Comprehensive laboratory diagnosis of Fanconi anaemia: comparison of cellular and molecular analysis.

BACKGROUND: Fanconi anaemia (FA) is a rare inherited bone marrow failure disease caused by germline pathogenic variants in any of the 22 genes involved in the FA-DNA interstrand crosslink (ICL) repair pathway. Accurate laboratory investigations are required for FA diagnosis for the clinical management of the patients. We performed chromosome breakage analysis (CBA), FANCD2 ubiquitination (FANCD2-Ub) analysis and exome sequencing of 142 Indian patients with FA and evaluated the efficiencies of these methods in FA diagnosis. METHODS: We performed CBA and FANCD2-Ub analysis in the blood cells and fibroblasts of patients with FA. Exome sequencing with improved bioinformatics to detect the single number variants and CNV was carried out for all the patients. Functional validation of the variants with unknown significance was done by lentiviral complementation assay. RESULTS: Our study showed that FANCD2-Ub analysis and CBA on peripheral blood cells could diagnose 97% and 91.5% of FA cases, respectively. Exome sequencing identified the FA genotypes consisting of 45 novel variants in 95.7% of the patients with FA. FANCA (60.2%), FANCL (19.8%) and FANCG (11.7%) were the most frequently mutated genes in the Indian population. A FANCL founder mutation c.1092G>A; p.K364=was identified at a very high frequency (~19%) in our patients. CONCLUSION: We performed a comprehensive analysis of the cellular and molecular tests for the accurate diagnosis of FA. A new algorithm for rapid and cost-effective molecular diagnosis for~90% of FA cases has been established.

Anticancer activity of quantum size carbon dots: opportunities and challenges.

Research into the anticancer activity of quantum-sized carbon dots (CDs) has emerged as a promising avenue in cancer research. This CDs delves into the opportunities and challenges associated with harnessing the potential of these nanostructures for combating cancer. Quantum-sized carbon dots, owing to their unique physicochemical properties, exhibit distinct advantages as potential therapeutic agents. Opportunities lie in their tunable size, surface functionalization capabilities, and biocompatibility, enabling targeted drug delivery and imaging in cancer cells. However, we include challenges, a comprehensive understanding of the underlying mechanisms, potential toxicity concerns, and the optimization of synthesis methods for enhanced therapeutic efficacy. A succinct summary of the state of the research in this area is given in this review, emphasizing the exciting possibilities and ongoing challenges in utilizing quantum-sized carbon dots as a novel strategy for cancer treatment.

Estrogen-Related Receptor Alpha (ERRα) Promotes Cancer Stem Cell-Like Characteristics in Breast Cancer.

Cancer stem cells drive tumor initiation, invasion, metastasis and recurrence. In the present study, we have evaluated the role of ERRα in the maintenance of breast cancer stem cells (BCSCs) using breast cancer cell lines. The inhibition of ERRα with the inverse agonist, XCT-790, or the knockdown of ERRα in breast cancer cells significantly reduced the mammosphere formation efficiency and mammosphere size along with a significant reduction in the CD44+/CD24- BCSCs. Treatment with XCT-790 significantly downregulated expression of the transcription factors involved in stem cell maintenance such as Oct4, Klf4, Sox2, Nanog and c-Myc in the mammosphere forming stem cells of MCF7 and MDA-MB-231. In addition, XCT-790 induced cell cycle arrest and apoptosis in the mammosphere-forming cells. The knockdown or inhibition of ERRα downregulated the expression of Notch1 and ß-catenin, whereas the overexpression of ERRα in MCF7 cells upregulated the expression of these proteins. Moreover, the inhibition of ERRα synergistically enhanced the efficacy of paclitaxel in inhibiting the BCSCs. These results show that ERRα is crucial for the maintenance of BCSCs and suggest that ERRα could be a potential target for breast cancer treatment.

Efficient deletion of microRNAs using CRISPR/Cas9 with dual guide RNAs.

MicroRNAs (miRNAs) are short non-coding RNAs that play crucial roles in gene regulation, exerting post-transcriptional silencing, thereby influencing cellular function, development, and disease. Traditional loss-of-function methods for studying miRNA functions, such as miRNA inhibitors and sponges, present limitations in terms of specificity, transient effects, and off-target effects. Similarly, CRISPR/Cas9-based editing of miRNAs using single guide RNAs (sgRNAs) also has limitations in terms of design space for generating effective gRNAs. In this study, we introduce a novel approach that utilizes CRISPR/Cas9 with dual guide RNAs (dgRNAs) for the rapid and efficient generation of short deletions within miRNA genomic regions. Through the expression of dgRNAs through single-copy lentiviral integration, this approach achieves over a 90% downregulation of targeted miRNAs within a week. We conducted a comprehensive analysis of various parameters influencing efficient deletion formation. In addition, we employed doxycycline (Dox)-inducible expression of Cas9 from the AAVS1 locus, enabling homogeneous, temporal, and stage-specific editing during cellular differentiation. Compared to miRNA inhibitory methods, the dgRNA-based approach offers higher specificity, allowing for the deletion of individual miRNAs with similar seed sequences, without affecting other miRNAs. Due to the increased design space, the dgRNA-based approach provides greater flexibility in gRNA design compared to the sgRNA-based approach. We successfully applied this approach in two human cell lines, demonstrating its applicability for studying the mechanisms of human erythropoiesis and pluripotent stem cell (iPSC) biology and differentiation. Efficient deletion of miR-451 and miR-144 resulted in blockage of erythroid differentiation, and the deletion of miR-23a and miR-27a significantly affected iPSC survival. We have validated the highly efficient deletion of genomic regions by editing protein-coding genes, resulting in a significant impact on protein expression. This protocol has the potential to be extended to delete multiple miRNAs within miRNA clusters, allowing for future investigations into the cooperative effects of the cluster members on cellular functions. The protocol utilizing dgRNAs for miRNA deletion can be employed to generate efficient pooled libraries for high-throughput comprehensive analysis of miRNAs involved in different biological processes.

Cerebral Venous Sinus Thrombosis Following Scrub Typhus Infection: A Case Report and a Review of the Literature.

Neurological manifestations of scrub typhus, a re-emerging infectious disease of tropic/subtropics caused by Orientia tsutsugamushi infection, have been ever-evolving. Several central nervous system infections have been acknowledged for the development of cerebral venous sinus thrombosis (CVT). Nevertheless, CVT has been a rarely described addendum to the ever-evolving "neuro-scrub" spectrum. Proposed pathogenesis for the development of CVT is disseminated endotheliitis resulting in the triad of venous stasis (due to raised intracranial pressure), cerebral vasculopathy (endothelial damage), and capillary perivasculitis (endothelial damage and resultant hypercoagulable state generated by inflammatory mediators). We herein report a case of a previously healthy young female from the Indian subcontinent who was diagnosed with CVT, following scrub typhus. She responded well to conventional therapy with antibiotics and anticoagulants. CVT is amid the few completely reversible neurological catastrophes if diagnosed and treated early. Again, scrub typhus infection is treated with commonly available and extremely "affordable" antibiotics therapy. Hence, the authors propose that all cases of acute febrile illness with neurological manifestations from scrub-typhus endemic zones (like several parts of India) should be tested for the presence of Orientia tsutsugamushi infection and treated accordingly.

Comprehensive Analysis of microRNAs in Human Adult Erythropoiesis.

MicroRNAs (miRNAs) are small non-coding RNAs, which play an important role in various cellular and developmental processes. The study of miRNAs in erythropoiesis is crucial to uncover the cellular pathways that are modulated during the different stages of erythroid differentiation. Using erythroid cells derived from human CD34+ hematopoietic stem and progenitor cells (HSPCs)and small RNA sequencing, our study unravels the various miRNAs involved in critical cellular pathways in erythroid maturation. We analyzed the occupancy of erythroid transcription factors and chromatin accessibility in the promoter and enhancer regions of the differentially expressed miRNAs to integrate miRNAs in the transcriptional circuitry of erythropoiesis. Analysis of the targets of the differentially expressed miRNAs revealed novel pathways in erythroid differentiation. Finally, we described the application of Clustered regularly interspaced short palindromic repeats-Cas9 (CRISPR-Cas9) based editing of miRNAs to study their function in human erythropoiesis.