Upper aerodigestive tract squamous cell carcinoma (UATSCC) is often easy to recognize in its conventional form. However, a subset of tumors loses histologic evidence of squamous differentiation and, instead, become sarcomatoid (spindled). These cases may be difficult or impossible to appropriately classify on biopsy material, even with immunohistochemistry. DNA methylation has emerged as a tool to help in classifying undifferentiated malignancies. We sought to explore whether conventional and sarcomatoid UATSCC harbor the same epigenetic signature. Cases of sarcomatoid UATSCC were collected and reviewed. The conventional and sarcomatoid components were documented. Only specimens with concomitant epithelial dysplasia and/or invasive SCC were included. A panel of keratins (CK AE1/3, CAM5.2 and CK5/6) was performed on the sarcomatoid component of each tumor and scored as follows: 0, < 1%; 1+, 1-25%; 2+, 26-50%, 3+, > 50%. DNA methylation profiling was performed and compared with profiles from publicly available UATSCC and a control cohort of pleomorphic malignancies including undifferentiated sarcoma, leiomyosarcoma, angiosarcoma, atypical fibroxanthoma/pleomorphic dermal sarcoma, and melanoma, as well as sarcomatoid carcinoma of cutaneous origin. 21 UATSCC samples from 15 patients were retrieved (9 M, 6 F; 47-88 years, median 71 years) from sites including tongue (7), larynx (5), pharynx (3), maxillary/mandibular alveolar ridge (3), "neck" (2), and nasal cavity (1). 12 samples were entirely sarcomatous, while six were conventional SCC and three contained both sarcomatoid and conventional components. Of the sarcomatous components, six were entirely negative (score = 0) for all keratins performed. Using dimensionality reduction analysis of DNA methylation data by Uniform Manifold Approximation and Projection (UMAP) and unsupervised hierarchical clustering, 11 of 12 sarcomatoid UATSCC samples, were positioned with conventional UATSCC. Methylomes of sarcomatoid carcinoma of upper aerodigestive tract and cutaneous origin overlapped. Copy number profile analysis did not show significant differences between conventional and sarcomatoid histologies or tumor time point. The epigenetic signatures of conventional and sarcomatoid UATSCC are similar, even in cases that lack expression of multiple keratins. DNA methylation profiling may be leveraged in biopsies and entirely sarcomatoid UATSCC to accurately diagnose these cases and differentiate them from pleomorphic sarcomas and melanoma.
The SUPREMO trial reported adjuvant chest wall radiotherapy had no effect on 10-year overall survival (primary endpoint) in patients with intermediate-risk breast cancer after mastectomy. The quality of life (QOL) substudy of SUPREMO (UK patients only) examines the effects of chest wall radiotherapy in patients with intermediate-risk breast cancer 1 year, 2 years, 5 years, and 10 years after treatment. Here, we report 5-year QOL results (a secondary endpoint), including prespecified subgroup analyses. SUPREMO is an international, parallel-group, randomised, controlled, phase 3 trial. Women aged 18 years or older with intermediate-risk breast cancer (defined as pT1-2N1 or pT3N0 or pT2N0 with histological grade 3, or lymphovascular invasion, or both) who had undergone mastectomy (and axillary surgery) were randomly assigned (1:1) to receive chest wall radiotherapy (40-50 Gy in 15-25 fractions) or no radiotherapy. Randomisation was in permuted blocks with varying block length, stratified by centre, without masking of patients or investigators. All UK patients were invited to the QOL substudy. Consenting patients completed validated questionnaires (European Organisation for Research and Treatment of Cancer Quality of Life Core 30-item questionnaire; European Organisation for Research and Treatment of Cancer Quality of Life Questionnaire-Breast Cancer Module, 23 items; Body Image Scale; and Hospital Anxiety and Depression Scale) before randomisation and at 1 year, 2 years, 5 years, and 10 years. Here, we report the prespecified primary QOL outcomes of global QOL, fatigue, physical function, chest wall symptoms, shoulder and arm symptoms, body image, anxiety, and depression in the intention to treat population, using repeated measures mixed-effects models. This trial is registered with the ISRCTN registry, ISRCTN61145589 and is now completed. Between Aug 4, 2006, and April 29, 2013, SUPREMO recruited 1691 patients internationally, of whom 1679 were randomly assigned to receive chest wall radiotherapy (n=845) or not (n=834). Of 1233 patients from UK centres (619 in the chest wall radiotherapy group and 614 in the no chest wall radiotherapy group), 989 (80%) consented to the QOL substudy: 947 (96%) of 989 returned baseline questionnaires and 620 (75%) of 832 patients returned the expected year-5 questionnaires. Chest wall symptoms were worse with chest wall radiotherapy (effect estimate 1·99 [95% CI 0·36-3·62]; p=0·017) with an improvement between years 1, 2, and 5. Chemotherapy was associated with less improvement over time (effect estimate 2·97 [95% CI 0·24-5·71]; p=0·033) but without interaction with chest wall radiotherapy. No statistically significant differences were observed in other QOL domains (arm symptoms, body image, fatigue, pain, overall QOL, physical functioning, or anxiety and depression). In the subgroup analyses, patients who had a sentinel lymph node biopsy followed up by an axillary lymph node clearance with chest wall radiotherapy had worse chest wall symptoms than those having a single axillary operation (difference chest wall radiotherapy vs no chest wall radiotherapy -5·29 [95%CI -8·53 to -2·05]; p=0·0015). The 5-year QOL findings showed worse chest wall toxicity and no effect on other QOL domains. The subgroup analyses suggested a potential negative effect of chemotherapy and axillary clearance after sentinel node biopsy. UK Medical Research Council, National Institute for Health and Care Research, European Organisation for Research and Treatment of Cancer, Edinburgh Trustees of Breast Cancer Institute in Edinburgh and the Edinburgh Cancer Centre NHS Endowment funds, The Dutch Cancer Society, Trustees of the Hong Kong and Shanghai Banking Corporation.
Maternal nutritional status before and during pregnancy is an important modifiable factor that may influence foetal development and long-term offspring health. Both undernutrition and excessive maternal body weight have been associated with adverse metabolic, cardiovascular, neurodevelopmental and immune-related outcomes in offspring. This narrative review summarises selected experimental, clinical and epidemiological evidence on maternal nutritional status, intrauterine programming and offspring health. Relevant literature was identified by searching the PubMed, Scopus and Web of Science databases covering publications from 2000 to 2026, with the majority of included papers published after 2015. The search terms included maternal nutrition, undernutrition, obesity, intrauterine programming, DOHaD, epigenetics, DNA methylation, microRNA and offspring health. The available evidence suggests that maternal undernutrition is associated with intrauterine growth restriction (IUGR) and an increased risk of cognitive and metabolic disorders. In contrast, maternal overweight or obesity is associated with macrosomia, as well as an increased risk of obesity, cardiovascular disease and metabolic dysfunction in later life. Both extremes of maternal nutritional status may also negatively affect the psychological health of the offspring. These associations suggest a potential role for epigenetic mechanisms; however, causal conclusions from observational studies in humans remain limited. Overall, the findings highlight the importance of optimising maternal nutritional and metabolic status before and during pregnancy as a key strategy for promoting favourable intrauterine programming and improving long-term offspring health. A better understanding of the underlying epigenetic mechanisms could facilitate the development of targeted nutritional and preventive interventions. As a narrative review, this analysis is limited by the associative nature of human observational studies and the inherent challenges in separating intrauterine effects from postnatal environmental confounders.
Myelination, driven by differentiation of oligodendrocyte precursor cells, is critical for metabolic and structural support and efficient axonal signal transmission in neurons. Loss of myelin is a hallmark of multiple sclerosis and other devastating demyelinating disorders. As demyelination persists, neurons become increasingly vulnerable, leading to neurodegeneration and chronic disability. Restoring myelin through endogenous repair mechanisms offers a promising therapeutic approach to mitigate progressive neuronal loss. One key regulator of myelination is the G protein-coupled receptor 17, GPR17, whose chronic upregulation in oligodendrocyte precursor cells is commonly seen with myelin injury. In line with single-nucleus transcriptomic data showing predominant expression of GPR17 in committed oligodendrocyte precursor cells, our postmortem immunohistochemical analyses of MS patient tissue revealed a significant upregulation of GPR17+/BCAS1+ oligodendrocyte precursor cells adjacent to and in demyelinated lesions. Importantly, remyelinated lesions lacked GPR17 immunoreactivity, consistent with a model in which sustained GPR17 expression is associated with demyelination and impaired oligodendrocyte precursor cell differentiation. To test the impact of pharmacological GPR17 inhibition on remyelination, we evaluated the effects of a novel, selective GPR17 antagonist in cuprizone-induced murine demyelination models. This toxin-induced approach has been widely used to study mechanisms of de- and remyelination, in the absence of the full inflammatory complexity of demyelinating diseases such as multiple sclerosis. We show that oral treatment results in robust functional recovery consistent with remyelination, as evidenced by improved spatial memory and recovery of visual evoked potential latency delays. GPR17 antagonism also accelerated structural remyelination in the corpus callosum and optic nerve. Together, these findings support a role for pharmacological GPR17 antagonism in promoting remyelination and highlight this G protein-coupled receptor as a promising therapeutic target for demyelinating disorders.
Despite advances in genetic testing, many 46,XY Disorders of sex development (DSD) cases remain unsolved after whole-exome sequencing (WES). This study intended to explore rare variants in patients with micropenis, cryptorchidism, or hypospadias using bioinformatics analysis to identify potential pathogenic contributors and pathways underlying 46,XY DSD. A total of 35 patients with specific phenotypes (micropenis/cryptorchidism/hypospadias) and negative whole-exome sequencing results were enrolled. Bioinformatics analysis methods (SKAT-O test and GO enrichment) were applied to identify the putative loss-of-function (pLoF) variation, including nonsense, frameshift, and canonical splice-site variants, and predicted deleterious missense variants (CADD Phred > 20). Literature was reviewed to explore the correlation of detected candidate genes/pathways and 46,XY disorder of sex development. After variant quality filtering, we identified 307,638 pLoF variants and 127,857 predicted deleterious missense variants across all samples. In subgroup A (micropenis, n = 21), we identified 146,268 pLoF variants and 104,746 predicted deleterious missense variants. In subgroup B (cryptorchidism, n = 10), we identified 111,172 pLoF variants and 77,244 predicted deleterious missense variants. In subgroup C (hypospadias, n = 4), we identified 50,198 pLoF variants and 23,111 predicted deleterious missense variants. Using SKAT-O with an initial screening threshold of p < 0.005 (FDR q < 0.05), we obtained 67 candidate genes from the pLoF variant set and 59 candidate genes from the predicted deleterious missense variant set in subgroup A; 81 and 11 candidate genes, respectively, in subgroup B; and 17 and 0 candidate genes, respectively, in subgroup C. Assessment of rare variants helps further explore the genetic contributors to 46,XY disorder of sex development and provide potential candidate genes and associated pathways.
Background and Objectives: Posterior fossa anomalies (PFAs) represent a heterogeneous group of congenital malformations involving the cerebellum and adjacent structures. Prenatal counseling remains challenging because neurodevelopmental outcomes vary substantially according to the specific anomaly. This study aimed to evaluate ultrasonographic and fetal magnetic resonance imaging (MRI) characteristics, associated anomalies, genetic test results, and perinatal and neurodevelopmental outcomes of prenatally diagnosed PFAs in a tertiary care population. Materials and Methods: This retrospective study included 115 fetuses diagnosed with PFAs between 2020 and 2024. PFAs were categorized into "simple" (mega cisterna magna, Blake's pouch cyst [BPC], and arachnoid cyst) and "complex" (Dandy-Walker malformation [DWM], vermian agenesis/hypoplasia, cerebellar hypoplasia, Joubert syndrome, Walker-Warburg syndrome, and pontocerebellar hypoplasia). Maternal characteristics, associated cerebral and extracerebral anomalies, fetal MRI findings, genetic test results, and pregnancy and postnatal outcomes were analyzed. Results: Among 115 cases, 44.3% were isolated, and 55.6% were non-isolated. Ventriculomegaly was detected in 35.6% of cases and was significantly more frequent in the complex group (47.9% vs. 14.2%). Fetal MRI was performed in 35.6% (n = 41) of cases and demonstrated a 24.3% discrepancy rate with ultrasound, mainly in differentiating DWM from BPC. Genetic testing was performed in 66.9% of cases, revealing chromosomal abnormalities in 15.6% of the total cohort. Complex anomalies, including cerebellar hypoplasia and vermian agenesis/hypoplasia, were more frequently associated with pathogenic copy-number variants and monogenic disorders. The simple group (n = 42) had favorable outcomes, with an 89.2% survival rate and neurodevelopmental delay in 15.2% of survivors. In contrast, the complex group (n = 73) had significantly poorer outcomes (p < 0.001), with a 38.1% survival rate, 42.5% rate of pregnancy termination, and neurodevelopmental delay in 56.3% of survivors. Conclusions: This study highlights the persistent diagnostic challenges associated with PFAs and underscores the importance of multimodal imaging and comprehensive genetic evaluation. The proposed simple-complex classification provides a clinically meaningful framework for prognostication and parental counseling. Future research should prioritize prospective multicenter studies with higher rates of comprehensive genetic testing coupled with standardized long-term neurodevelopmental follow-up to refine the understanding of the natural history and prognostic trajectory of these complex brain malformations.
Eggerthella lenta (E. lenta) is an opportunistic anaerobic pathogen associated with severe systemic infections, yet rapid and accurate diagnostic tools remain limited. To address this challenge, we developed a highly sensitive and specific dual-readout diagnostic platform integrating recombinase-aided amplification (RAA) with the CRISPR/Cas13a system, targeting the highly conserved rsmG gene of E. lenta. The assay offers two detection modalities: a real-time fluorescence readout and a visually interpretable lateral flow strip. Analytical evaluation demonstrated that the fluorescence-based assay achieved a limit of detection (LOD) of 4.4 copies per reaction (95% CI: 3.7-5.6 copies/reaction), while the instrument-free lateral flow assay yielded an LOD of 10⁴ copies per reaction. The platform exhibited exceptional specificity, showing no cross-reactivity with 10 common non-target bacterial species. Clinical validation was performed using 24 synovial fluid samples, all confirmed positive for E. lenta by Sanger sequencing. The fluorescence assay successfully detected all 24 samples, achieving a detection rate of 100% (24/24). In parallel, the lateral flow assay detected 21 of the 24 positive samples, yielding a detection rate of 87.5% (21/24). The three samples undetected by the lateral flow strip were verified as true positives by sequencing, indicating that the discrepancy was due to the lower analytical sensitivity of the strip format rather than a lack of specificity. In conclusion, this dual-mode RAA-CRISPR/Cas13a platform serves as a robust and practical tool for rapid clinical diagnosis and point-of-care (POC) triaging of E. lenta infections. The fluorescence format is optimal for high-sensitivity laboratory testing, whereas the lateral flow variant provides a deployable alternative for rapid, point-of-care screening in resource-limited environments.
Patient serum for anti-HLA antibodies are tested with purified antigens covalently affixed to beads as targets. Serologic nomenclature from the World Health Organization (WHO) is currently used to specify the reactivity of antibodies, although target antigens are defined at a finer allelic level. We investigated whether different reactivity patterns could be present at the allele level of a single WHO serologic antigen. We analyzed 2389 serum samples from 1215 patients waiting for solid-organ transplant by using class II single antigen beads for detection of HLA antibodies. We used principal component analysis to identify unique antibody reactivity patterns against the beads. We particularly focused on patterns within a WHO antigen that suggested the presence of new serologic class II HLA specificities. We used the HLA DQ7 data to illustrate the principle of the discovery methodology. Four assay target beads representing different DQA1 alleles were classified as DQ7. Two principal components accounted for about 98.6%of the DQ7 data variance. The dominant principal component, which accounted for 83.9% of the variance, represented a reaction pattern in which sera reacted equally with all 4 beads. The second principal component (14.7% of the variance) represented sera that reacted selectively with DQA1*03:01, DQB1*03:01. Four of 10 DR and 6 of 7 DQ WHO serologic specificities that were studied showed more than 5% nonconcordant patterns revealed by principal component analysis. Principal component analysis revealed differential reactivity patterns of the alleles of single WHO serological specificity. This method could be a powerful tool for identifying patterns of reactivity, which may represent new serological specificities.
Sudden Unexplained Death in Youth (SUDY) requires thorough investigation to identify underlying causes and guide prevention strategies. In the Netherlands, cases are investigated using the standardized Postmortem Evaluation of Sudden Unexplained Death in Infants and Children (PESUDIC), in which autopsy is offered as a standard component. However, its invasive nature and associated time burden may limit parental acceptance. This study evaluated to what extent the cause of death can be established using a limited set of diagnostic tests compared to the standard procedure including autopsy. In this observational study, children > 2 years of age who died suddenly and unexpectedly and underwent PESUDIC, including imaging and autopsy, were included. Two expert panels, consisting of a forensic and a pediatric specialist, independently assessed early diagnostic tests available before autopsy. Cases were classified by level of diagnostic certainty and need for autopsy. Panel conclusions were compared with the reference standard: a multidisciplinary audit incorporating all available information, including autopsy findings. Sixty-six cases were included (median age 12 years (IQR 4-14.7), 63% male). Panels identified indicative information for a cause of death in 60 patients (91%). Nevertheless, autopsy was required in most cases (n = 59) to confirm the diagnosis. In 7 cases (10.6%), panels were sufficiently confident to establish the cause of death without autopsy with complete agreement with the reference standard. Causes included obstructive gastrointestinal pathology (n = 5) and diabetic ketoacidosis with dehydration (n = 2).  Only a small proportion of children > 2 years of age with sudden unexpected death have a cause of death that can be established with sufficient certainty at an early stage. In the vast majority of cases, the cause of death remains uncertain, supporting the recommendation to perform an autopsy. • Autopsy in sudden unexplained death in youth is worldwide recognized as the gold standard for postmortem examination. However, its invasive nature and associated time burden may limit parental acceptance. • In 7 of 60 sudden and unexplained deceased minors (10%), sufficient certainty regarding the cause of death can be obtained by other minimal invasive diagnostic test, so without the need of an autopsy. Causes without the need of an autopsy included obstructive gastrointestinal pathology and diabetic ketoacidosis with dehydration.
A lateral flow analysis (LFA) strip for detecting dual miRNAs (miR155 and miR21) was constructed based on the "AND" logic gate and DNA self-assembly. The ternary synergistic strategy of "logic gating-signal amplification-visual output" enables highly sensitive and visual detection of miR21 and miR155 for breast cancer diagnosis. Only if both miR21 and miR155 are present can the trigger DNA (TDNA) in the sensor specifically dissociate and activate the "AND" logic gate (output signal = "1"); in the absence of either miRNA, the system remains in an inactive state (output signal = "0"). TDNA initiates catalytic hairpin assembly (CHA), and the product of the CHA reaction cascaded triggers the hybridization chain reaction (HCR) to achieve dual signal amplification. Gold nanoparticles (AuNPs) were used to label DNA for the construction of the LFA strip, allowing naked-eye observation of the detection results of miR21 and miR155. Detection results showed that the signal intensity of the test line (T line) had a positive correlation with the concentration of miRNAs (with a miR155:miR21 ratio of 1:1). Within the concentration range of 0.1-10 nM, the average grayscale value of the T line exhibited a good correlation with the logarithm of miRNA concentration (correlation coefficient r = 0.9983), and the limit of detection (LOD) was 30.98 pM. This LFA strip has been preliminarily validated to the detection of human serum samples, and the results indicated that the levels of miR155 and miR21 in the serum of breast cancer patients were significantly higher than those in normal individuals. Free from the limitation of large-scale instruments, this strip holds great commercialization potential.
This study describes the development and analytical validation of a DNA-based quantitative PCR (qPCR) assay for detecting the NPM1 Type A mutation (NPM1-A mut) in acute myeloid leukemia (AML). The assay employs a modified wild-type blocker (WTB) to preferentially suppress amplification of wild-type (WT) alleles, thereby enriching mutant targets. The modified WTB was synthesized as a 3'-ddC-terminated LNA/DNA chimera and used together with a 6-FAM-MGB-Eclipse hydrolysis probe. The analytical performance of the WTB-qPCR assay was compared with that of an otherwise identical qPCR assay lacking WTB. Key parameters such as linearity, precision, limit of detection (LoD), and limit of quantification (LoQ) were evaluated following the guidelines outlined in CLSI EP17-A2. Concurrently, modified WTB was implemented in Sanger sequencing to improve the sensitivity of mutation detection. Incorporation of WTB yielded an LoD of 0.00302% and an LoQ of 0.00528%, corresponding to approximately 3.02 and 5.28 NPM1-Amut copies, respectively, per 100,000 WT copies. This represented a clear improvement over the non-WTB assay. The WTB-qPCR showed excellent linearity (R2 > 0.999) and precision (CV < 5%), with 100% analytical specificity and no detectable cross-reactivity. Application of modified WTB in Sanger sequencing increased analytical sensitivity to ~ 0.1% mutant allele frequency. The modified WTB-qPCR assay offers a rapid, highly sensitive, and cost-effective DNA-based method for MRD monitoring in NPM1-A-mutated AML. Its strong analytical performance and straightforward workflow support its implementation as a routine diagnostic tool in clinical laboratories.
Patients with cancer are at increased risk of severe outcomes from COVID-19. Yet, the immunological determinants underlying this vulnerability remain incompletely understood, particularly in low- and middle-income settings. Moreover, the impact of the severe viral disease surge and its compensatory mechanisms, such as stressed myelopoiesis, on this population needs further elucidation. This study aims to characterize the cytokine and lymphocyte profiles of cancer patients with COVID-19, correlate these profiles with disease severity, and compare them to those of non-cancer patients with COVID-19. Plasma cytokine, chemokine, and growth factor levels were quantified using Luminex technology, and immune cell subsets were characterized by flow cytometry. A total of 67 patients were analyzed: 40 with cancer (26 mild cases and 14 severe cases) and 27 without cancer (12 mild cases and 15 severe cases). Clinical outcomes showed an 86% mortality rate in cancer patients due to severe COVID-19. This contrasted with a 3.8% mortality rate in cancer patients with mild COVID-19, all unrelated to the infection. Our findings revealed elevated CXCL10 (IP-10) and reduced MIF levels in cancer patients with COVID-19, distinguished by disease severity. Compared with that in cancer patients with mild COVID-19, the level of CXCL10 in cancer patients with severe COVID-19 was further elevated. Additionally, cancer patients with COVID-19 presented reduced CD3+ T lymphocytes, expansion of CD4+CD25+FoxP3+ regulatory cells and CD56BRIGHT NK cells, a shift from effector memory to central memory T-cells, and increased numbers of exhausted (PD-1+) T lymphocytes. In conclusion, our data suggest a distinct immunological profile observed in cancer patients with COVID-19. Especially in severe cases, viral surge-related suppressor cells and proinflammatory cytokines were accompanied by a compensatory immunosuppressive state, with decreased effector function and increased exhaustion. This may negatively impact clinical outcomes and highlight potential implications for the management of cancer patients.
Ovarian cancer stem cells (OvCSCs) are one of the main factors contributing to post-treatment recurrence and the poor prognosis of patients with ovarian cancer. Therefore, the development of therapeutic strategies targeting OvCSCs is needed to improve patient survival. We previously reported the high expression of survivin/BIRC5 in OvCSCs and also that targeting pathways regulating survivin expression effectively suppressed OvCSC survival. In the present study, we tested a panel of agents consisting of FDA-approved drugs and compounds under clinical studies for their ability to suppress survivin expression in OvCSCs and identified clotrimazole (CTZ) as a potent candidate. The effects of CTZ on survivin expression were examined by RT-PCR and Western blot analyses. The effects of CTZ alone or in combination with anticancer agents on OvCSCs were evaluated using WST-8, PI uptake, and colony formation assays. CTZ preferentially impaired OvCSC survival by suppressing the c-myc-survivin axis without affecting normal fibroblasts and enhanced the efficacy of platinum- and taxane-based chemotherapeutic agents. These results suggest that CTZ suppresses survivin expression in OvCSCs and enhances the effects of conventional ovarian cancer chemotherapeutic agents, supporting further investigations of this approach as a potential strategy for ovarian cancer treatment.
Due to shared ß-hexosamindase A deficiencies and significant clinical overlap, the predominating subtypes of late-onset GM2 gangliosidosis, late-onset Tay-Sachs (LOTS) and late-onset Sandhoff disease (LOSD) have been considered essentially indistinguishable. However, growing evidence supports several distinctions between the two entities. We highlight these distinctions through the cross-sectional evaluation of 27 (21 LOTS and 6 LOSD) late-onset GM2 gangliosidosis participants. Study protocol included physical examinations, assessments of gait, balance, muscle strength, ataxia, and nerve conduction velocities, and brain magnetic resonance imaging. Lower limb weakness and later development of upper limb weakness was highly prevalent in both diseases. Accompanying gait disturbances, balance issues, and dysmetria were also prevalent in both cohorts. Strength evaluations showed weakness in both the LOTS and LOSD cohorts compared to controls primarily impacting extensor muscles. In contrast, BARS scores for dysarthria and oculomotor dysfunction were present and heterogenous in LOTS participants and absent in LOSD participants. Twenty-four percent of LOTS participants and none of the LOSD participants had a history of neuropsychiatric symptoms. Cerebellar volume including lobules V and VI were lower in LOTS compared to LOSD and normative data. However, severe length-dependent sensory neuropathy was present in all LOSD participants but not in LOTS participants. The finding of lower cerebellar volume in LOTS suggests the distinctive features of the LOTS phenotype are related to cerebellar dysfunction. However, the cause of the phenotypic differences between LOTS and LOSD remains a mystery, and the molecular and biochemical basis for the dichotomy requires further investigation.
Congenital disorders of glycosylation (CDG) are a group of inherited metabolic diseases rapidly growing due to the discovery of new subtypes. As with many genetic conditions, their diagnosis can be challenging, impairing proper patient care and causing additional suffering to patients and their families. We have developed an N-glycomics strategy that can provide insightful information towards diagnosing CDG type II (CDG-II). N-glycans released from the plasma of healthy individuals were labeled with deuterated iodomethane, mixed with samples from known or suspected CDG-II individuals, which were derivatized with standard iodomethane, and analyzed by liquid chromatography-mass spectrometry. After identification, relative quantification of 65 glycans was performed, revealing considerable alterations in the N-glycome of several patients. Notably, reduced fucosylation was observed in patients with FUT8-CDG and SLC35C1-CDG. Additionally, individuals with mutations in the MAN1B1 gene exhibited increased amounts of hybrid and oligomannosidic structures, whereas patients with the Golgi homeostasis disorders COG1-CDG and ATP6V0A2-CDG presented marked increases in hypogalactosylated and hyposialylated structures. Multivariate statistical analysis indicated two undiagnosed patients with alterations similar to ATP6V0A2-CDG patients and another two with a profile similar to MAN1B1-CDG patients. Genetic sequencing (targeted gene panel or whole exome sequencing) of these undiagnosed patients revealed variants in the corresponding genes, confirming the diagnosis obtained from the N-glycomics analysis. Our results demonstrate how the analysis of total plasma N-glycans can be used to identify metabolic disorders and diagnose conditions based on their molecular effects on the glycoproteome.
Digital immunoassay allows for the detection of proteins at fg/mL levels by leveraging the principles of Poisson distribution. Nevertheless, the inherent Poisson noise restricts digital sensitivity to targets exceeding 100 molecules, rendering standard digital assays incapable of detecting targets below this threshold. To overcome this constraint, we introduce an integrated CRISPR/Cas12a-digital immunoassay technology. In this system, the CRISPR/Cas12a machinery first acts as a molecular amplifier, converting each target protein into multiple enzymatic reporters. These enzyme molecules are then individually quantified via a digitized readout system, enabling ultrasensitive protein detection. By harnessing the catalytic amplification of CRISPR/Cas12a, the method achieves detection of proteins at copy numbers below 100, effectively surpassing the conventional sensitivity barrier of digital immunoassays. We validated this approach through highly selective and accurate detection of CD44 protein. A linear response was observed across a concentration range of 0.05 to 5 fg/mL, conforming to the calibration model: P(X > 0) = 0.1191c + 0.0036. The limit of detection was determined to be 0.018 fg/mL, equivalent to approximately 36 molecules of CD44. The method was further applied to quantify CD44 in plasma samples from colorectal cancer patients, demonstrating its strong potential for clinical use in early cancer diagnosis and treatment monitoring.
This systematic literature review (SLR) aims to update the evidence regarding therapeutic strategies in difficult-to-treat rheumatoid arthritis (D2T RA), building on the previous SLR informing the European Alliance of Associations for Rheumatology (EULAR) points to consider for the management of D2T RA. Three research questions addressed efficacy or safety of treatments in patients with RA with (1) active disease and limited treatment options; (2) active disease with ≥2 prior biologic/targeted synthetic disease-modifying antirheumatic drugs (b/tsDMARDs) and (3) poor health-related quality of life and low objective disease activity (non-pharmacological interventions). MEDLINE, Embase and Cochrane Library were searched until July 2025. Meta-analysis and meta-regression were conducted. We screened 8589 records and included 131 studies. For research question (RQ)1, evidence on DMARD efficacy and safety was synthesised across a wide spectrum of comorbidities including obesity, cardiovascular disease, history of malignancy and respiratory comorbidities. For RQ2, all b/tsDMARDs except otilimab demonstrated better efficacy than placebo (mostly high risk of bias). Meta-regression showed efficacy was maintained for Janus kinase inhibitors (JAKi) despite increasing number of prior bDMARD failures (1 to ≥3). Safety results confirmed the increased occurrence of infection and malignancy with JAKi. For RQ3, limited evidence suggested orthopaedic surgical intervention as a potential non-pharmacological option in patients with D2T RA. This SLR summarised evidence supporting DMARD efficacy/safety across multiple comorbidities. In patients with active RA with ≥2 prior bDMARDs, JAKi offer substantial clinical benefits but require careful risk stratification given cardiovascular and malignancy risks. Furthermore, standardised reporting and dedicated studies on non-pharmacological interventions are urgently needed. CRD42024593584.
Cervical cancer is the second most prevalent cancer among women in developing nations, including India. This study aims to assess the knowledge, attitude, and practice (KAP)  regarding cervical cancer screening, risk factors, and prevention strategies, along with the  barriers to participation in screening activities, among married tribal women in  Sirohi district, Rajasthan, to identify areas for improvement in this essential aspect of women's health. A cross-sectional, community-based study was conducted among 170 married tribal women recruited from the Tribal Sub-Plan (TSP) zone in Sirohi district, Rajasthan. A  standardised, fixed-response questionnaire was used to obtain data on participants' KAP regarding cervical cancer screening. The mean age of the study population was 32.81 ± 10.5 years. Out of 170, 28 (16.5%) women had heard of cervical cancer, and 20 (11.8%) women were aware of cervical cancer screening. Only 19.4% of women agreed to undergo screening for cervical cancer. A significant proportion, 86.5% of participants, had poor knowledge, 84.1% had a negative attitude, and only 0.6% demonstrated good practices regarding cervical cancer screening. Knowledge scores were significantly associated with age group, education, socioeconomic class, and number of children (p < 0.05). Attitude scores were significantly associated with age, contraceptive method used, and tobacco use. Practice scores were significantly associated with education and menstrual cycle characteristics (p < 0.05). Identified barriers to screening included lack of awareness, financial constraints, anxiety about vaginal examinations, feelings of embarrassment, and communication difficulties. The study findings indicate a critical deficiency in knowledge, attitude, and practice as well as low willingness to participate in cervical cancer screening among tribal women. Educational interventions  through culturally sensitive awareness programs should be conducted in the local tribal language to enhance preventive healthcare in the tribal population.
Maternal diabetes during pregnancy increases the risk of metabolic and cardiac disorders in offspring. Nevertheless, the mechanism by which intrauterine hyperglycemia affects neonatal cardiac remodeling remains uncertain. This study aims to characterize the prenatal environment in the context of gestational diabetes mellitus and to identify the corresponding fetal changes. Using an intrauterine hyperglycemia rodent model, we observe cardiac remodeling and inflammatory responses in offspring hearts. Moreover, the O-GlcNAcylation levels are increased in neonatal hearts exposed to gestational diabetes. Further mechanistic investigations, supported by RNA sequencing and mitochondrial functional analyses, reveal that gestational diabetes triggers O-GlcNAcylation-dependent activation of CaMKIIδ during the embryonic stage. This activation leads to the release of mitochondrial DNA (mtDNA) from the mitochondrial matrix into the cytosol, which subsequently activates STING signaling and triggers an inflammatory response in neonatal cardiomyocytes. Pharmacological or genetic inhibition of O-GlcNAcylation attenuates mtDNA-induced myocardial inflammation and improves cardiac function in neonatal offspring subjected to intrauterine hyperglycemia. Together, these findings identify a previously unrecognized CaMKIIδ/mtDNA/STING axis in which CaMKIIδ O-GlcNAcylation leads to mtDNA-dependent activation of cardiac remodeling, suggesting that plasma mtDNA levels could serve as a predictive biomarker in neonatal cardiac inflammatory injury.
Glucagon-like peptide-1 receptor agonists (GLP-1RAs), including semaglutide, produce robust and sustained weight loss, yet the central mechanisms supporting their long-term efficacy remain incompletely understood. Agouti-related peptide (AgRP) neurons of the arcuate nucleus are classically activated by negative energy balance and promote feeding and energy conservation. Based on this framework, GLP-1RAs have generally been expected to suppress or bypass AgRP neuron activity. Here, we report that AgRP neuron activation is required for the full weight-lowering effects of GLP-1RAs in female mice. Across complementary AgRP loss-of-function models, disruption of AgRP circuit integrity reduced the full weight-lowering effects of GLP-1RAs. This requirement varies with sex, diet, and mode of AgRP disruption. We found that GLP-1RA treatment is associated with increased markers of neuronal activation, mitochondrial engagement, and synaptic remodeling in AgRP neurons. We further identify a glucocorticoid-to-AgRP signaling axis as an important pathway mediating this functional recruitment of AgRP neurons. Together, these findings reveal that contrary to prevailing assumptions, GLP-1RA engages AgRP neurons to sustain weight loss, highlighting an unexpected role for these neurons in coordinating adaptive metabolic responses to pharmacologically induced negative energy balance.