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Targeted knockout, mutations, or knock-in of genomic DNA fragments in model organisms have been used widely for functional and cell-tracking studies. The desired genetic perturbation is often accomplished by recombination-based or CRISPR/Cas9-based genome engineering. For validating the intended genetic modification, a local region surrounding the targeted locus is typically examined based on enzymatic cleavage and consequent length patterns, e.g. in a Southern analysis. Despite its wide use, this approach is open to incomplete and ambiguous readouts. With decreasing costs of high-throughput sequencing, it is becoming feasible to consider a large-scale validation of a new strain after a targeted genetic perturbation. Here we describe a dataset of whole-genome sequences and the variant analysis results from four novel reporter mouse strains. This served to validate the strains and identified all the off-target effects on the genome, thereby increasing the genetic diversity of genomic sequences over those represented in the public databases for inbred mice., Competing Interests: Competing interests The authors declare no competing interests., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Sheet-like colloidal assemblies represent model systems to investigate the structure and properties of two-dimensional materials. Here, we report a simple yet versatile method for the preparation of colloidal monolayer sheet-like assemblies that affords control over the size, crystalline order, flexibility, and defect density. The protocol that we report relies on self-assembly of colloids as a sessile drop of dispersion is evaporated on an oil-covered substrate. In this case, the contact line continually moves as the drop shrinks. Polyethyleneimine polymer-covered micrometer-sized colloidal particles are transported to the air-water interface and assemble to form a monolayer sheet as the drop dries. Cross-linking the polymer renders the colloidal assembly permanent. Interestingly, monodisperse colloidal particles form disordered assemblies when dried from low concentration dispersions, while polycrystalline ordered assemblies form at higher concentrations. We demonstrate that increasing the cross-linker to polymer ratio decreases the flexibility of the assembly. Introduction of different-sized colloidal particles in a sheet leads to increased disorder. Removal of sacrificial particles from the sheet allowed the introduction of "holes" in the sheets. Thus, these colloidal sheets are models for probing the effects of disorder, doping, and vacancies in two-dimensional systems.

Limited information exists about the types of immune cells present in Kaposi sarcoma (KS) lesions, especially in KS in the gastrointestinal tract. Using previously reported RNA-sequencing results from Kaposi sarcoma lesions in skin and gastrointestinal tract with normal matched tissues from the same patients at the same time, we investigated changes in lymphocytes in these tissues. We employed a computational method that determines changes in cell type distributions using KS lesion transcriptome data compared to a reference set of RNA expression patterns of purified immune cells. Since secreted cytokines and chemokines from KSHV-infected cells may influence the microenvironment of Kaposi sarcoma lesions, we performed cytokine profiling of conditioned media from KSHV-infected primary human dermal lymphatic endothelial cells. We also measured how this conditioned media altered the differentiation of macrophages in cell culture assays. These results suggested that factors in conditioned media from KSHV-infected endothelial cells promoted differentiation of a promonocytic cell line to proinflammatory macrophages., Competing Interests: The authors have no conflicts of interest to disclose., (2024, National Center for Global Health and Medicine.)

A novel series of 20 compounds containing 4-aminopyrazolo[3,4- d ]pyrimidine core were synthesized, characterized and their chemical structures confirmed using spectroscopic techniques such as 1 H NMR, 13 C NMR, IR, and HRMS. The compound's growth inhibitory activities were evaluated against 60 human tumor cell lines from nine panels: leukemia, non-small cell lung cancer (NSCLC), colon, central nervous system (CNS), melanoma, ovarian, renal, prostate, and breast cancer. Among all the compounds, 11, 12c, 12d, 12f, and 12j are active against different cancer cell lines. Between all the cell lines, compounds 12c, 12d, 12f, 12j, and 11 showed good inhibitory activity against renal cancer cell lines. From the five-dose study, based on IC 50 values, the order of activity of compounds against renal cancer cell lines was found to be 12c > 12f > 12c > 12j > 11 with 12c being the most potent, was better than sunitinib and sorafenib. Having been recognized as initial hits, these substances need additional pharmacological investigation., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Background Burn injuries pose a significant global public health challenge, causing substantial morbidity and mortality, particularly in low- and middle-income countries. Timely and effective wound coverage is critical in treating severe burns to prevent infection, reduce pain, and promote healing. Cadaveric skin grafts (allografts) have become essential in treating extensive burn wounds, serving as temporary biological dressings to prepare the wound bed for autografting. This study aims to comprehensively analyze the process of cadaveric skin graft harvest and usage in a tertiary care setting. It seeks to evaluate the procedures, challenges, and outcomes associated with cadaveric skin grafts, contributing to optimizing burn care practices and improving patient outcomes. Methods  This observational study was conducted at a tertiary care hospital and burns center with a skin bank, involving 44 cadaveric skin graft harvests and 27 applications between July 1, 2011, and June 30, 2013. The study focused on prospective donors and recipients needing cadaveric skin grafts. Inclusion criteria for donors included consent from the next of kin and the absence of infections or septicemia, while exclusion criteria included prolonged post-mortem intervals and medico-legal cases. The procedures adhered to the Euro Skin Bank protocols, encompassing retrieval, processing, storage, and usage. Data were analyzed using Epi-Info version 7.2.1 software, employing descriptive statistics for categorical variables. Ethical clearance was obtained from the university ethical committee, with mandatory written informed consent for skin donation. Results Out of 519 deaths in the tertiary care hospital, significant barriers to skin donation included septicemia, skin changes, late reporting, young age, medico-legal issues, and positive viral markers. Notably, 114 (21.97%) of next of kin refused consent. Cadaveric skin was harvested in five (11.36%) cases, with potential donors identified in 78 (15.02%) of deaths. Donors were predominantly older males, with efficient procurement processes ensuring timely harvests. The tertiary burns center facilitated 39 (88.63%) cases of cadaveric skin harvests with a skin bank, either at the donor's home or other hospitals notified to the burns center. Cadaveric skin grafts were applied in 27 cases, primarily for burns, with high graft uptake observed over 10 days. Non-healing ulcers showed 100% graft uptake. The survival rate among burn patients was 20 (74%), with deaths mainly due to sepsis and multi-organ failure. Significant barriers to obtaining consent included a lack of awareness, superstitions, social stigmas, and religious objections. Conclusion  The study highlights the critical role of cadaveric skin in managing extensive wounds, particularly burns. Despite challenges in obtaining consent and limited donor availability, cadaveric skin grafts effectively prepared wound beds for autografting, contributing to improved patient outcomes. Increasing community awareness and addressing superstitions and social stigmas are essential for improving donation rates., Competing Interests: Human subjects: Consent was obtained or waived by all participants in this study. Ethics Committee of Maharashtra University of Health Sciences issued approval MUHS/PG-T/E1/T-Ap/1620/2012. Indian Naval Hospital Ship, Asvini, is a naval institute with postgraduate seats in general surgery affiliated with Maharashtra University of Health Sciences Nashik. The Ethics Committee of the university grants approval to all theses conducted at affiliated institutes. Animal subjects: All authors have confirmed that this study did not involve animal subjects or tissue. Conflicts of interest: In compliance with the ICMJE uniform disclosure form, all authors declare the following: Payment/services info: All authors have declared that no financial support was received from any organization for the submitted work. Financial relationships: All authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work. Other relationships: All authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work., (Copyright © 2024, B et al.)

Giant basal cell carcinoma (GBCC) is a rare and more aggressive variant of basal cell carcinoma. We present a case of GBCC with an overview of the challenges it presents.A man in his 60s presented to the tertiary care unit with a history of an ulcer over the posterior aspect of his left heel for the past 1 year. Examination revealed an ulceroproliferative lesion of 10×8 cm on the posterior aspect of the left heel and lower Tendo-Achilles region. A wedge biopsy of the lesion was performed twice, which demonstrated basal cell carcinoma. The patient underwent excision of the lesion with 10 mm margins. A split-thickness skin graft was placed and secured over the resultant wound with the application of a negative pressure wound dressing.The correlation between tumour size and tumour behaviour is examined. Additionally, the significance of tumour location, width of margins, incidence recurrence or metastasis is also studied., Competing Interests: Competing interests: None declared., (© BMJ Publishing Group Limited 2024. No commercial re-use. See rights and permissions. Published by BMJ.)

Recent FDA modernization act 2.0 has led to increasing industrial R&D investment in advanced in vitro 3D models such as organoids, spheroids, organ-on-chips, 3D bioprinting, and in silico approaches. Liver-related advanced in vitro models remain the prime area of interest, as liver plays a central role in drug clearance of compounds. Growing evidence indicates the importance of recapitulating the overall liver microenvironment to enhance hepatocyte maturity and culture longevity using liver-on-chips (LoC) in vitro . Hence, pharmaceutical industries have started exploring LoC assays in the two of the most challenging areas: accurate in vitro - in vivo extrapolation (IVIVE) of hepatic drug clearance and drug-induced liver injury. We examine the joint efforts of commercial chip manufacturers and pharmaceutical companies to present an up-to-date overview of the adoption of LoC technology in the drug discovery. Further, several roadblocks are identified to the rapid adoption of LoC assays in the current drug development framework. Finally, we discuss some of the underexplored application areas of LoC models, where conventional 2D hepatic models are deemed unsuitable. These include clearance prediction of metabolically stable compounds, immune-mediated drug-induced liver injury (DILI) predictions, bioavailability prediction with gut-liver systems, hepatic clearance prediction of drugs given during pregnancy, and dose adjustment studies in disease conditions. We conclude the review by discussing the importance of PBPK modeling with LoC, digital twins, and AI/ML integration with LoC., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)

Background: Cancer is one of the most common reasons for mortality in the world. A continuous effort to develop effective anti-cancer drugs with minimum side effects has become necessary. The use of small-molecule drugs has revolutionized cancer research by inhibiting cancer cell survival and proliferation. Quinazolines are a class of bioactive heterocyclic compounds with active pharmacophores in several anti-cancer drugs. Such small molecule inhibitors obstruct the significant signals responsible for cancer cell development, thus blocking these cell signals to prevent cancer development and spread., Objective: In the current study, novel quinazoline derivatives structurally similar to erlotinib were synthesized and explored as novel anti-cancer agents., Methods: All the synthesized molecules were confirmed by spectroscopic techniques like 1 H NMR, 13 C NMR, and ESI-MS. Various techniques were applied to study the protein-drug interaction, DFT analysis, Hirshfeld surface, and target prediction. The molecules were screened in vitro for their anti-cancer properties against 60 human tumor cell lines. The growth inhibitory properties of a few compounds were studied against the MCF7 breast cancer cell line., Results: The activity of compounds 9f, 9o, and 9s were found to be active. However, compound 9f is more active when compared with other compounds., Conclusion: Some synthesized compounds were active against different cancer cell lines. The in-vitro study results were found to be in agreement with the predictions from in-silico data. The selected molecules were further subjected to get the possible mechanism of action against different cancer cells., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Venetoclax is a potent BCL-2 inhibitor that is used for the treatment of several blood cancers. During the oxidative stress degradation of venetoclax, we observed the formation of two potential impurities at levels of about 8-10%, which have similar molecular weights. The two impurities were isolated and identified as 4-(3-((1 H -pyrrolo[2,3- b ]pyridin-5-yl)oxy)-4-(((3-nitro-4-(((tetrahydro-2 H -pyran-4-yl)methyl)amino)phenyl)sulfonyl)carbamoyl)phenyl)-1-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl)methyl)piperazine 1-oxide (venetoclax N -oxide, VNO ) and 2-((1 H -pyrrolo[2,3- b ]pyridin-5-yl)oxy)-4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl)methoxy)piperazin-1-yl)- N -((3-nitro-4-(((tetrahydro-2 H -pyran-4-yl)methyl)amino)phenyl)sulfonyl)benzamide (venetoclax hydroxylamine impurity, VHA ). To confirm these two compounds, we have synthesized each impurity individually and analyzed it by high-performance liquid chromatography, mass spectrometry, 1 H NMR, 13 C NMR, and 2D NMR. VNO was synthesized by the oxidation of venetoclax using m -CPBA in dichloromethane to get the required N -oxide impurity. After the confirmation of the VNO impurity, the VNO impurity was heated with water at reflux in a sealed tube for 36 h to get the VHA impurity of about 6-8% after 36 h. After thorough analysis, it was confirmed that venetoclax N -oxide undergoes [1,2] Meisenheimer rearrangement to form the venetoclax hydroxylamine impurity. These two impurities may be relevant reference standards in manufacturing venetoclax Active Pharmaceutical Ingredient (API) (or) tablets., Competing Interests: The authors declare no competing financial interest., (© 2023 The Authors. Published by American Chemical Society.)

Background and Hypothesis: Schizophrenia and comorbid substance use disorders (SUDs) are associated with poor treatment outcomes but differences between the associations of different SUDs with clinical outcomes are poorly characterized. This study examines the associations of comorbid SUDs with clinical outcomes in schizophrenia using a largescale electronic health record (EHR) database., Design: Real-world data (RWD) analysis using the NeuroBlu database; de-identified EHR data were analysed. Multivariable logistic regression, Poisson and CoxPH models were used to compare the associations of specific comorbid SUDs with outcome variables., Results: Comorbid SUD was significantly different on all outcome measures compared to no SUD (U = 1.44e 7 -1.81e 7 , all ps < .001), except number of unique antipsychotics (U = 1.61e 7 , p = .43). Cannabis (OR = 1.58, p < .001) and polysubstance (OR = 1.22, p = .007) use disorders were associated with greater CGI-S. Cannabis (IRR = 1.13, p = .003) and polysubstance (IRR = 1.08, p = .003) use disorders were associated with greater number of unique antipsychotics prescribed, while cocaine (HR = 1.87, p < .001), stimulants (HR = 1.64, p = .024), and polysubstance (HR = 1.46, p < .001) use disorders were associated with a shorter time to antipsychotic discontinuation. Conversely, alcohol use (IRR = 0.83, p < .001), cocaine use (IRR = 0.61, p < .001), opioid use (IRR = 0.61, p < .001), stimulant use (IRR = 0.57, p < .001) and polysubstance use (IRR = 0.87, p < .001) disorders were associated fewer inpatient days., Conclusion: Comorbid SUDs were generally associated with greater CGI-S and poorer clinical outcomes in patients with schizophrenia. Treatment strategies should target not only schizophrenia symptoms but also comorbid SUD to improve management of both conditions., Competing Interests: Declaration of competing interest All authors report current or previous employment with Holmusk Technologies, Inc. RP has received grant funding from the National Institute of Health Research (NIHR301690), the Medical Research Council (MR/S003118/1), the Academy of Medical Sciences (SGL015/1020) and Janssen, and consulting fees from Holmusk, Akrivia Health, Columbia Data Analytics, Otsuka and Boehringer Ingelheim., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Kaposi sarcoma (KS) is a multicentric tumor caused by Kaposi sarcoma herpesvirus (KSHV) that leads to morbidity and mortality among people with HIV worldwide. KS commonly involves the skin but can occur in the gastrointestinal tract (GI) in severe cases., Methods: RNA sequencing was used to compare the cellular and KSHV gene expression signatures of skin and GI KS lesions in 44 paired samples from 19 participants with KS alone or with concurrent KSHV-associated diseases. Analyses of KSHV expression from KS lesions identified transcriptionally active areas of the viral genome., Results: The transcript of an essential viral lytic gene, ORF75, was detected in 91% of KS lesions. Analyses of host genes identified 370 differentially expressed genes (DEGs) unique to skin KS and 58 DEGs unique to GI KS lesions as compared to normal tissue. Interleukin (IL)-6 and IL-10 gene expression were higher in skin lesions as compared to normal skin but not in GI KS lesions. Twenty-six cellular genes were differentially expressed in both skin and GI KS tissues: these included Fms-related tyrosine kinase 4 (FLT4), encoding an angiogenic receptor, and Stanniocalcin 1 (STC1), a secreted glycoprotein. FLT4 and STC1 were further investigated in functional studies using primary lymphatic endothelial cells (LECs). In these models, KSHV infection of LECs led to increased tubule formation that was impaired upon knock-down of STC1 or FLT4., Conclusions: This study of transcriptional profiling of KS tissue provides novel insights into the characteristics and pathogenesis of this unique virus-driven neoplasm., (© 2023. BioMed Central Ltd., part of Springer Nature.)