SAKE (Single-cell RNA-Seq Analysis and Klustering Evaluation) Identifies Markers of Resistance to Targeted BRAF Inhibitors in Melanoma Cell Populations

Single-cell RNA-Seq’s (scRNA-Seq) unprecedented cellular resolution at a genome wide scale enables us to address questions about cellular heterogeneity that are inaccessible using methods that average over bulk tissue extracts. However, scRNA-Seq datasets also present additional challenges such as high transcript dropout rates, stochastic transcription events, and complex population substructures. Here, we present SAKE (Single-cell RNA-Seq Analysis and Klustering Evaluation): a robust method for scRNA-Seq analysis that provides quantitative statistical metrics at each step of the scRNA-Seq analysis pipeline including metrics for: the determination of the number of clusters present, the likelihood that each cell belongs to a given cluster, and the association of each gene marker in determining cluster membership. Comparing SAKE to multiple single-cell analysis methods shows that most methods perform similarly across a wide range cellular contexts, with SAKE outperforming these methods in the case of large complex populations. We next applied the SAKE algorithms to identify drug-resistant cellular populations as human melanoma cells respond to targeted BRAF inhibitors. Single-cell RNA-Seq data from both the Fluidigm C1 and 10x Genomics platforms were analyzed with SAKE to dissect this problem at multiple scales. Data from both platforms indicate that BRAF inhibitor resistant cells can emerge from rare populations already present before drug application, with SAKE identifying both novel and known markers of resistance. In addition, we compare integrated genomic and transcriptomic markers to show that resistance can arise stochastically within multiple distinct clonal populations

TGF-beta reduces DNA ds-break repair mechanisms to heighten genetic diversity and adaptability of CD44+/CD24- cancer cells

Many lines of evidence have indicated that both genetic and non-genetic determinants can contribute to intra-tumor heterogeneity and influence cancer outcomes. Among the best described sub-population of cancer cells generated by non-genetic mechanisms are cells characterized by a CD44+/CD24- cell surface marker profile. Here, we report that human CD44+/CD24- cancer cells are genetically highly unstable due to intrinsic defects in their DNA repair capabilities. In fact, in CD44+/CD24- cells constitutive activation of the TGF-beta axis was both necessary and sufficient to reduce the expression of genes that are critical in coordinating DNA damage repair mechanisms. Consequently, we observed that cancer cells that reside in a CD44+/CD24- state are characterized by increased accumulation of DNA copy number alterations, greater genetic diversity and improved adaptability to drug treatment. Together, these data suggest that the transition into a CD44+/CD24- cell state can promote intra-tumor genetic heterogeneity, spur tumor evolution and increase tumor fitness

Unresolved endoplasmic reticulum stress engenders immune-resistant, latent pancreatic cancer metastases

The majority of patients with pancreatic ductal adenocarcinoma (PDA) develop metastatic disease after resection of their primary tumor. We found that livers from patients and mice with PDA harbor single, disseminated cancer cells (DCCs) lacking expression of cytokeratin-19 (CK19) and major histocompatibility complex class I (MHCI). We created a mouse model to determine how these DCCs develop. Intra-portal injection of immunogenic PDA cells into pre-immunized mice seeded livers only with single, non-replicating DCCs that were CK19(-) and MHCI(-) The DCCs exhibited an endoplasmic reticulum (ER) stress response but, paradoxically lacked both inositol-requiring enzyme 1alpha activation and expression of the spliced form of transcription factor XBP1 (XBP1s). Inducible expression of XBP1s in DCCs, in combination with T cell-depletion, stimulated the outgrowth of macro-metastatic lesions that expressed CK19 and MHCI. Thus, unresolved ER stress enables DCCs to escape immunity and establish latent metastases

Cost-effectiveness of non-invasive methods for assessment and monitoring of liver fibrosis and cirrhosis in patients with chronic liver disease: systematic review and economic evaluation

BACKGROUND: Liver biopsy is the reference standard for diagnosing the extent of fibrosis in chronic liver disease; however, it is invasive, with the potential for serious complications. Alternatives to biopsy include non-invasive liver tests (NILTs); however, the cost-effectiveness of these needs to be established. OBJECTIVE: To assess the diagnostic accuracy and cost-effectiveness of NILTs in patients with chronic liver disease. DATA SOURCES: We searched various databases from 1998 to April 2012, recent conference proceedings and reference lists. METHODS: We included studies that assessed the diagnostic accuracy of NILTs using liver biopsy as the reference standard. Diagnostic studies were assessed using the Quality Assessment of Diagnostic Accuracy Studies (QUADAS-2) tool. Meta-analysis was conducted using the bivariate random-effects model with correlation between sensitivity and specificity (whenever possible). Decision models were used to evaluate the cost-effectiveness of the NILTs. Expected costs were estimated using a NHS perspective and health outcomes were measured as quality-adjusted life-years (QALYs). Markov models were developed to estimate long-term costs and QALYs following testing, and antiviral treatment where indicated, for chronic hepatitis B (HBV) and chronic hepatitis C (HCV). NILTs were compared with each other, sequential testing strategies, biopsy and strategies including no testing. For alcoholic liver disease (ALD), we assessed the cost-effectiveness of NILTs in the context of potentially increasing abstinence from alcohol. Owing to a lack of data and treatments specifically for fibrosis in patients with non-alcoholic fatty liver disease (NAFLD), the analysis was limited to an incremental cost per correct diagnosis. An analysis of NILTs to identify patients with cirrhosis for increased monitoring was also conducted. RESULTS: Given a cost-effectiveness threshold of £20,000 per QALY, treating everyone with HCV without prior testing was cost-effective with an incremental cost-effectiveness ratio (ICER) of £9204. This was robust in most sensitivity analyses but sensitive to the extent of treatment benefit for patients with mild fibrosis. For HBV [hepatitis B e antigen (HBeAg)-negative)] this strategy had an ICER of £28,137, which was cost-effective only if the upper bound of the standard UK cost-effectiveness threshold range (£30,000) is acceptable. For HBeAg-positive disease, two NILTs applied sequentially (hyaluronic acid and magnetic resonance elastography) were cost-effective at a £20,000 threshold (ICER: £19,612); however, the results were highly uncertain, with several test strategies having similar expected outcomes and costs. For patients with ALD, liver biopsy was the cost-effective strategy, with an ICER of £822. LIMITATIONS: A substantial number of tests had only one study from which diagnostic accuracy was derived; therefore, there is a high risk of bias. Most NILTs did not have validated cut-offs for diagnosis of specific fibrosis stages. The findings of the ALD model were dependent on assuptions about abstinence rates assumptions and the modelling approach for NAFLD was hindered by the lack of evidence on clinically effective treatments. CONCLUSIONS: Treating everyone without NILTs is cost-effective for patients with HCV, but only for HBeAg-negative if the higher cost-effectiveness threshold is appropriate. For HBeAg-positive, two NILTs applied sequentially were cost-effective but highly uncertain. Further evidence for treatment effectiveness is required for ALD and NAFLD. STUDY REGISTRATION: This study is registered as PROSPERO CRD42011001561. FUNDING: The National Institute for Health Research Health Technology Assessment programme

A Molecular Clock Infers Heterogeneous Tissue Age Among Patients with Barrett's Esophagus

National Institutes of Health (www.nih.gov) and the National Cancer Institute (www.cancer.gov) under grants U01CA182940 (BG-U01) (to EGL, CJW, WDH, WMG, and KC), 5P30CA015704 (to WMG and CJW), 5U01CA152756 (to WMG and CJW), 5U54CA163060 (to AC), and NIH1P50CA150964-01A1 (to JEW

A study of onychomycosis in Krishna district of Andhra Pradesh, India

Introduction: Onychomycosis is a chronic infection of nails caused by fungi such as dermatophytes, yeasts or nondermatophyte moulds. It is the most prevalent of all the nail ailments and affects 3- 5% of the population worldwide and represents 20-40% of onychopathies and about 30% of mycotic cutaneous infections. Aims: 1)To isolate and identify the etiological fungi and to assess the prevalence of onychomycosis. 2)To analyse the epidemiological and mycological features of onychomychosis. Methods: 109 nail samples were collected from 102 clinically suspected cases of onychomycosis and further analysed. Results: Of 102 cases, the commonest age group was 41-50 years 38 (37.25%); males were 63 (61.76%) and females 39 (38.24%); involvement of toe nails in 73 (71.57%), finger nails 25 (24.51%) and both 4 (3.92%); 56 (54.90%) belonged to low socio-economic status, middle 31 (30.39%) and high 15 (14.71%). Labourers were 14 (13.73%), farmers and office personnel 10 (9.80%). Of 109 samples, direct microscopy by KOH mount was positive in 82 (75.23%) and fungal culture in 52 (47.71%) of which 29 (26.61%) yielded dermatophytes, NDM’s 11(21.15%), Candida spp. 8 (15.38%) and mixed growth 4 (7.68%). Dermatophytes 25(48.08%) were the predominant group isolated from toe nails and Candida spp. 6 (11.54%) from the finger nails respectively. Among the 56 isolates, dermatophytes were the predominant group 31 (55.36%) followed by NDM’s 15 (26.78%) and Candida spp.10 (17.86%). Conclusion: Onychomycosis is a frequent cause of nail infection. The mycological study and the identification of etiological agents of onychomycosis are needed to confirm the clinical diagnosis and for the choice of therap

dr “MUCOR MYCOSIS AND OTHER FUNGAL INFECTIONS IN COVID-19 PATIENTS DURING SECOND WAVE OF PANDEMIC AT A TERTIARY CARE HOSPITAL”

ABSTRACT In India there has been a sudden Surge in Mucor mycosis cases during second wave of covid-19, made Government of India to declare it as a notifiable disease. Aim: To know the fungal etiology of the suspected Mucor mycosis cases. Material and methods: In a retrospective study, we examined different samples from 112 Covid-19 positive, in-patients with suspected Mucor mycosis. History of risk factors was taken. KOH mount was done for all samples followed by culture on SDA. Results: 81.25% of total cases were from 31–60-year age group. Diabetes mellitus, Steroid therapy and ICU admissions were the risk factors observed. Fungal filaments were observed in 37 samples in KOH mount (33%) and culture was positive in 45 samples (40.17%). Mixed infection was seen in two cases (4.44%). Most common fungus isolated was Mucor in 23 samples (48.93%) followed by Aspergillus in 7 cases (14.89%; A.niger in four cases,  A. fumigatus in 2 cases & A. flavus in 1 case) and Candida spp in 6 cases (12.76%). Among mixed infections, Mucor & Aspergillus niger in one case and Candida & Aspergillus niger in another case were isolated. Rhyzopus, Syncephalastrum, Penicillium and Fusarium were the other fungi isolated. Conclusion: Mucorales were the most common species isolated (63.82%) followed by Aspergillus species. High suspicion and diagnosis of Mucor mycosis in Covid-19 patients is very important to initiate antifungal treatment, to reduce the mortality

Drug interaction presenting as acute abdomen

Warfarin is the most common oral anticoagulant prescribed around the world. Adverse drug interactions with warfarin are a huge problem especially in the elderly and in patients who take multiple medications. Most adverse drug interactions involve concomitantly prescribed oral or intravenous medications. Occasionally, topical or mucosally absorbed drugs can interact, leading to fluctuations in warfarin levels with adverse consequences. In this case report, we describe a case of intestinal intramural hematoma, a rare but known consequence of a supra therapeutic international normalized ratio (INR). The supra therapeutic INR was a consequence of mucosally absorbed miconazole, prescribed for vaginal candidiasis. We wish to highlight this rare and potentially fatal drug interaction, along with the need for frequent INR monitoring when new drugs are added or removed in patients taking warfarin

A comparative study of carrier state of Candida and its speciation in oral flora – Among healthy individuals, persons with DM and HIV sero positive individuals

The aim was to determine colonization rate of candida in oral cavity of healthy individuals, diabetics and HIV seropositive individuals.Material and methods: Samples were collected from oral cavity of 50 HIV sero positive individuals, 50 diabetics and 50 healthy individuals by swabbing palatal mucosa, dorsum of tongue and buccal mucosa with a sterile swab. Samples were processed by inoculating on Hi Chrome Agar and speciation was done by growth on Hi Chrome agar, germ tube test, chlamydospore formation on CMA, pellicle formation in SDA broth and growth at 45ºc.Results: 27 HIV sero positive individuals (54%) carried candida in their oral cavities (single strain in 44% and combination of strains in 10%). Whereas it was 44% in diabetics (single species in 38% and a combination of species in 6%) and 24% in healthy individuals (only single species). Candida albicans accounts for 41.66% in healthy individuals, 68% in diabetics and 42.42% in HIV seropositive persons. other species isolated were Candida tropicalis, Candida parapsilosis, Candida glabrata in all 3 groups in addition to Candida dubliniensis in healthy and HIV seropositive individuals and C.krusei in HIV seropositive persons only. P value- less than 0.05 between healthy persons & diabetics and between healthy persons & HIV seropositives (significant).Conclusion: candidal carriage was higher in diabetics and HIV seropositive individuals. Candida albicans was the commonest species in all three groups. Candida krusei was seen only in HIV positive persons

Isolation and characterisation of Candida species from oropharyngeal secretions of HIV positive individuals

Material and methods: Oropharyngeal secretions were collected from 100 HIV positive individuals with CD4 counts less than 500, over a period of 4 months from June-September 2010. Samples were processed by standard methods for isolation of candida. Speciation was done by the color of growth on HiCrome agar, germ tube test, pellicle formation on SDA broth, chlamydospore production on CMA, growth at 450c and growth on Pal’s agar. Results: Out of 100, 63 samples were positive for fungal growth. Among 63, 17 samples yielded mixed growth and 46 samples yielded single isolates. Total isolates from 63 samples were 80. C. albicans was the predominant species both in mixed cultures (17 out of 34 mixed isolates i.e 50%) & single isolates (18out of 46 samples i.e 39%) and also in total isolates 35 out of 80 isolates, (43.7%) followed by C. tropicalis (17.6%, 20.9%, 18.7%), C. parapsilosis (20.5%, 6.9% and 12.5%), C. dubliniensis (11.8, 8.7%, 10%) in mixed, single and total isolates respectively. C. krusei and C. glabrata were obtained as single isolates. More positive cases were seen in individuals with CD4 count less than 200/cumm. Cultures (17 out of 34 mixed isolates i.e 50%) and single isolates (18 out of 46 samples i.e 39%) and also in total isolates (43.7%) followed by C. tropicalis (17.6%, 20.9%, 18.7%), C. parapsilosis (20.5%, 6.9%, 12.5%), C. dubliniensis (11.8, 8.7%, 10%) in mixed, single and total isolates respectively. C. krusei and C. glabrata were isolated as single isolates. More positive cases were seen in individuals with CD4 count less than 200/cumm