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Recent innovations in single-cell RNA-sequencing (scRNA-seq) provide the technology to investigate biological questions at cellular resolution. Pooling cells from multiple individuals has become a common strategy, and droplets can subsequently be assigned to a specific individual by leveraging their inherent genetic differences. An implicit challenge with scRNA-seq is the occurrence of doublets—droplets containing two or more cells. We develop Demuxafy, a framework to enhance donor assignment and doublet removal through the consensus intersection of multiple demultiplexing and doublet detecting methods. Demuxafy significantly improves droplet assignment by separating singlets from doublets and classifying the correct individual.

The mechanisms by which DNA alleles contribute to disease risk, drug response, and other human phenotypes are highly context-specific, varying across cell types and different conditions. Human induced pluripotent stem cells are uniquely suited to study these context-dependent effects but cell lines from hundreds or thousands of individuals are required. Village cultures, where multiple induced pluripotent stem lines are cultured and differentiated in a single dish, provide an elegant solution for scaling induced pluripotent stem experiments to the necessary sample sizes required for population-scale studies. Here, we show the utility of village models, demonstrating how cells can be assigned to an induced pluripotent stem line using single-cell sequencing and illustrating that the genetic, epigenetic or induced pluripotent stem line-specific effects explain a large percentage of gene expression variation for many genes. We demonstrate that village methods can effectively detect induced pluripotent stem line-specific effects, including sensitive dynamics of cell states. Village cultures, where multiple stem cell lines are cultured in a single dish, provide an elegant solution for population-scale studies. Here, authors show the utility of village models – showing that expression heterogeneity is largely a result of line-specific effects and not village cultures.

Background Human papillomavirus (HPV)-associated oropharyngeal cancer (OPC) is increasing in prevalence, but the drivers of metastasis remain poorly understood, which impacts the ability to personalise management decisions. Much of the genomic research to date focuses on the HPV-negative population. Here, we utilise single-cell and spatial single-cell techniques to understand the drivers of metastasis. Methods Patients with HPV-positive OPC and cervical lymph node metastases treated with curative surgery had matched samples from the primary and lymph nodes collected for research. Single-cell RNA sequencing, single-cell spatial sequencing (Visium) and in-situ spatial platforms were performed. Cancer clones were delineated using inferred copy number variation. Expression phenotypes and interactions with the tumour microenvironment were compared between the metastasising and non-metastasising cancer clones. Results Individual cancer clones have varied abilities to metastasise and undergo clonal expansion in the lymph node, with only a subset of clones present in the primary expanding in the lymph node. Four mechanisms were identified as defining the metastatic phenotype, including protein translation adaptation, immunoproteasome dysfunction and immune evasion, suppression of the IFN immune response and cap-independent protein translation. Conclusions This research elucidates multiple mechanisms driving the expansion of cancer clones in HPV-positive oropharyngeal cancer. By detailing the roles of translational adaptation, immunoproteasome dysfunction, suppression of the interferon immune response and cap-independent protein translation, we provide insights into how these processes contribute to immune evasion and tumour survival.

Adult-type diffuse gliomas are a family of aggressive brain tumours with few effective treatments. Their complex cellular makeup adds to the challenge of finding successful therapies. This intratumoural heterogeneity is fuelled by a subpopulation of glioma stem-like cells (GSCs) that drive tumour growth and resistance to standard treatments. Previous research focused on the three glioma types (astrocytoma, oligodendroglioma, glioblastoma) individually revealed malignant cells mimic the transcriptional profiles of normal brain cell types. Whether these diverse cellular states stem from a shared biological origin is unknown. Here, we show through single-cell RNA sequencing of 40 glioma tumours that all gliomas are described by seven recurring cell states. We also identify a shared astrocyte-like GSC population. Our unique method of identifying GSCs, based on reconstructed tumour phylogenies, repositions astrocyte-like cells at the apex of a differentiation hierarchy in glioma. Our findings indicate the transcriptional heterogeneity observed in gliomas stems from a GSC population recapitulating lineages of healthy adult neural stem cells. These results suggest a shared lineage drives the intratumoural heterogeneity observed in adult-type diffuse gliomas. We anticipate that a deeper understanding of the molecular mechanisms maintaining the GSC state will provide a new framework for future therapeutic development and research into glioma cell biology. Recurring cell states are shared across adult-type diffuse gliomas Reconstructed tumour phylogenies identify an astrocyte-like glioma stem cell population Tumour subclones are segregated non-randomly across cell states

Head and neck cancers, representing the seventh most common malignancy globally, have seen a shift in causative factors from traditional smoking and alcohol use to human papillomavirus (HPV) infection, now accounting for up to 80% of oropharyngeal cancers. We identify the cellular and clonal mechanisms underlying immune avoidance and metastasis by analysing single-cell and spatial genomic data from primary and metastatic cancers. We first map the clonal evolution of malignant cells based on the accumulation of mutations. We identify metastasising clones based on mutational similarity scores between cells in the primary and lymph node metastasis. Genomic analysis of metastasising and non-metastasising clones identified virally mediated protein translation relief (P=4.24x10-24) pathway underlying metastatic expansion. We show that in metastatic clones, this process is driven through upregulation of transition-initiating factors, EIF4E (P=1.5x10-13) and EIFG1 (P<2.22x10-16), and suppression of regulatory kinases EIF4EBP1 (P=2.1x10), EIF2AK2 (P<2.22x10-16), and EIF2S1 (P<2.22x10-16). We subsequently identify that metastatic clones have a corresponding downregulation of the JAK/STAT pathway and immunoproteasome genes PSMB8 (P<2.22x10- 16) and PSMB9 (P<2.22x10-16), suggesting these clones escape immune surveillance through decreased INF inflammatory response and antigen presentation. We validate these results using spatial RNA-seq data, where metastatic cancer clones show decreased cell-to-cell interactions with CD4 T-effector memory cells (CD4TEM) (P=0.0077), CD8 T-exhausted cells (CD8Ex) (P=0.0191), and innate lymphoid cells (ILC) (P=0.04). Finally, we demonstrate that the upregulation of cap-independent translational drives cell proliferation in metastatic clones through the expression of translation initiation factors (EIF4G1: P<2.22x10-16). Our results provide evidence of the mechanisms by which virally induced cancer clones lead to advanced disease and poor prognosis in patients.

Germline ATM gene variations result in phenotypic heterogeneity characterized by a variable degree of disease severity. We retrospectively collected clinical, genetic, and immunological data of 26 cases with A-T. Clinical manifestations included oculocutaneous telangiectasia (100%), ataxia (100%), fever, loose stools or infection (67%), cerebellar atrophy (50%), nystagmus (8%), dysarthria (15.38%), and visual impairment (8%). Genetic analysis confirmed  ATM  gene variations in 16 unrelated cases. The most common type of variation was stopgain variants (56%). Immunoglobulin profile indicated reduced IgA, IgG, and IgM in 94%, 50%, and 20% cases, respectively. T cell lymphopenia was observed in 80% of cases among those investigated. Unusual presentations included an EBV-associated smooth muscle tumour located in the liver in one case and Hyper IgM syndrome-like presentation in two cases. Increased immunosenescence was observed in T-cell subsets (CD4+CD57+ and CD8+CD57+). T-cell receptor excision circles (TRECs) were reduced in 3/8 (37.50%) cases.

Gynecomastia, the abnormal enlargement of male breast tissue, is a rare side effect associated with dasatinib. This drug is used in the treatment of chronic myeloid leukemia (CML). We present a case of dasatinib-induced gynecomastia in a 52-year-old gentleman with CML who developed bilateral breast enlargement and tenderness after approximately four months of dasatinib treatment. The patient's hormonal profile was within normal limits, including testosterone, estradiol, prolactin, and follicle-stimulating hormone levels. While the exact pathophysiology remains unclear, it is postulated that dasatinib's inhibition of various kinases, including src family kinases and receptor kinases, may contribute to developing gynecomastia. The reported incidence of dasatinib-induced gynecomastia is low, and the onset of symptoms can vary widely. Management strategies for dasatinib-induced gynecomastia are not well-established, but options include androgen support, tamoxifen, or switching to an alternative tyrosine kinase inhibitor. This case report highlights the importance of monitoring patients on dasatinib therapy for developing gynecomastia and other hormonal abnormalities. Clinicians should educate the patients about the possibility of this potential adverse effect, emphasizing the need to report any symptoms indicative of low testosterone syndromes. Further research is warranted to better understand the underlying mechanisms, risk factors, and optimal management strategies for dasatinib-induced gynecomastia.

BackgroundDyspnea is one of the common symptoms patients present to the emergency department (ED). The broad spectrum of differentials often requires laboratory and radiological testing in addition to clinical evaluation, causing unnecessary delay. Point of care ultrasound (PoCUS) has shown promising results in accurately diagnosing patients with dyspnea, thus, becoming a popular tool in ED while saving time and maintaining safety standards. Our study aimed to determine the utilization of point of care ultrasound in patients with acute dyspnea as an initial diagnostic tool in our settings.MethodologyThe study was conducted at the emergency department of a tertiary healthcare center in Northern India. Adult patients presenting with acute dyspnea were prospectively enrolled. They were clinically evaluated and necessarily investigated, and a provisional diagnosis was made. Another EP, trained in PoCUS, performed the scan, blinded to the laboratory investigations (not the clinical parameters), and made a PoCUS diagnosis. Our gold standard was the final composite diagnosis made by two Emergency Medicine consultants (who had access to all investigations). Accuracy and concordance of the ultrasound diagnosis to the final composite diagnosis were calculated. The time to formulate a PoCUS diagnosis and final composite diagnosis was compared.ResultsTwo hundred thirty-seven patients were enrolled. The PoCUS and final composite diagnosis showed good concordance (κ = 0.668). PoCUS showed a high sensitivity for acute pulmonary edema, pleural effusion, pneumothorax, pneumonia, pericardial effusion, and low sensitivity for acute exacerbation of chronic obstructive pulmonary disease (AECOPD) and acute respiratory distress syndrome (ARDS)/acute lung injury (ALI). High overall specificity was seen. A high positive predictive value for all except left ventricular dysfunction, pericardial effusion, non-cardiopulmonary causes of dyspnea, and a low negative predictive value was seen for pneumonia. The median time to make a PoCUS diagnosis was 16 (5–264) min compared to the 170 (8–1346) min taken for the final composite diagnosis. Thus, time was significantly lower for PoCUS diagnosis (p value <0.001).ConclusionBy combining the overall accuracy of PoCUS, the concordance with the final composite diagnosis, and the statistically significant reduction in time taken to formulate the diagnosis, PoCUS shows immense promise as an initial diagnostic tool that may expedite the decision-making in ED for patients’ prompt management and disposition with reliable accuracy.

Background: Biodosimetry is the quantification of absorbed radiation dose using biological material obtained from an exposed individual. Radiation can cause different types of chromosomal aberrations, including stable aberrations like translocations and unstable ones like micronuclei, dicentric chromosomes (DC), acentric, and ring forms. Dicentric chromosome assay has become the \"gold standard\" for cytogenetic biodosimetry due to its reproducibility, specificity (low baseline rates), and sensitivity to low doses. Using existing calibration curves and models obtained from in vitro irradiation of blood, the yield of DCs can be used to estimate the average whole-body absorbed dose. Purpose: To evaluate and compare the in vivo dose-response relation of DC aberration formation in peripheral blood lymphocytes of head and neck cancer (HNC) patients undergoing radiotherapy (RT) alone, cisplatin-based chemoradiation (CCRT), accelerated fractionation RT (AFRT), and CCRT with gefitinib (GCRT). Methodology: This prospective observational and analytical study was conducted from 2018 to 2021 in the Department of Radiation Oncology and Genetic Lab of tertiary care, teaching hospital after approval from the Institutional Ethics Committee. Biodosimetric analysis was done weekly in patients undergoing RT (n = 20) versus CCRT (n = 20), CCRT (n = 12) versus AFRT (n = 12), and CCRT (n = 6) versus GCRT (n = 6). The yield of DCs was measured in blood samples taken before starting treatment, that is, day 0 and during RT on days 6, 11, and 16 in RT alone versus CCRT; on days 7 and 13 in CCRT versus AFRT; and days 6 and 11 in CCRT versus GCRT from a blood sample drawn 1-2 h after RT. Phytohemagglutinin-stimulated lymphocytes were cultured using heparinized blood in RPMI-1640 medium supplemented with fetal bovine serum. Cells were arrested at metaphase using demecolcine, harvested by centrifugation, mounted, and stained with Giemsa. Cytogenetic analysis was performed by analyzing at least 100 metaphases with well-spread chromosomes. DC aberrations and acentric fragments were identified and recorded. To standardize the findings as per the customized field for every patient, the mean DC yield per cm2 of the irradiated area was calculated and compared. Results: The mean yield of DC/cm2 in the CCRT group was greater than the RT alone group by 16.33%, 28.57%, and 18.68% on days 6, 11, and 16 of treatment, respectively. This difference between the two groups at day 6 (P = 0.001), day 11 (P < 0.001), and day 16 (P < 0.001) was found to be statistically significant. The mean yield of DC/cm2 in the CCRT group was greater than the AFRT group by 7.9% and 18.3% on days 7 and 13 of treatment, respectively. This difference at day 7 (P < 0.001) and day 13 (P < 0.001) was found to be statistically significant. The mean yield of DC/cm2 in the CCRT group was greater than the GCRT group by 22.7% and 21.8% on days 6 and 11 of treatment, respectively. The difference at day 6 (P = 0.01) was statistically significant but, on day 11 (P = 0.065) this difference was found insignificant. Conclusion: There is a dose-dependent increase in the yield of DCs in lymphocytes of HNC patients undergoing RT with subsequent fractions. Cisplatin-based chemoradiation is the superior method of treatment intensification radio-biologically proven by higher DC yield.

Penetrating facial trauma can be a life-threatening condition, especially due to its impact on the airway. In a facial trauma, there is a distortion in the basic anatomy of the affected, making it a particularly difficult situation for managing the airway. Challenging intubation scenarios have been widely explored in the literature; however, difficult to ventilate situations have been undermined. We describe a case of a 35-year-old female who presented with a history of animal attack on the face. The extent of penetrating facial trauma warranted the need to secure the airway. Preserving spontaneous breathing and using an oral endotracheal tube for oxygenation saved the airway manager from cannot intubate and cannot oxygenate situation in a facial trauma patient. Difficult to mask ventilate while arranging for a definitive airway can be more pressing and challenging for the emergency physician. It also jeopardizes the patient's life, whose survival may only depend on acquiring the patency of the airway. Facial trauma patients may be conscious and spontaneously breathing, leading to the missed or delayed intervention in the airway; hence, prompt assessment and management of the airway in all facial trauma are of utmost importance.