contribute to follicle development and ovulation of young-adult PCOS rats.

Smartphone-based sensitive detection of SARS-CoV-2 from saline gargle samples via flow profile analysis on a paper microfluidic chip.

Respiratory viruses, especially coronaviruses, have resulted in worldwide pandemics in the past couple of decades. Saliva-based paper microfluidic assays represent an opportunity for noninvasive and rapid screening, yet both the sample matrix and test method come with unique challenges. In this work, we demonstrated the rapid and sensitive detection of SARS-CoV-2 from saliva samples, which could be simpler and more comfortable for patients than existing methods. Furthermore, we systematically investigated the components of saliva samples that affected assay performance. Using only a smartphone, an antibody-conjugated particle suspension, and a paper microfluidic chip, we made the assay user-friendly with minimal processing. Unlike the previously established flow rate assays that depended solely on the flow rate or distance, this unique assay analyzes the flow profile to determine infection status. Particle-target immunoagglutination changed the surface tension and subsequently the capillary flow velocity profile. A smartphone camera automatically measured the flow profile using a Python script, which was not affected by ambient light variations. The limit of detection (LOD) was 1 fg/µL SARS-CoV-2 from 1% saliva samples and 10 fg/µL from simulated saline gargle samples (15% saliva and 0.9% saline). This method was highly specific as demonstrated using influenza A/H1N1. The sample-to-answer assay time was <15 min, including <1-min capillary flow time. The overall accuracy was 89% with relatively clean clinical saline gargle samples. Despite some limitations with turbid clinical samples, this method presents a potential solution for rapid mass testing techniques during any infectious disease outbreak as soon as the antibodies become available.

Detrimental Effects of IFN-γ on an Epidermolysis Bullosa Simplex Cell Model and Protection by a Humanized Anti-IFN-γ Monoclonal Antibody.

Epidermolysis bullosa is a group of severe skin blistering disorders, which currently have no cure. The pathology of epidermolysis bullosa is recognized as having an inflammatory component, but the role of inflammation in different epidermolysis bullosa disorders is unclear. Epidermolysis bullosa simplex (EBS) is primarily caused by sequence variants in keratin genes; its most severe form, EBS generalized severe, is characterized by aggregates of keratin proteins, and cell models of EBS generalized severe show constitutively elevated stress. IFN-γ is a major mediator of inflammation, and we show that the addition of IFN-γ alone to disease model keratinocytes promotes keratin aggregation, decreases cell-cell junctions, delays wound closure, and reduces cell proliferation. IFN-γ exposure weakens the intercellular cohesion of monolayers on mechanical stress, with IFN-γ-treated EBS monolayers more fragmented than IFN-γ-treated wild-type monolayers. A humanized monoclonal antibody to IFN-γ neutralized the detrimental effects on keratinocytes, restoring cell proliferation, increasing cell-cell adhesion, accelerating wound closure in the presence of IFN-γ, and reducing IFN-γ-mediated keratin aggregation in EBS cells. These suggest that treatment with IFN-γ blocking antibodies may constitute a promising new therapeutic strategy for patients with EBS and may also have ameliorating effects on other inflammatory skin diseases.

Direct capture and smartphone quantification of airborne SARS-CoV-2 on a paper microfluidic chip.

SARS, a new type of respiratory disease caused by SARS-CoV, was identified in 2003 with significant levels of morbidity and mortality. The recent pandemic of COVID-19, caused by SARS-CoV-2, has generated even greater extents of morbidity and mortality across the entire world. Both SARS-CoV and SARS-CoV-2 spreads through the air in the form of droplets and potentially smaller droplets (aerosols) via exhaling, coughing, and sneezing. Direct detection from such airborne droplets would be ideal for protecting general public from potential exposure before they infect individuals. However, the number of viruses in such droplets and aerosols is too low to be detected directly. A separate air sampler and enough collection time (several hours) are necessary to capture a sufficient number of viruses. In this work, we have demonstrated the direct capture of the airborne droplets on the paper microfluidic chip without the need for any other equipment. 10% human saliva samples were spiked with the known concentration of SARS-CoV-2 and sprayed to generate liquid droplets and aerosols into the air. Antibody-conjugated submicron particle suspension is then added to the paper channel, and a smartphone-based fluorescence microscope isolated and counted the immunoagglutinated particles on the paper chip. The total capture-to-assay time was <30 min, compared to several hours with the other methods. In this manner, SARS-CoV-2 could be detected directly from the air in a handheld and low-cost manner, contributing to slowing the spread of SARS-CoV-2. We can presumably adapt this technology to a wide range of other respiratory viruses.

Biosensor detection of airborne respiratory viruses such as SARS-CoV-2.

Airborne SARS-CoV-2 transmission represents a significant route for possible human infection that is not yet fully understood. Viruses in droplets and aerosols are difficult to detect because they are typically present in low amounts. In addition, the current techniques used, such as RT-PCR and virus culturing, require large amounts of time to get results. Biosensor technology can provide rapid, handheld, and point-of-care systems that can identify virus presence quickly and accurately. This paper reviews the background of airborne virus transmission and the characteristics of SARS-CoV-2, its relative risk for transmission even at distances greater than the currently suggested 6 feet (or 2 m) physical distancing. Publications on biosensor technology that may be applied to the detection of airborne SARS-CoV-2 and other respiratory viruses are also summarized. Based on the current research we believe that there is a pressing need for continued research into handheld and rapid methods for sensitive collection and detection of airborne viruses. We propose a paper-based microfluidic chip and immunofluorescence assay as one method that could be investigated as a low-cost and portable option.