Patient-derived organoid biobank identifies epigenetic dysregulation of intestinal epithelial MHC-I as a novel mechanism in severe Crohn's Disease.

OBJECTIVE: Epigenetic mechanisms, including DNA methylation (DNAm), have been proposed to play a key role in Crohn's disease (CD) pathogenesis. However, the specific cell types and pathways affected as well as their potential impact on disease phenotype and outcome remain unknown. We set out to investigate the role of intestinal epithelial DNAm in CD pathogenesis. DESIGN: We generated 312 intestinal epithelial organoids (IEOs) from mucosal biopsies of 168 patients with CD (n=72), UC (n=23) and healthy controls (n=73). We performed genome-wide molecular profiling including DNAm, bulk as well as single-cell RNA sequencing. Organoids were subjected to gene editing and the functional consequences of DNAm changes evaluated using an organoid-lymphocyte coculture and a nucleotide-binding oligomerisation domain, leucine-rich repeat and CARD domain containing 5 (NLRC5) dextran sulphate sodium (DSS) colitis knock-out mouse model. RESULTS: We identified highly stable, CD-associated loss of DNAm at major histocompatibility complex (MHC) class 1 loci including NLRC5 and cognate gene upregulation. Single-cell RNA sequencing of primary mucosal tissue and IEOs confirmed the role of NLRC5 as transcriptional transactivator in the intestinal epithelium. Increased mucosal MHC-I and NLRC5 expression in adult and paediatric patients with CD was validated in additional cohorts and the functional role of MHC-I highlighted by demonstrating a relative protection from DSS-mediated mucosal inflammation in NLRC5-deficient mice. MHC-I DNAm in IEOs showed a significant correlation with CD disease phenotype and outcomes. Application of machine learning approaches enabled the development of a disease prognostic epigenetic molecular signature. CONCLUSIONS: Our study has identified epigenetically regulated intestinal epithelial MHC-I as a novel mechanism in CD pathogenesis.

Surgical Management of Submucous Cleft Palate by Radical Muscle Dissection Veloplasty: Speech Outcomes in Patients with 22q11.2 Deletion Syndrome.

OBJECTIVE: To evaluate the benefit of anatomical muscle dissection repair for velopharyngeal insufficiency (VPI) in patients with submucous cleft palate (SMCP) with 22q11.2 deletion syndrome. DESIGN: Retrospective blinded randomised analysis of a surgeon's management over 10 years. SETTING: The study was performed at a specialised Paediatric hospital in the United Kingdom. PATIENTS: Children with SMCP and 22q11.2 deletion syndrome. INTERVENTIONS: All participants underwent radical muscle dissection veloplasty. OUTCOMES MEASURED: Pre- and post- operative measurements included severity of anatomical defect, speech samples and lateral images which were digitised, randomised then externally and blindly analysed using validated techniques. Stata software was used to perform statistical analysis. RESULTS: 57 children with 22q11.2 deletion syndrome were included in this analysis. Intra-operatively, the majority of cases were identified as SMCP Grade I anomalies. Post-operatively, a statistically significant improvement in hypernasality, resting palate length, palate length at maximum closure, palate excursion and gap size at maximum closure was observed. Secondary surgery was performed for 59% of patients by ten years. CONCLUSION: Muscle dissection repair improves hypernasality, palate closure function and the closure gap in patients with 22q11.2 deletion syndrome. Although over 50% of patients may require further surgery, muscle dissection repair should be a first step due to its utility at a younger age, when invasive investigations are impossible, its lower morbidity, speech and language benefits or altering the plans for less obstructive secondary surgery when it lead to reduced velo-pharyngeal gap and improved palate mobility even when adequate velo-pharyngeal closure was not achieved.

Chronic activation of Toll-like receptor 2 induces an ichthyotic skin phenotype.

BACKGROUND: Ichthyosis defines a group of chronic conditions that manifest phenotypically as a thick layer of scales, often affecting the entire skin. While the gene mutations that lead to ichthyosis are well documented, the actual signalling mechanisms that lead to scaling are poorly characterized; however, recent publications suggest that common mechanisms are active in ichthyotic tissue and in analogous models of ichthyosis. OBJECTIVES: To determine common mechanisms of hyperkeratosis that may be easily targeted with small-molecule inhibitors. METHODS: We combined gene expression analysis of gene-specific short hairpin RNA (shRNA) knockdowns in rat epidermal keratinocytes (REKs) of two genes mutated in autosomal recessive congenital ichthyosis (ARCI), Tgm1 and Alox12b, and proteomic analysis of skin scale from patients with ARCI, as well as RNA sequencing data from rat epidermal keratinocytes treated with the Toll-like receptor 2 (TLR2) agonist Pam3CSK4. RESULTS: We identified common activation of the TLR2 pathway. Exogenous TLR2 activation led to increased expression of important cornified envelope genes and, in organotypic culture, caused hyperkeratosis. Conversely, blockade of TLR2 signalling in keratinocytes from patients with ichthyosis and our shRNA models reduced the expression of keratin 1, a structural protein overexpressed in ichthyosis scale. A time course of TLR2 activation in REKs revealed that although there was rapid initial activation of innate immune pathways, this was rapidly superseded by widespread upregulation of epidermal differentiation-related proteins. Both nuclear factor kappa B phosphorylation and GATA3 upregulation was associated with this switch, and GATA3 overexpression was sufficient to increase keratin 1 expression. CONCLUSIONS: Taken together, these data define a dual role for TLR2 activation during epidermal barrier repair that may be a useful therapeutic modality in treating diseases of epidermal barrier dysfunction.

Phosphorylation of NTRK1 at Y674/Y675 induced by TP53-dependent repression of PTPN6 expression: a potential novel prognostic marker for breast cancer.

We have identified a ligand-independent mechanism whereby the tumor suppressor, TP53, induces nerve growth factor receptor, NTRK1, phosphorylation at Y674/Y675 (NTRK1-pY674/pY675), via the repression of the NTRK1-phosphatase, PTPN6. This results in suppression of breast cancer cell proliferation. In this investigation, we aimed to establish whether perturbation of the wild-type TP53-NTRK1-pY674/pY675-PTPN6 pathway has an impact on disease-free survival of breast cancer patients without neo-adjuvant treatment. A total of 308 tumor samples were stained for NTRK1, NTRK1-pY674/pY675, PTPN6, and TP53 expression. Association between expression levels and disease-free survival was determined by the univariate/multivariate and Kaplan-Meir methods of analysis. DNA from tumors was sequenced to identify mutant or wild-type TP53. Tumors expressing NTRK1-pY674/pY675 but with undetectable or low levels of PTPN6 and TP53 were associated with prolonged 5, 10, and 15 years' disease-free survival by 48%, 36%, and 37%, respectively, in the multivariate analysis (P<0.05). A similar result was observed in tumors expressing wild-type TP53, NTRK1-pY674/pY675, and low or undetectable levels of PTPN6. Given that estrogen receptor-positive breast cancers encode wild-type TP53, we analyzed this expression pattern in these tumors. Multivariate analysis showed that it was significantly and independently predictive of prolonged survival by 66%, 70%, and 84%, respectively, (P<0.05). The Kaplan-Meir method demonstrated that NTRK1-pY674/pY675 together with undetectable or low levels of PTPN6 correlated with 59% probability of disease-free survival (median survival 15 years), compared with 7% probability of disease-free survival (median survival 4.5 years) when absent. In luminal A tumors, the presence of this pattern was estimated to have a 61% probability of disease-free survival (median survival 15 years), compared with 6% probability of disease-free survival (median survival 3 years) when it was absent. These results strongly suggest that expression of NTRK1-pY674/pY675 together with wild-type TP53 and low levels of PTPN6 expression are predictors of improved disease-free survival and that they could be useful biomarkers to predict clinical outcome.

Debulking corneal biopsy with tectonic amniotic membrane transplantation in refractory clinically presumed fungal keratitis.

The treatment of fungal keratitis (FK) is challenging due to the subacute indolent course, and initial misdiagnosis. In this retrospective case series, we highlight both the diagnostic and therapeutic roles of corneal biopsy together with amniotic membrane transplantation (AMT) in patients with refractory clinically presumed FK. Debulking biopsy and tectonic AMT were performed during the initial presentation. Biopsy specimens were sent for KOH smears and cultures. After KOH smears confirmed the presence of fungal elements, topical voriconazole 1% was prescribed for the first 72 h then tailored according to the clinical response and the culture results. The outcome measures were complete resolution of infection and restoration of corneal integrity. Cases associated with culture proven bacterial keratitis were excluded. Twelve cases were included in the study. KOH smears confirmed the presence of fungal growth in all specimens. Cultures grew Aspergillus in 6/12 cases, sensitive to voriconazole (5/6) and amphotericin (3/6); Fusarium (4/12), sensitive to both voriconazole and amphotericin; and no growth in 2/12 cases. Amphotericin 0.15% eye drops were added to the 7 cases with proven sensitivity and to the remaining 2 culture negative cases. Gradual resolution of infection was seen in all cases after 35.6 ± 7.8 days. In FK, a debulking biopsy simultaneously with AMT help decrease the microbial load, suppress the inflammatory process, support the corneal integrity, confirm the presence of fungal pathogen.

Pattern of eye trauma among pediatric ophthalmic patients in upper and lower Egypt: A prospective two-center medicolegal study.

Background: Ocular trauma is the leading cause of monocular blindness in children. This study aimed to identify and compare pediatric ocular injuries at two national university hospitals: Benha University Hospital (BUH) and South Valley University Hospital (SVUH) while evaluating the predictive utility of the Ocular Trauma Score (OTS) for post-traumatic permanent visual infirmity. Design and methods: This multicenter observational study enrolled children under 18 years at BUH and SVUH Ophthalmology Departments from May 31 to December 31, 2022. Comprehensive medical histories and detailed data on eye trauma were obtained, with each undergoing thorough eye examinations at the initial presentation and during the 6-month follow-up period. The final visual outcome was permanent visual infirmity that was defined as the Best Corrected Visual Acuity in the better eye worse than 3/60 or near vision worse than N6 according to the International Classification of Diseases 11. Results: The study included 120 cases evenly distributed between the two hospitals, with a mean age of 10.03 (±3.30) years, 74.17% male and 51.67% urban residents. Blunt, sharp, and foreign objects were the most common causes (39.17%, 33.33%, and 11.67%, respectively). Closed-globe injuries prevailed at BUH (83.33%) than SVUH (40.0%). Most injuries were accidental and outdoors, with an average interval of 9.93 (±23.57) hours to seek medical care. Lack of education, open-globe injuries, poor initial visual acuity, longer intervals from admission to intervention, and lower OTS were associated with visual impairment.

The protein phosphatase 2A regulatory subunit Ppp2r2a is required for Connexin-43 dephosphorlyation during epidermal barrier acquisition.

Epidermal barrier acquisition during late mammalian development is a prerequisite for terrestrial existence. Over a 24-h period, the epidermis goes from being a barrier-deficient, dye permeable epithelium to a barrier-competent epithelium. We have previously shown that Akt signalling is necessary for barrier acquisition in the mouse and that the protein phosphatase 2A regulatory subunit Ppp2r2a causes barrier acquisition by dephosphorylation of cJun. Here, we demonstrate that there is transient interaction between the gap junction protein Connexin 43 (Cx43) and Zonula occludins-1 (Zo-1) during epidermal barrier acquisition. Ppp2r2a knockdown prevented plasma membrane co-localisation and interaction between the two proteins. Ppp2r2a knockdown also increased phosphorylation at Serine 368 of Connexin 43. Cx43 phosphorlyation at Serine368 occurred just prior to the interaction between Connexin 43 and Zo-1. We therefore propose a model in which Ppp2r2a is required both for the initial interaction between Zo-1 and Cx43 and the consequent dephosphorylation of Connexin 43, preventing interaction of Zo-1 and allowing Zo-1 to initiate tight junction formation and barrier acquisition.

Disseminating cells in human oral tumours possess an EMT cancer stem cell marker profile that is predictive of metastasis in image-based machine learning.

Cancer stem cells (CSCs) undergo epithelial-mesenchymal transition (EMT) to drive metastatic dissemination in experimental cancer models. However, tumour cells undergoing EMT have not been observed disseminating into the tissue surrounding human tumour specimens, leaving the relevance to human cancer uncertain. We have previously identified both EpCAM and CD24 as CSC markers that, alongside the mesenchymal marker Vimentin, identify EMT CSCs in human oral cancer cell lines. This afforded the opportunity to investigate whether the combination of these three markers can identify disseminating EMT CSCs in actual human tumours. Examining disseminating tumour cells in over 12,000 imaging fields from 74 human oral tumours, we see a significant enrichment of EpCAM, CD24 and Vimentin co-stained cells disseminating beyond the tumour body in metastatic specimens. Through training an artificial neural network, these predict metastasis with high accuracy (cross-validated accuracy of 87-89%). In this study, we have observed single disseminating EMT CSCs in human oral cancer specimens, and these are highly predictive of metastatic disease.

When oral cancers metastasise ­ that is, when tumour cells invade other parts of the body ­ they typically do so by first colonizing the lymph nodes present in the neck. As this event significantly reduces chances of survival, oral cancer patients often have their neck lymph nodes removed to prevent the spread of the disease. However, this surgery carries risks and leads to longer hospital stays, stressing the need for better ways to predict which oral tumours will metastasise. Evidence from lab-grown cells and mice studies suggest that, in oral cancer, metastasis occurs when some cells in the original tumour go through a process called the epithelial-mesenchymal transition (EMT for short). This transformation allows the cells to detach from the tumour and become invasive. However, it has so far been difficult to observe this process in actual human tumours; this is partly because cells undergoing EMT stop producing the proteins that scientists rely on to distinguish cancer and healthy cells. To address this knowledge gap, Youssef et al. focused on three proteins: two tumour markers, EpCAM and CD24; and Vimentin, which is produced in greater quantities in the invasive mesenchymal state. Previous work had shown that a specific population of oral tumour cells can continue to express all three proteins even when adopting a mesenchymal identity through EMT. Based on this knowledge, Youssef et al. hypothesised that tracking Vimentin, EpCAM and CD24 using fluorescence microscopy would allow them to identify metastasising cells in human samples. An analysis of over 12,000 images from 74 tumours obtained from surgeries revealed that, in the metastatic samples, the cells detaching from primary tumours were more likely to express these three proteins. Finally, Youssef et al. used these images to train a machine learning algorithm. When applied to data from new oral cancer patients, the programme was able to predict whether their tumours were likely to spread with 89% accuracy. If confirmed by further work, and in particular on larger samples, these findings could in the future help clinicians decide which patients with oral cancer would benefit the most from surgery to remove neck lymph nodes.

dialogi: Utilising NLP With Chemical and Disease Similarities to Drive the Identification of Drug-Induced Liver Injury Literature.

Drug-Induced Liver Injury (DILI), despite its low occurrence rate, can cause severe side effects or even lead to death. Thus, it is one of the leading causes for terminating the development of new, and restricting the use of already-circulating, drugs. Moreover, its multifactorial nature, combined with a clinical presentation that often mimics other liver diseases, complicate the identification of DILI-related (or "positive") literature, which remains the main medium for sourcing results from the clinical practice and experimental studies. This work-contributing to the "Literature AI for DILI Challenge" of the Critical Assessment of Massive Data Analysis (CAMDA) 2021- presents an automated pipeline for distinguishing between DILI-positive and negative publications. We used Natural Language Processing (NLP) to filter out the uninformative parts of a text, and identify and extract mentions of chemicals and diseases. We combined that information with small-molecule and disease embeddings, which are capable of capturing chemical and disease similarities, to improve classification performance. The former were directly sourced from the Chemical Checker (CC). For the latter, we collected data that encode different aspects of disease similarity from the National Library of Medicine's (NLM) Medical Subject Headings (MeSH) thesaurus and the Comparative Toxicogenomics Database (CTD). Following a similar procedure as the one used in the CC, vector representations for diseases were learnt and evaluated. Two Neural Network (NN) classifiers were developed: a baseline model that accepts texts as input and an augmented, extended, model that also utilises chemical and disease embeddings. We trained, validated, and tested the classifiers through a Nested Cross-Validation (NCV) scheme with 10 outer and 5 inner folds. During this, the baseline and extended models performed virtually identically, with F1-scores of 95.04 ± 0.61% and 94.80 ± 0.41%, respectively. Upon validation on an external, withheld, dataset that is meant to assess classifier generalisability, the extended model achieved an F1-score of 91.14 ± 1.62%, outperforming its baseline counterpart which received a lower score of 88.30 ± 2.44%. We make further comparisons between the classifiers and discuss future improvements and directions, including utilising chemical and disease embeddings for visualisation and exploratory analysis of the DILI-positive literature.

DILI C : An AI-Based Classifier to Search for Drug-Induced Liver Injury Literature.

Drug-induced liver injury (DILI) is a class of adverse drug reactions (ADR) that causes problems in both clinical and research settings. It is the most frequent cause of acute liver failure in the majority of Western countries and is a major cause of attrition of novel drug candidates. Manual trawling of the literature is the main route of deriving information on DILI from research studies. This makes it an inefficient process prone to human error. Therefore, an automatized AI model capable of retrieving DILI-related articles from the huge ocean of literature could be invaluable for the drug discovery community. In this study, we built an artificial intelligence (AI) model combining the power of natural language processing (NLP) and machine learning (ML) to address this problem. This model uses NLP to filter out meaningless text (e.g., stop words) and uses customized functions to extract relevant keywords such as singleton, pair, and triplet. These keywords are processed by an apriori pattern mining algorithm to extract relevant patterns which are used to estimate initial weightings for a ML classifier. Along with pattern importance and frequency, an FDA-approved drug list mentioning DILI adds extra confidence in classification. The combined power of these methods builds a DILI classifier (DILI C ), with 94.91% cross-validation and 94.14% external validation accuracy. To make DILI C as accessible as possible, including to researchers without coding experience, an R Shiny app capable of classifying single or multiple entries for DILI is developed to enhance ease of user experience and made available at https://researchmind.co.uk/diliclassifier/. Additionally, a GitHub link (https://github.com/sanjaysinghrathi/DILI-Classifier) for app source code and ISMB extended video talk (https://www.youtube.com/watch?v=j305yIVi_f8) are available as supplementary materials.

Case Series of Perforated Keratomycosis after Laser-Assisted In Situ Keratomileusis.

BACKGROUND: Fungal keratitis is an extremely rare complication of laser vision correction resulting in poor visual outcomes. Amniotic membrane transplantation should be kept in mind in eyes with corneal perforation prior to penetrating keratoplasty. AIM: To assess the outcomes of multilayered fresh amniotic membrane transplantation (MLF-AMT) in patients with severe keratomycosis after laser-assisted in situ keratomileusis (LASIK). Study design. Hospital-based prospective interventional case series. METHODS: Five eyes of 5 patients were included in the study. All cases underwent microbiological scrapings from residual bed and intrastromal injections of amphotericin (50 mcg/mL), with flap amputation if needed, followed by topical 5% natamycin and 0.15% amphotericin. MLF-AMT was performed after corneal perforation. Later, penetrating keratoplasty (PK) was performed when corneal opacity compromised visual acuity. The outcome measures were complete resolution of infection, corneal graft survival, and best-corrected visual acuity (BCVA). RESULTS: The mean age of patients was 22 ± 1.2 years with 4/5 (80%) were females. The mean interval between LASIK and symptom onset was 8.8 ± 1 day, and the mean interval between symptom onset and referral was 14 ± 1.4 days. Potassium hydroxide (KOH) smears showed filamentous fungi, and Sabouraud's medium grew Aspergillus in all cases. Melted flaps were amputated in 4 (80%) cases. MLF-AMT was performed in all cases due to corneal perforation after a mean time of 12.4 ± 1.2 days of antifungals. In all cases, complete resolution of infection was seen 26 ± 1.8 days after MLF-AMT, and optical PK was done at a mean of 2.4 months later. No postoperative complications after MLF-AMT or PK were observed, with a 0% incidence of corneal graft rejection, and a final BCVA ranged from 20/20 to 20/80 after a mean follow-up of 14 ± 1.1 months. CONCLUSION: MLF-AMT is a safe and valid option to manage corneal perforation during keratmycosis treatment to avoid emergency therapeutic keratoplasty.

The presence of Y674/Y675 phosphorylated NTRK1 via TP53 repression of PTPN6 expression as a potential prognostic marker in neuroblastoma.

The tumor suppressor TP53 promotes nerve growth factor receptor (NTRK1) -Y674/Y675 phosphorylation (NTRK1-pY674/pY675) via repression of the NTRK1 phosphatase PTPN6 in a ligand-independent manner, resulting in suppression of breast cancer cell proliferation. Moreover, NTRK1-pY674/pY675 together with low levels of PTPN6 and TP53 expression is associated with favorable disease-free survival of breast cancer patients. We determined whether in neuroblastoma this protein expression pattern impacts relapse-free survival (RFS). NTRK1-pY674/pY675, PTPN6, and TP53 expression was assessed in 98 neuroblastoma samples by immunohistochemistry. Association between expression levels and RFS was investigated by multivariate and Kaplan-Meier analysis. Mutant or wild-type TP53 was identified by sequencing tumor DNA. Tumors expressing NTRK1-pY674/pY675 and low or undetectable levels of PTPN6 and TP53 were significantly associated with 5-year RFS (P = .014) when the dataset was stratified by MYCN amplification, segmental chromosomal abnormalities and histology. Similar results were observed with tumors expressing wild-type TP53, NTRK1-pY674/pY675 and low or undetectable levels of PTPN6. Kaplan-Meier analysis demonstrated a significant correlation (P = .004), with a 50% probability of RFS (median survival 4.73 years) when present compared with 19.51% (median survival 11.63 months) when absent. Similar results were seen with non-amplified MYCN or unfavorable/undifferentiating samples and tumors from patients aged 18 months or less. Importantly, NTRK1-pY674/pY675 is an independent predictor of improved RFS. These results strongly suggest that NTRK1-pY674/pY675 together with wild-type TP53 and undetectable or low levels of PTPN6 expression is a potential biomarker of improved RFS of neuroblastoma patients. The predictive value of NTRK1-pY674/pY675 together with wild-type TP53 and low PTPN6 expression could contribute to neuroblastoma patient prognosis.

Ovine pulmonary adenocarcinoma: a large animal model for human lung cancer.

Lung cancer is the leading cause of cancer deaths worldwide. Recent progress in understanding the molecular pathogenesis of this disease has resulted in novel therapeutic strategies targeting specific groups of patients. Further studies are required to provide additional advances in diagnosis and treatment. Animal models are valuable tools for studying oncogenesis in lung cancer, particularly during the early stages of disease where tissues are rarely available from human cases. Mice have traditionally been used for studying lung cancer in vivo, and a variety of spontaneous and transgenic models are available. However, it is recognized that other species may also be informative for studies of cancer. Ovine pulmonary adenocarcinoma (OPA) is a naturally occurring lung cancer of sheep caused by retrovirus infection and has several features in common with adenocarcinoma of humans, including a similar histological appearance and activation of common cell signaling pathways. Additionally, the size and organization of human lungs are much closer to those of sheep lungs than to those of mice, which facilitates experimental approaches in sheep that are not available in mice. Thus OPA presents opportunities for studying lung tumor development that can complement conventional murine models. Here we describe the potential applications of OPA as a model for human lung adenocarcinoma with an emphasis on the various in vivo and in vitro experimental systems available.