Development of an in vitro platform for the analysis of contractile and calcium dynamics in single human myotubes.

In vitro myotube cultures are widely used as models for studying muscle pathophysiology, but their limited maturation and heterogeneity pose significant challenges for functional analyses. While they remain the gold standard for studying muscle function in vitro, myotube cultures do not fully recapitulate the complexity and native features of muscle fibers, which may compromise their ability to predict in vivo outcomes. To promote maturation and decrease heterogeneity, we have incorporated engineered structures into myotube cultures, based on a PDMS thin layer with micrometer-sized grooves (µGrooves) placed over a glass substrate. Different sizes and shapes of µGrooves were tested for their ability to promote alignment and fusion of myoblasts and enhance their differentiation into myotubes. A 24 hour electrical field stimulation protocol (4 V, 6 ms, 0.1 Hz) was used to further promote myotube maturation, after which several myotube features were assessed, including myotube alignment, width, fusion index, contractile function, and calcium handling. Our results indicate superior calcium and contractile performance in µGrooved myotubes, particularly with the 100 µm-width 700 µm-long geometry (7 : 1). This platform generated homogeneous and isolated myotubes that reproduced native muscle features, such as excitation-contraction coupling and force-frequency responses. Overall, our 2D muscle platform enables robust high-content assays of calcium dynamics and contractile readouts with increased sensitivity and reproducibility compared to traditional myotube cultures, making it particularly suitable for screening therapeutic candidates for different muscle pathologies.

Dupilumab treatment in paediatric atopic dermatitis (2 to 18 years): Spanish multicentre retrospective real-life study.

BACKGROUND: Moderate-to-severe atopic dermatitis (AD) can be difficult to manage in paediatric patients, with few licensed treatments in this age group. Dupilumab is approved for AD in children older than 6 months. OBJECTIVES: To assess the effectiveness and safety of dupilumab in a real-life cohort of paediatric AD patients in Spain. METHODS: A multicentre, retrospective real-life study on the effectiveness and safety of dupilumab in patients aged 2 to 18 years old with moderate-to-severe AD was conducted. Demographic and clinical characteristics were analysed, and effectiveness (EASI, IGA, DLQI, NRS itch), safety, and drug survival measures were assessed. A comparison of our results with other real-world outcomes and with clinical trials was made. RESULTS: Data from 243 patients from 19 centres was collected, with a mean follow-up of 85 weeks. Dupilumab exhibited significant effectiveness, with marked reductions in severity scores from week 4. By week 16, 79.4% of patients reached EASI75 and 40.5% reached EASI90. Mean percentage reduction in EASI was 79.7%. Increasing improvements were observed until week 52, with 85.8% and 49.6% achieving EASI75 and EASI90, respectively. Forty-three patients developed adverse events (AE) (43/243, 17.7%), being the most frequent ocular surface diseases (20/243, 8.2%), injection site reactions (8/243, 3.3%) and facial redness (7/243, 2.9%). Drug survival was high (96.9% and 93.1% after 1 and 2 years of follow-up, respectively), with only 19 (19/243, 7.8%) patients interrupting treatment: 7 (7/243, 2.9%) due to AE, 2 (2/243, 0.82%) due to secondary failure, 5 (5/243, 2.1%) were lost to follow-up and 5 (5/243, 2.1%) entered remission and stopped treatment. CONCLUSION: Real-life use of dupilumab in paediatric AD showcased sustained effectiveness, high drug survival, and acceptable safety profiles. Longer-term studies are crucial for AE surveillance and how to manage disease remission.

ECAD study: Evaluating agreement degree among paediatricians in hip dysplasia assessment by the Graf ultrasound method.

Developmental dysplasia of the hip (DDH) is the most common musculoskeletal anomaly of childhood. This study analyses paediatrician's concordance performing hip ultrasound according to Graf's method. An analytical cross-sectional study is designed. Patients' hips are evaluated by Graf's ultrasound method at 4-6 and 12-16 weeks of age. Demographic characteristics, pregnancy history, factors related to DDH, hip type, and α and ß angles are collected. To assess the agreement degree of hip diagnosis and hip types Kappa index (I. kappa) is used. Intraclass correlation coefficient (ICC) evaluates the concordance of α and ß angles. A p value < 0.05 is considered statistically significant. All results are represented with their 95% confidence interval (95% CI). Four hundred sixty-seven neonates are recruited and meet the inclusion criteria. A total of 3182 images were analysed. Agreement between paediatricians according to hip diagnosis and different types ranges from moderate to almost perfect I. kappa 0.6-1.0 (95% CI 0.5-0.7; 95% CI 1.0-1.0). α angle ICC for paediatricians is between 0.75 and 0.88 (95% CI 0.61-0.86; 95% CI 0.80-0.92). ß angle value agreement degree among paediatricians at both visits is moderate. The Graf method is the most popular ultrasound technique for hip assessment. In our setting, paediatricians carry out children follow-up; therefore, they should perform universal screening. In this study agreement degree between paediatricians varies between substantial and almost perfect. Conclusion: The Graf ultrasound procedure performed by paediatricians is a reliable test and can be used for DDH screening. What is Known • Developmental dysplasia of the hip (DDH) is the most common congenital musculoskeletal anomaly of childhood. Early diagnosis and treatment improve DDH prognosis. Lack of detection can cause complications such as lameness, early osteoarthritis and need for hip replacement at an early age. What is New • Today, the best screening method is still subject of debate. But it seems that with selective screening many pathological hips go unnoticed. Universal screening implementation may be a challenge in some countries. But the question is if this could be carried out by paediatricians as part of healthy child follow-up.

Schistosomiasis in migrant children and adolescents in a paediatric tropical referral unit in Spain: diagnosis and long-term management challenges.

Globalisation and population movement have led to an increasing number of migrant children residing in areas non-endemic for schistosomiasis. However, diagnosing and managing schistosomiasis in children remain controversial. This study aims to investigate the prevalence of schistosomiasis in migrant children and to describe the diagnostic approach and management strategies, including long-term follow-up, to explore the potential role of serological tests in evaluating treatment response. We conducted a retrospective descriptive study spanning from January 2014-July 2021 at a referral unit for Paediatric Tropical Diseases in Madrid (Spain). The study included patients under 18 years diagnosed with schistosomiasis. Of 679 children screened for schistosomiasis, 73 (10.8%) tested positive. The median age was 16.3 years [IQR 9-17.6], 74% male. The majority originated from Sub-Saharan Africa (47%) and Asia (47%). Only 40% presented with symptoms, with gastrointestinal (18%) and cutaneous (17%) manifestations being the most common. Eosinophilia was observed in 43% (median [IQR]: 1103/mm3 [671-1536]), and ova were visualised in the urine of 2/50 (4.0%). Praziquantel treatment was administered to 92%, and 5 patients required retreatment. Follow-up data were available for 58 (80%) over a median period of 9 months [IQR 6-19.8], revealing a progressive decline in eosinophil count, IgE titres, and ELISA optical density.    Conclusion: In this series, the prevalence of schistosomiasis among migrant children was significant (10%), highlighting the importance of including serological tests in migrant health screening. The disease is largely asymptomatic, eosinophilia is often absent, and visualisation of ova in urine is exceedingly rare. Eosinophil count, IgE titres, and ELISA optical density could prove valuable as an initial approach for monitoring inflammation during follow-up assessments. What is Known: • The burden of disease related to schistosomiasis is significant, particulary in children, and it is advisable to screen this vulnerable population. What is New: • Eosinophilia may not be present in parasitic infections, so serological tests are crucial for screening migrant children. • Serological monitoring facilitates long-term management of migrant children with schistosomiasis.

Self-regulation training generalizability using the regulation of craving task. An fMRI study.

Individual differences in reward salience may relate to the difficulty in regulating the effects of multiple substances (e.g., nicotine and food). Increased brain activation in reward and self-regulation (SR) regions has been evidenced while adults view appetitive cues (e.g., food pictures) to test substance use disorder treatment response. Enhancing SR with behavioral interventions may increase brain activation in SR regions and reduce responses in reward regions. Our primary analysis demonstrated increased brain activation in SR regions to smoking cues among individuals who practiced SR by delaying their first cigarette of the day for 2 weeks. However, little is known about the generalizability of SR between appetitive cues. This secondary analysis explored the influence of adherence to a SR behavioral intervention by examining the impact of practicing smoking SR on brain activation to food cues among adults who smoke. Participants (N = 65) were randomly assigned to practice SR by delaying their first daily cigarette or smoking as usual for 2-weeks. Functional magnetic resonance imaging data were collected while people were told to think of "negative" or "positive" associations with the cue. The results indicated that practicing smoking SR was linked with increased activation in the dorsolateral prefrontal cortex (dlPFC) when viewing food cues. There was no correlation between delaying smoking adherence and brain activation in the dlPFC. Exploratory analyses suggested higher dlPFC activation when people thought about "positive" associations with the food cues instead of "negative" ones. We concluded that practicing smoking SR is related to increased brain activation to food cues, suggesting potential generalizability of SR practice from smoking cues to food cues.

Systemic inflammation following traumatic injury and its impact on neuroinflammatory gene expression in the rodent brain.

BACKGROUND: Trauma can result in systemic inflammation that leads to organ dysfunction, but the impact on the brain, particularly following extracranial insults, has been largely overlooked. METHODS: Building upon our prior findings, we aimed to understand the impact of systemic inflammation on neuroinflammatory gene transcripts in eight brain regions in rats exposed to (1) blast overpressure exposure [BOP], (2) cutaneous thermal injury [BU], (3) complex extremity injury, 3 hours (h) of tourniquet-induced ischemia, and hind limb amputation [CEI+tI+HLA], (4) BOP+BU or (5) BOP+CEI and delayed HLA [BOP+CEI+dHLA] at 6, 24, and 168 h post-injury (hpi). RESULTS: Globally, the number and magnitude of differentially expressed genes (DEGs) correlated with injury severity, systemic inflammation markers, and end-organ damage, driven by several chemokines/cytokines (Csf3, Cxcr2, Il16, and Tgfb2), neurosteroids/prostaglandins (Cyp19a1, Ptger2, and Ptger3), and markers of neurodegeneration (Gfap, Grin2b, and Homer1). Regional neuroinflammatory activity was least impacted following BOP. Non-blast trauma (in the BU and CEI+tI+HLA groups) contributed to an earlier, robust and diverse neuroinflammatory response across brain regions (up to 2-50-fold greater than that in the BOP group), while combined trauma (in the BOP+CEI+dHLA group) significantly advanced neuroinflammation in all regions except for the cerebellum. In contrast, BOP+BU resulted in differential activity of several critical neuroinflammatory-neurodegenerative markers compared to BU. t-SNE plots of DEGs demonstrated that the onset, extent, and duration of the inflammatory response are brain region dependent. Regardless of injury type, the thalamus and hypothalamus, which are critical for maintaining homeostasis, had the most DEGs. Our results indicate that neuroinflammation in all groups progressively increased or remained at peak levels over the study duration, while markers of end-organ dysfunction decreased or otherwise resolved. CONCLUSIONS: Collectively, these findings emphasize the brain's sensitivity to mediators of systemic inflammation and provide an example of immune-brain crosstalk. Follow-on molecular and behavioral investigations are warranted to understand the short- to long-term pathophysiological consequences on the brain, particularly the mechanism of blood-brain barrier breakdown, immune cell penetration-activation, and microglial activation.

Exposure to bisphenol A in European women from 2007 to 2014 using human biomonitoring data - The European Joint Programme HBM4EU.

BACKGROUND: Bisphenol A (BPA; or 4,4'-isopropylidenediphenol) is an endocrine disrupting chemical. It was widely used in a variety of plastic-based manufactured products for several years. The European Food Safety Authority (EFSA) recently reduced the Tolerable Daily Intake (TDI) for BPA by 20,000 times due to concerns about immune-toxicity. OBJECTIVE: We used human biomonitoring (HBM) data to investigate the general level of BPA exposure from 2007 to 2014 of European women aged 18-73 years (n = 4,226) and its determinants. METHODS: Fifteen studies from 12 countries (Austria, Belgium, Denmark, France, Germany, Greece, Israel, Luxembourg, Slovenia, Spain, Sweden, and the United Kingdom) were included in the BPA Study protocol developed within the European Joint Programme HBM4EU. Seventy variables related to the BPA exposure were collected through a rigorous post-harmonization process. Linear mixed regression models were used to investigate the determinants of total urine BPA in the combined population. RESULTS: Total BPA was quantified in 85-100 % of women in 14 out of 15 contributing studies. Only the Austrian PBAT study (Western Europe), which had a limit of quantification 2.5 to 25-fold higher than the other studies (LOQ=2.5 µg/L), found total BPA in less than 5 % of the urine samples analyzed. The geometric mean (GM) of total urine BPA ranged from 0.77 to 2.47 µg/L among the contributing studies. The lowest GM of total BPA was observed in France (Western Europe) from the ELFE subset (GM=0.77 µg/L (0.98 µg/g creatinine), n = 1741), and the highest levels were found in Belgium (Western Europe) and Greece (Southern Europe), from DEMOCOPHES (GM=2.47 µg/L (2.26 µg/g creatinine), n = 129) and HELIX-RHEA (GM=2.47 µg/L (2.44 µg/g creatinine), n = 194) subsets, respectively. One hundred percent of women in 14 out of 15 data collections in this study exceeded the health-based human biomonitoring guidance value for the general population (HBM-GVGenPop) of 0.0115 µg total BPA/L urine derived from the updated EFSA's BPA TDI. Variables related to the measurement of total urine BPA and those related to the main socio-demographic characteristics (age, height, weight, education, smoking status) were collected in almost all studies, while several variables related to BPA exposure factors were not gathered in most of the original studies (consumption of beverages contained in plastic bottles, consumption of canned food or beverages, consumption of food in contact with plastic packaging, use of plastic film or plastic containers for food, having a plastic floor covering in the house, use of thermal paper…). No clear determinants of total urine BPA concentrations among European women were found. A broader range of data planned for collection in the original questionnaires of the contributing studies would have resulted in a more thorough investigation of the determinants of BPA exposure in European women. CONCLUSION: This study highlights the urgent need for action to further reduce exposure to BPA to protect the population, as is already the case in the European Union. The study also underscores the importance of pre-harmonizing HBM design and data for producing comparable data and interpretable results at a European-wide level, and to increase HBM uptake by regulatory agencies.

Pregnancy as a window of opportunity for dementia prevention: a narrative review.

Dementia is a debilitating condition with a disproportionate impact on women. While sex differences in longevity contribute to the disparity, the role of the female sex as a biological variable in disease progression is not yet fully elucidated. Metabolic dysfunctions are drivers of dementia etiology, and cardiometabolic diseases are among the most influential modifiable risk factors. Pregnancy is a time of enhanced vulnerability for metabolic disorders. Many dementia risk factors, such as hypertension or blood glucose dysregulation, often emerge for the first time in pregnancy. While such cardiometabolic complications in pregnancy pose a risk to the health trajectory of a woman, increasing her odds of developing type 2 diabetes or chronic hypertension, it is not fully understood how this relates to her risk for dementia. Furthermore, structural and functional changes in the maternal brain have been reported during pregnancy suggesting it is a time of neuroplasticity for the mother. Therefore, pregnancy may be a window of opportunity to optimize metabolic health and support the maternal brain. Healthy dietary patterns are known to reduce the risk of cardiometabolic diseases and have been linked to dementia prevention, yet interventions targeting cognitive function in late life have largely been unsuccessful. Earlier interventions are needed to address the underlying metabolic dysfunctions and potentially reduce the risk of dementia, and pregnancy offers an ideal opportunity to intervene. This review discusses current evidence regarding maternal brain health and the potential window of opportunity in pregnancy to use diet to address neurological health disparities for women.

PIM1 is a potential therapeutic target for the leukemogenic effects mediated by JAK/STAT pathway mutations in T-ALL/LBL.

Precursor T-cell neoplasms (T-ALL/LBL) are aggressive hematological malignancies that arise from the malignant transformation of immature thymocytes. Despite the JAK/STAT pathway is recurrently altered in these neoplasms, there are not pharmacological inhibitors officially approved for the treatment of T-ALL/LBL patients that present oncogenic JAK/STAT pathway mutations. In the effort to identify potential therapeutic targets for those patients, we followed an alternative approach and focused on their transcriptional profile. We combined the analysis of molecular data from T-ALL/LBL patients with the generation of hematopoietic cellular models to reveal that JAK/STAT pathway mutations are associated with an aberrant transcriptional profile. Specifically, we demonstrate that JAK/STAT pathway mutations induce the overexpression of the PIM1 gene. Moreover, we show that the pan-PIM inhibitor, PIM447, significantly reduces the leukemogenesis, as well as the aberrant activation of c-MYC and mTOR pathways in cells expressing different JAK/STAT pathway mutations, becoming a potential therapeutic opportunity for a relevant subset of T-ALL/LBL patients.

Histological deep margins in cutaneous squamous cell carcinoma of the scalp and risk of recurrence.

BACKGROUND: Consensus is lacking on adequate deep histological margins in cutaneous squamous cell carcinoma (cSCC). Deep clearance for tumours located on the scalp is limited by anatomic constraints. OBJECTIVE: To determine whether clear but close deep histological margins (<1 mm) confer a higher risk of recurrence in cSCCs of the scalp treated by wide local excision, compared to deep histological margins ≥1 mm. METHODS: Multicentre retrospective observational cohort study and multivariate competing risk analysis to evaluate risk factors for recurrence. RESULTS: In total, 295 patients with 338 cSCCs were included. Close deep histological margins were not associated with an increased cumulative incidence of recurrence (subhazard ratio [SHR] 1.96 [95% CI 0.87-4.41]). However, an increased risk of recurrence was observed for those tumours that presented concurrent invasion of the galea aponeurotica and close deep margins, as opposed to patients without these factors (SHR 3.52 [1.24-10.01]). Tumours with clear but close peripheral margins (<1 mm) also had higher risk of recurrence (SHR 5.01 [1.68-14.97]). LIMITATIONS: Retrospective observational study based on pathology reports. CONCLUSION: Deep histological margins <1 mm do not confer a greater risk of recurrence as long as the tumour is completely excised and the galea aponeurotica is not involved. Surgical excision of cSCC on the scalp should include the galea to ensure proper assessment of deep margins.

Vaccine value profile for invasive non-typhoidal Salmonella disease.

Invasive non-typhoidal Salmonella (iNTS) disease is an under-recognized high-burden disease causing major health and socioeconomic issues in sub-Saharan Africa (sSA), predominantly among immune-naïve infants and young children, including those with recognized comorbidities such as HIV infection. iNTS disease is primarily caused by Salmonella enterica serovar Typhimurium sequence type (ST) 313 and 'African-restricted clades' of Salmonella Enteritidis ST11 that have emerged across the African continent as a series of epidemics associated with acquisition of new antimicrobial resistance. Due to genotypes with a high prevalence of antimicrobial resistance and scarcity of therapeutic options, these NTS serovars are designated by the World Health Organization as a priority pathogen for research and development of interventions, including vaccines, to address and reduce NTS associated bacteremia and meningitis in sSA. Novel and traditional vaccine technologies are being applied to develop vaccines against iNTS disease, and the results of the first clinical trials in the infant target population should become available in the near future. The "Vaccine Value Profile" (VVP) addresses information related predominantly to invasive disease caused by Salmonella Enteritidis and Salmonella Typhimurium prevalent in sSA. Information is included on stand-alone iNTS disease candidate vaccines and candidate vaccines targeting iNTS disease combined with another invasive serotype, Salmonella Typhi, that is also common across sSA. Out of scope for the first version of this VVP is a wider discussion on either diarrheagenic NTS disease (dNTS) also associated with Salmonella Enteritidis and Salmonella Typhimurium or the development of a multivalent Salmonella vaccines targeting key serovars for use globally. This VVP for vaccines to prevent iNTS disease is intended to provide a high-level, holistic assessment of the information and data that are currently available to inform the potential public health, economic, and societal value of pipeline vaccines and vaccine-like products. Future versions of this VVP will be updated to reflect ongoing activities such as vaccine development strategies and a "Full Vaccine Value Assessment" that will inform the value proposition of an iNTS disease vaccine. This VVP was developed by a working group of subject matter experts from academia, non-profit organizations, public private partnerships, and multi-lateral organizations, and in collaboration with stakeholders from the World Health Organization African Region. All contributors have extensive expertise on various elements of the iNTS disease VVP and collectively aimed to identify current research and knowledge gaps. The VVP was developed using only existing and publicly available information.

TB PCR in BAL and EBUS-TBNA samples for the diagnosis of pulmonary and mediastinal lymph node TB: retrospective TRiBE study.

INTRODUCTION: The role of Xpert Ultra in bronchoalveolar lavage (BAL) and endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) samples for pulmonary and mediastinal lymph node tuberculosis (TB) remains unclear. METHODS: This was a retrospective observational service evaluation at a tertiary TB centre in a low-incidence setting. The diagnostic indices of Xpert Ultra, smear and culture (with cytology for EBUS-TBNA samples) were compared with culture positivity or a composite reference standard of clinical TB diagnosis. Trace readouts, a new category of results for Xpert Ultra indicating low bacillary load, were analysed in two ways as a true positive or true negative result. 282 BAL and 139 EBUS-TBNA samples were included in the analysis. RESULTS: BAL: sensitivity with 95% CI against culture-confirmed pulmonary TB from BAL samples for Xpert Ultra (trace as positive) was 0.91 (0.82 to 0.98), Xpert Ultra (trace as negative) was 0.76 (0.69 to 0.83), smear was 0.38 (p=0.0009) and culture was 1.00 (0.91 to 1.00). Specificities for all the tests were ≥0.99 (0.98 to 1.00). The addition of smear to Xpert Ultra did not improve the diagnostic accuracy.EBUS-TBNA: sensitivity against culture-confirmed TB from EBUS-TBNA samples for Xpert Ultra (trace as positive) was 0.71 (0.63 to 0.78), Xpert Ultra (trace as negative) was 0.59 (0.54 to 0.63), smear was 0.12 (p=0.002), culture was 1.00 (0.89 to 1.00), cytology was 0.87 (0.76 to 0.98) and rapid on-site evaluation of cytology (ROSE) was 0.92 (0.78 to 1.00). Specificities were 0.99 (0.97 to 1.00), 0.99 (0.97 to 1.00), 1.00 (0.98 to 1.00), 1.00 (0.98 to 1.00), 0.67 (0.67 to 0.68) and 0.42, respectively. CONCLUSION: Xpert Ultra had a significantly higher sensitivity compared with smear in both BAL and EBUS-TBNA samples. Xpert Ultra had a lower sensitivity compared with culture but comparable specificity with results being available within <24 hours. Trace readings in our low-incidence setting were associated with culture positivity in all BAL samples.

[Prevalence of physical inactivity and risk of sarcopenia in primary care. Cross-sectional study]. / Prevalencia de inactividad física y riesgo de sarcopenia en atención primaria. Estudio transversal.

OBJECTIVES: Assess the prevalence of physical inactivity and risk of sarcopenia in primary care patients and their companions. DESIGN: Cross-sectional study. We carried out an anonymous survey of primary care users (patients and companions) in primary care consultations and stands coinciding with a community health activity for World Physical Activity Day. SITE: Five primary care centers (CAPs) of the South Metropolitan health region: in Cornellà de Llobregat (CAP Jaume Soler), in l'Hospitalet de Llobregat (CAP Florida Nord, CAP Florida Sud and CAP Bellvitge) and in Viladecans (CAP Maria Bernades) between 27 March to April 6, 2023 (coinciding with World Physical Activity Day). PARTICIPANTS: Primary care population consists of patients and their companions over 18 years of age. INTERVENTIONS: The health workers administered questionnaires to users and companions. MAIN MEASUREMENTS: We evaluated physical inactivity with the BPAAT questionnaire, risk of sarcopenia with SARC-F screening test, sex and age range. We performed an univariate descriptive analysis to report prevalence. RESULTS: Nine hundred ninety-eight participants were surveyed. Physical inactivity was present in 38.9% of the participants. Among those over 50 years (665 participants), 15.4% were at risk of sarcopenia (9.58% men, 19.2% women). CONCLUSIONS: The prevalence of physical inactivity and risk of sarcopenia (in individuals over 50 years old) in the studied population is high. Women have greater physical inactivity and a greater risk of sarcopenia than men.

INF2 formin variants linked to human inherited kidney disease reprogram the transcriptome, causing mitotic chaos and cell death.

Mutations in the human INF2 gene cause autosomal dominant focal segmental glomerulosclerosis (FSGS)-a condition characterized by podocyte loss, scarring, and subsequent kidney degeneration. To understand INF2-linked pathogenicity, we examined the effect of pathogenic INF2 on renal epithelial cell lines and human primary podocytes. Our study revealed an increased incidence of mitotic cells with surplus microtubule-organizing centers fostering multipolar spindle assembly, leading to nuclear abnormalities, particularly multi-micronucleation. The levels of expression of exogenous pathogenic INF2 were similar to those of endogenous INF2. The aberrant nuclear phenotypes were observed regardless of the expression method used (retrovirus infection or plasmid transfection) or the promoter (LTR or CMV) used, and were absent with exogenous wild type INF2 expression. This indicates that the effect of pathogenic INF2 is not due to overexpression or experimental cell manipulation, but instead to the intrinsic properties of pathogenic INF2. Inactivation of the INF2 catalytic domain prevented aberrant nuclei formation. Pathogenic INF2 triggered the translocation of the transcriptional cofactor MRTF into the nucleus. RNA sequencing revealed a profound alteration in the transcriptome that could be primarily attributed to the sustained activation of the MRTF-SRF transcriptional complex. Cells eventually underwent mitotic catastrophe and death. Reducing MRTF-SRF activation mitigated multi-micronucleation, reducing the extent of cell death. Our results, if validated in animal models, could provide insights into the mechanism driving glomerular degeneration in INF2-linked FSGS and may suggest potential therapeutic strategies for impeding FSGS progression.

The relationship between nanoscale genome organization and gene expression in mouse embryonic stem cells during pluripotency transition.

During early development, gene expression is tightly regulated. However, how genome organization controls gene expression during the transition from naïve embryonic stem cells to epiblast stem cells is still poorly understood. Using single-molecule microscopy approaches to reach nanoscale resolution, we show that genome remodeling affects gene transcription during pluripotency transition. Specifically, after exit from the naïve pluripotency state, chromatin becomes less compacted, and the OCT4 transcription factor has lower mobility and is more bound to its cognate sites. In epiblast cells, the active transcription hallmark, H3K9ac, decreases within the Oct4 locus, correlating with reduced accessibility of OCT4 and, in turn, with reduced expression of Oct4 nascent RNAs. Despite the high variability in the distances between active pluripotency genes, distances between Nodal and Oct4 decrease during epiblast specification. In particular, highly expressed Oct4 alleles are closer to nuclear speckles during all stages of the pluripotency transition, while only a distinct group of highly expressed Nodal alleles are in close proximity to Oct4 when associated with a nuclear speckle in epiblast cells. Overall, our results provide new insights into the role of the spatiotemporal genome remodeling during mouse pluripotency transition and its correlation with the expression of key pluripotency genes.

Modeling of Intracellular Taurine Levels Associated with Ovarian Cancer Reveals Activation of p53, ERK, mTOR and DNA-Damage-Sensing-Dependent Cell Protection.

Taurine, a non-proteogenic amino acid and commonly used nutritional supplement, can protect various tissues from degeneration associated with the action of the DNA-damaging chemotherapeutic agent cisplatin. Whether and how taurine protects human ovarian cancer (OC) cells from DNA damage caused by cisplatin is not well understood. We found that OC ascites-derived cells contained significantly more intracellular taurine than cell culture-modeled OC. In culture, elevation of intracellular taurine concentration to OC ascites-cell-associated levels suppressed proliferation of various OC cell lines and patient-derived organoids, reduced glycolysis, and induced cell protection from cisplatin. Taurine cell protection was associated with decreased DNA damage in response to cisplatin. A combination of RNA sequencing, reverse-phase protein arrays, live-cell microscopy, flow cytometry, and biochemical validation experiments provided evidence for taurine-mediated induction of mutant or wild-type p53 binding to DNA, activation of p53 effectors involved in negative regulation of the cell cycle (p21), and glycolysis (TIGAR). Paradoxically, taurine's suppression of cell proliferation was associated with activation of pro-mitogenic signal transduction including ERK, mTOR, and increased mRNA expression of major DNA damage-sensing molecules such as DNAPK, ATM and ATR. While inhibition of ERK or p53 did not interfere with taurine's ability to protect cells from cisplatin, suppression of mTOR with Torin2, a clinically relevant inhibitor that also targets DNAPK and ATM/ATR, broke taurine's cell protection. Our studies implicate that elevation of intracellular taurine could suppress cell growth and metabolism, and activate cell protective mechanisms involving mTOR and DNA damage-sensing signal transduction.

Taste of common prebiotic oligosaccharides: impact of molecular structure.

Prebiotic oligosaccharides are naturally occurring nondigestible carbohydrates with demonstrated health benefits. They are also a chemically diverse class of nutrients, offering an opportunity to investigate the impact of molecular structure on oligosaccharide taste perception. Accordingly, a relevant question is whether these compounds are detected by the human gustatory system, and if so, whether they elicit sweet or "starchy" taste. Here, in 3 psychophysical experiments, we investigated the taste perception of 3 commercially popular prebiotics [fructooligosaccharides (FOS), galactooligosaccharides (GOS), xylooligosaccharides (XOS)] in highly pure form. Each of these classes of prebiotics differs in the type of glycosyl residue, and position and type of bond between those residues. In experiments I and II, participants were asked to discriminate a total of 9 stimuli [FOS, GOS, XOS; degree of polymerization (DP) of 2, 3, 4] prepared at 75 mM in the presence and absence of lactisole, a sweet receptor antagonist. We found that all 9 compounds were detectable (P < 0.05). We also found that GOS and XOS DP 4 were discriminable even with lactisole, suggesting that their detection was not via the canonical sweet receptor. Accordingly, in experiment III, the taste of GOS and XOS DP 4 were directly compared with that of MOS (maltooligosaccharides) DP 4-6, which has been reported to elicit "starchy" taste. We found that GOS and MOS were perceived similarly although narrowly discriminable, while XOS was easily discriminable from both GOS and MOS. The current findings suggest that the molecular structure of oligosaccharides impacts their taste perception in humans.

Integrated miRNA-mRNA Analysis Reveals Critical miRNAs and Targets in Diet-Induced Obesity-Related Glomerulopathy.

This study aimed to investigate obesity-related glomerulopathy (ORG) at cellular, structural, and transcriptomic levels. Thirty Wistar rats were randomized into two groups: 15 rats were fed with a standard diet (SD-rats), and 15 rats were fed with a high-fat diet (HFD-rats). After 10 weeks, the weight, kidney function, histological features, and transcriptomic changes were assessed. HFD-rats gained significantly more weight (55.8% vs. 29.2%; p < 0.001) and albuminuria (10,384.04 ng/mL vs. 5845.45 ng/mL; p < 0.001) compared to SD-rats. HFD-rats exhibited early stages of ORG, with predominant mesangial matrix increase and podocyte hypertrophy (PH). These lesions correlated with differentially expressed (DE) genes and miRNAs. Functional analysis showed that miR-205, which was DE in both the kidneys and urine of HFD-rats, negatively regulated the PTEN gene, promoting lipid endocytosis in podocytes. The downregulation of PTEN was proved through a higher PTEN/nephrin ratio in the SD-rats and the presence of lipid vacuoles in HFD-podocytes. This study has found a specific targetome of miRNAs and gene expression in early stages of ORG. Also, it emphasizes the potential value of miR-205 as a urinary biomarker for detecting podocyte injury in ORG, offering a tool for early diagnosis, and opening new avenues for future therapeutic research of obesity-related glomerulopathy.

Immortalized human myotonic dystrophy type 1 muscle cell lines to address patient heterogeneity.

Historically, cellular models have been used as a tool to study myotonic dystrophy type 1 (DM1) and the validation of therapies in said pathology. However, there is a need for in vitro models that represent the clinical heterogeneity observed in patients with DM1 that is lacking in classical models. In this study, we immortalized three DM1 muscle lines derived from patients with different DM1 subtypes and clinical backgrounds and characterized them at the genetic, epigenetic, and molecular levels. All three cell lines display DM1 hallmarks, such as the accumulation of RNA foci, MBNL1 sequestration, splicing alterations, and reduced fusion. In addition, alterations in early myogenic markers, myotube diameter and CTCF1 DNA methylation were also found in DM1 cells. Notably, the new lines show a high level of heterogeneity in both the size of the CTG expansion and the aforementioned molecular alterations. Importantly, these immortalized cells also responded to previously tested therapeutics. Altogether, our results show that these three human DM1 cellular models are suitable to study the pathophysiological heterogeneity of DM1 and to test future therapeutic options.

Clinician Prediction of Survival vs Calculated Prediction Scores in Patients Requiring Extracorporeal Membrane Oxygenation.

BACKGROUND: Determining appropriate extracorporeal membrane oxygenation (ECMO) candidacy ensures appropriate utilization of this costly resource. The current ECMO survival prediction scores do not consider clinician assessment of patient viability. This study compared clinician prediction of survival to hospital discharge versus prediction scores. OBJECTIVES: The aim of this study was to compare clinician prediction of patients' survival to hospital discharge versus prognostic prediction scores (Respiratory ECMO Survival Prediction [RESP] or Survival After Veno-Arterial ECMO [SAVE] score) to actual survival. METHODS: This was an observational descriptive study from January 2020 to November 2021 conducted with interviews of nurses, perfusionists, and physicians who were involved during the initiation of ECMO within the first 24 hours of cannulation. Data were retrieved from the medical record to determine prediction scores and survival outcomes at hospital discharge. Accuracy of clinician prediction of survival was compared to the RESP or SAVE prediction scores and actual survival to hospital discharge. RESULTS: Accurate prediction of survival to hospital discharge for veno-venous ECMO by nurses was 47%, 64% by perfusionists, 45% by physicians, and 45% by the RESP score. Accurate predictions of patients on veno-arterial ECMO were correct in 54% of nurses, 77% of physicians, and 14% by the SAVE score. Physicians were more accurate than the SAVE score, P = .021, and perfusionists were significantly more accurate than the RESP score, P = .044. There was no relationship between ECMO specialists' years of experience and accuracy of predications. CONCLUSION: Extracorporeal membrane oxygenation clinicians may have better predictions of survival to hospital discharge than the prediction scores. Further research is needed to develop accurate prediction tools to help determine ECMO eligibility.