Correction: Satam et al. Next-Generation Sequencing Technology: Current Trends and Advancements. Biology 2023, 12, 997.

We are very thankful to the commentator for pointing out the issues in the review article by Satam et al [...].

Identification of WNK1 as a therapeutic target to suppress IgH/MYC expression in multiple myeloma.

Multiple myeloma (MM) remains an incurable hematological malignancy demanding innovative therapeutic strategies. Targeting MYC, the notorious yet traditionally undruggable oncogene, presents an appealing avenue. Here, using a genome-scale CRISPR-Cas9 screen, we identify the WNK lysine-deficient protein kinase 1 (WNK1) as a regulator of MYC expression in MM cells. Genetic and pharmacological inhibition of WNK1 reduces MYC expression and, further, disrupts the MYC-dependent transcriptional program. Mechanistically, WNK1 inhibition attenuates the activity of the immunoglobulin heavy chain (IgH) enhancer, thus reducing MYC transcription when this locus is translocated near the MYC locus. WNK1 inhibition profoundly impacts MM cell behaviors, leading to growth inhibition, cell-cycle arrest, senescence, and apoptosis. Importantly, the WNK inhibitor WNK463 inhibits MM growth in primary patient samples as well as xenograft mouse models and exhibits synergistic effects with various anti-MM compounds. Collectively, our study uncovers WNK1 as a potential therapeutic target in MM.

Targeting the GPI transamidase subunit GPAA1 abrogates the CD24 immune checkpoint in ovarian cancer.

CD24 is frequently overexpressed in ovarian cancer and promotes immune evasion by interacting with its receptor Siglec10, present on tumor-associated macrophages, providing a "don't eat me" signal that prevents targeting and phagocytosis by macrophages. Factors promoting CD24 expression could represent novel immunotherapeutic targets for ovarian cancer. Here, using a genome-wide CRISPR knockout screen, we identify GPAA1 (glycosylphosphatidylinositol anchor attachment 1), a factor that catalyzes the attachment of a glycosylphosphatidylinositol (GPI) lipid anchor to substrate proteins, as a positive regulator of CD24 cell surface expression. Genetic ablation of GPAA1 abolishes CD24 cell surface expression, enhances macrophage-mediated phagocytosis, and inhibits ovarian tumor growth in mice. GPAA1 shares structural similarities with aminopeptidases. Consequently, we show that bestatin, a clinically advanced aminopeptidase inhibitor, binds to GPAA1 and blocks GPI attachment, resulting in reduced CD24 cell surface expression, increased macrophage-mediated phagocytosis, and suppressed growth of ovarian tumors. Our study highlights the potential of targeting GPAA1 as an immunotherapeutic approach for CD24+ ovarian cancers.

EZH2 inhibition reactivates epigenetically silenced FMR1 and normalizes molecular and electrophysiological abnormalities in fragile X syndrome neurons.

Fragile X Syndrome (FXS) is a neurological disorder caused by epigenetic silencing of the FMR1 gene. Reactivation of FMR1 is a potential therapeutic approach for FXS that would correct the root cause of the disease. Here, using a candidate-based shRNA screen, we identify nine epigenetic repressors that promote silencing of FMR1 in FXS cells (called FMR1 Silencing Factors, or FMR1- SFs). Inhibition of FMR1-SFs with shRNAs or small molecules reactivates FMR1 in cultured undifferentiated induced pluripotent stem cells, neural progenitor cells (NPCs) and post-mitotic neurons derived from FXS patients. One of the FMR1-SFs is the histone methyltransferase EZH2, for which an FDA-approved small molecule inhibitor, EPZ6438 (also known as tazemetostat), is available. We show that EPZ6438 substantially corrects the characteristic molecular and electrophysiological abnormalities of cultured FXS neurons. Unfortunately, EZH2 inhibitors do not efficiently cross the blood-brain barrier, limiting their therapeutic use for FXS. Recently, antisense oligonucleotide (ASO)-based approaches have been developed as effective treatment options for certain central nervous system disorders. We therefore derived efficacious ASOs targeting EZH2 and demonstrate that they reactivate FMR1 expression and correct molecular and electrophysiological abnormalities in cultured FXS neurons, and reactivate FMR1 expression in human FXS NPCs engrafted within the brains of mice. Collectively, our results establish EZH2 inhibition in general, and EZH2 ASOs in particular, as a therapeutic approach for FXS.

Loss of TDP-43 function contributes to genomic instability in amyotrophic lateral sclerosis.

A common pathological hallmark of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) is the cytoplasmic mislocalization and aggregation of the DNA/RNA-binding protein TDP-43, but how loss of nuclear TDP-43 function contributes to ALS and FTD pathogenesis remains largely unknown. Here, using large-scale RNAi screening, we identify TARDBP, which encodes TDP-43, as a gene whose loss-of-function results in elevated DNA mutation rate and genomic instability. Consistent with this finding, we observe increased DNA damage in induced pluripotent stem cells (iPSCs) and iPSC-derived post-mitotic neurons generated from ALS patients harboring TARDBP mutations. We find that the increase in DNA damage in ALS iPSC-derived neurons is due to defects in two major pathways for DNA double-strand break repair: non-homologous end joining and homologous recombination. Cells with defects in DNA repair are sensitive to DNA damaging agents and, accordingly, we find that ALS iPSC-derived neurons show a marked reduction in survival following treatment with a DNA damaging agent. Importantly, we find that increased DNA damage is also observed in neurons with nuclear TDP-43 depletion from ALS/FTD patient brain tissues. Collectively, our results demonstrate that ALS neurons with loss of nuclear TDP-43 function have elevated levels of DNA damage and contribute to the idea that genomic instability is a defining pathological feature of ALS/FTD patients with TDP-43 pathology.

Next-Generation Sequencing Technology: Current Trends and Advancements.

The advent of next-generation sequencing (NGS) has brought about a paradigm shift in genomics research, offering unparalleled capabilities for analyzing DNA and RNA molecules in a high-throughput and cost-effective manner. This transformative technology has swiftly propelled genomics advancements across diverse domains. NGS allows for the rapid sequencing of millions of DNA fragments simultaneously, providing comprehensive insights into genome structure, genetic variations, gene expression profiles, and epigenetic modifications. The versatility of NGS platforms has expanded the scope of genomics research, facilitating studies on rare genetic diseases, cancer genomics, microbiome analysis, infectious diseases, and population genetics. Moreover, NGS has enabled the development of targeted therapies, precision medicine approaches, and improved diagnostic methods. This review provides an insightful overview of the current trends and recent advancements in NGS technology, highlighting its potential impact on diverse areas of genomic research. Moreover, the review delves into the challenges encountered and future directions of NGS technology, including endeavors to enhance the accuracy and sensitivity of sequencing data, the development of novel algorithms for data analysis, and the pursuit of more efficient, scalable, and cost-effective solutions that lie ahead.

Emerging Trends in Immunotherapy for Cancer.

Recent advances in cancer immunology have enabled the discovery of promising immunotherapies for various malignancies that have shifted the cancer treatment paradigm. The innovative research and clinical advancements of immunotherapy approaches have prolonged the survival of patients with relapsed or refractory metastatic cancers. Since the U.S. FDA approved the first immune checkpoint inhibitor in 2011, the field of cancer immunotherapy has grown exponentially. Multiple therapeutic approaches or agents to manipulate different aspects of the immune system are currently in development. These include cancer vaccines, adoptive cell therapies (such as CAR-T or NK cell therapy), monoclonal antibodies, cytokine therapies, oncolytic viruses, and inhibitors targeting immune checkpoints that have demonstrated promising clinical efficacy. Multiple immunotherapeutic approaches have been approved for specific cancer treatments, while others are currently in preclinical and clinical trial stages. Given the success of immunotherapy, there has been a tremendous thrust to improve the clinical efficacy of various agents and strategies implemented so far. Here, we present a comprehensive overview of the development and clinical implementation of various immunotherapy approaches currently being used to treat cancer. We also highlight the latest developments, emerging trends, limitations, and future promises of cancer immunotherapy.

Macrophages as a Potential Immunotherapeutic Target in Solid Cancers.

The revolution in cancer immunotherapy over the last few decades has resulted in a paradigm shift in the clinical care of cancer. Most of the cancer immunotherapeutic regimens approved so far have relied on modulating the adaptive immune system. In recent years, strategies and approaches targeting the components of innate immunity have become widely recognized for their efficacy in targeting solid cancers. Macrophages are effector cells of the innate immune system, which can play a crucial role in the generation of anti-tumor immunity through their ability to phagocytose cancer cells and present tumor antigens to the cells of adaptive immunity. However, the macrophages that are recruited to the tumor microenvironment predominantly play pro-tumorigenic roles. Several strategies targeting pro-tumorigenic functions and harnessing the anti-tumorigenic properties of macrophages have shown promising results in preclinical studies, and a few of them have also advanced to clinical trials. In this review, we present a comprehensive overview of the pathobiology of TAMs and their role in the progression of solid malignancies. We discuss various mechanisms through which TAMs promote tumor progression, such as inflammation, genomic instability, tumor growth, cancer stem cell formation, angiogenesis, EMT and metastasis, tissue remodeling, and immunosuppression, etc. In addition, we also discuss potential therapeutic strategies for targeting TAMs and explore how macrophages can be used as a tool for next-generation immunotherapy for the treatment of solid malignancies.

Newer Horizon of Mesenchymal Stem Cell-Based Therapy in the Management of SARS-CoV-2-Associated Mucormycosis: A Safe Hope for Future Medicine.

SARS-CoV-2-infected patients are reported to show immunocompromised behavior that gives rise to a wide variety of complications due to impaired innate immune response, cytokine storm, and thrombo-inflammation. Prolonged use of steroids, diabetes mellitus, and diabetic ketoacidosis (DKA) are some of the factors responsible for the growth of Mucorales in such immunocompromised patients and, thus, can lead to a life-threatening condition referred to as mucormycosis. Therefore, an early diagnosis and cell-based management cosis is the need of the hour to help affected patients overcome this severe condition. In addition, extended exposure to antifungal drugs/therapeutics is found to initiate hormonal and neurological complications. More recently, mesenchymal stem cells (MSCs) have been used to exhibit immunomodulatory function and proven to be beneficial in a clinical cell-based regenerative approach. The immunomodulation ability of MSCs in mucormycosis patient boosts the immunity by the release of chemotactic proteins. MSC-based therapy in mucormycosis along with the combination of short-term antifungal drugs can be utilized as a prospective approach for mucormycosis treatment with promising outcomes. However, preclinical and in mucormyIn mucormycosis, the hyphae of clinical trials are needed to establish the precise mechanism of MSCs in mucormycosis treatment.

FBXL20 promotes breast cancer malignancy by inhibiting apoptosis through degradation of PUMA and BAX.

Apoptosis is a programmed cell death that efficiently removes damaged cells to maintain tissue homeostasis. Defect in apoptotic machinery can lead to tumor development, progression, and resistance to chemotherapy. PUMA (p53 upregulated modulator of apoptosis) and BAX (BCL2-associated X protein) are among the most well-known inducers of apoptosis. It has been reported that expression levels of BAX and PUMA are controlled at the posttranslational level by phosphorylation. However, the posttranslational regulation of these proapoptotic proteins remains largely unexplored. In this study, using biochemical, molecular biology, flow cytometric, and immunohistochemistry techniques, we show that PUMA and BAX are the direct target of the F-box protein FBXL20, which restricts their cellular levels. FBXL20 directs the proteasomal degradation of PUMA and BAX in a protein kinase AKT1-dependent manner to promote cancer cell proliferation and tumor growth. Interestingly, inactivation of AKT1 results in activation of another protein kinase GSK3α/ß, which facilitates the proteasomal degradation of FBXL20 by another F-box protein, FBXO31. Thus, a switch between two signaling kinases AKT1 and GSK3α/ß modulates the functional activity of these proapoptotic regulators, thereby determining cell survival or death. RNAi-mediated ablation of FBXL20 results in increased levels of PUMA as well as BAX, which further enhances the sensitivity of cancer cells to chemotherapeutic drugs. We showed that high level expression of FBXL20 in cancer cells reduces therapeutic drug-induced apoptosis and promotes chemoresistance. Overall, this study highlights the importance of targeting FBXL20 in cancers in conjunction with chemotherapy and may represent a promising anticancer strategy to overcome chemoresistance.

Mesenchymal Stromal Cell-Derived Tailored Exosomes Treat Bacteria-Associated Diabetes Foot Ulcers: A Customized Approach From Bench to Bed.

Exosomes are nano-vesicles of endosomal origin inherited with characteristics of drug delivery and cargo loading. Exosomes offer a diverse range of opportunities that can be exploited in the treatment of various diseases post-functionalization. This membrane engineering is recently being used in the management of bacteria-associated diabetic foot ulcers (DFUs). Diabetes mellitus (DM) is among the most crippling disease of society with a large share of its imposing economic burden. DM in a chronic state is associated with the development of micro- and macrovascular complications. DFU is among the diabetic microvascular complications with the consequent occurrence of diabetic peripheral neuropathy. Mesenchymal stromal cell (MSC)-derived exosomes post-tailoring hold promise to accelerate the diabetic wound repair in DFU associated with bacterial inhabitant. These exosomes promote the antibacterial properties with regenerative activity by loading bioactive molecules like growth factors, nucleic acids, and proteins, and non-bioactive substances like antibiotics. Functionalization of MSC-derived exosomes is mediated by various physical, chemical, and biological processes that effectively load the desired cargo into the exosomes for targeted delivery at specific bacterial DFUs and wound. The present study focused on the application of the cargo-loaded exosomes in the treatment of DFU and also emphasizes the different approaches for loading the desired cargo/drug inside exosomes. However, more studies and clinical trials are needed in the domain to explore this membrane engineering.

Autism-Associated Vigilin Depletion Impairs DNA Damage Repair.

Vigilin (Vgl1) is essential for heterochromatin formation, chromosome segregation, and mRNA stability and is associated with autism spectrum disorders and cancer: vigilin, for example, can suppress proto-oncogene c-fms expression in breast cancer. Conserved from yeast to humans, vigilin is an RNA-binding protein with 14 tandemly arranged nonidentical hnRNP K-type homology (KH) domains. Here, we report that vigilin depletion increased cell sensitivity to cisplatin- or ionizing radiation (IR)-induced cell death and genomic instability due to defective DNA repair. Vigilin depletion delayed dephosphorylation of IR-induced γ-H2AX and elevated levels of residual 53BP1 and RIF1 foci, while reducing Rad51 and BRCA1 focus formation, DNA end resection, and double-strand break (DSB) repair. We show that vigilin interacts with the DNA damage response (DDR) proteins RAD51 and BRCA1, and vigilin depletion impairs their recruitment to DSB sites. Transient hydroxyurea (HU)-induced replicative stress in vigilin-depleted cells increased replication fork stalling and blocked restart of DNA synthesis. Furthermore, histone acetylation promoted vigilin recruitment to DSBs preferentially in the transcriptionally active genome. These findings uncover a novel vigilin role in DNA damage repair with implications for autism and cancer-related disorders.

Therapeutic strategies against hDOT1L as a potential drug target in MLL-rearranged leukemias.

Therapeutic intervention of proteins participating in chromatin-mediated signaling with small-molecules is a novel option to reprogram expression networks for restraining disease states. Protein methyltransferases form the prominent family of such proteins regulating gene expression via epigenetic mechanisms thereby representing novel targets for pharmacological intervention. Disruptor of telomeric silencing, hDot1L is the only non-SET domain containing histone methyltransferase that methylates histone H3 at lysine 79. H3K79 methylation mediated by hDot1L plays a crucial role in mixed lineage leukemia (MLL) pathosis. MLL fusion protein mediated mistargeting of DOT1L to aberrant gene locations results in ectopic H3K79 methylation culminating in aberrant expression of leukemogenic genes like HOXA9 and MEIS1. hDOT1L has thus been proposed as a potential target for therapeutic intervention in MLL. This review presents the general overview of hDOT1L and its functional role in distinct biological processes. Furthermore, we discuss various therapeutic strategies against hDOT1L as a promising drug target to vanquish therapeutically challenging MLL.

Management of Intussusception in the Era of Ultrasound-Guided Hydrostatic Reduction: A 3-Year Experience from a Tertiary Care Center.

INTRODUCTION: Ultrasound-guided hydrostatic reduction (HSR) is currently the initial management tool in the treatment of intussusception. HSR is, however, confronted with failures besides there are still a number of patients who primarily undergo surgical intervention for the management of intussusception. We undertook this study to assess the efficacy of HSR and also to look for factors demanding the surgical exploration in patients with intussusception. MATERIALS AND METHODS: A total of 215 patients with intussusception from June 2014 to June 2017 were prospectively studied. HSR was carried out in 203 patients, which was successful in 187 and unsuccessful in 16. These two groups were compared using the Student's t-test. Significance was set at P < 0.05. Twelve patients undergoing surgery primarily were also assessed for the factors affecting the decision-making. RESULTS: HSR was successful in 187 and unsuccessful in 16. The failed group was more likely to have symptoms over 24 h, appearance of crescent, and ≥10-cm length on ultrasonography (USG). Two of these patients had ischemic bowel, two had ileoileal intussusception, and eight had pathological lead points, whereas no obvious cause could be identified in the rest of the four patients. Among the 12 patients who were primarily operated, four patients had peritonitis and other four patients were neonates. Laparoscopic reduction was done in four patients. CONCLUSION: HSR is a safe and effective treatment modality for intussusception. However, it is met with higher failure rates in patients with risk factors such as delayed presentation, appearance of crescent on USG, and length >10 cm. The role of HSR is also dubious in situations such as neonatal intussusception, small-bowel intussusception, and multiple intussusceptions and also in preventing the future recurrence. Such patients ought to be managed by laparotomy or where feasible by laparoscopy. Furthermore, before embarking on HSR, peritonitis and bowel ischemia should be ruled out clinically and radiologically. In the suspicious cases of bowel ischemia, USG Doppler may be helpful.

Vigilin protein Vgl1 is required for heterochromatin-mediated gene silencing in Schizosaccharomyces pombe.

Heterochromatin is a conserved feature of eukaryotic genomes and regulates various cellular processes, including gene silencing, chromosome segregation, and maintenance of genome stability. In the fission yeast Schizosaccharomyces pombe, heterochromatin formation involves methylation of lysine 9 in histone H3 (H3K9), which recruits Swi6/HP1 proteins to heterochromatic loci. The Swi6/HP1-H3K9me3 chromatin complex lies at the center of heterochromatic macromolecular assemblies and mediates many functions of heterochromatin by recruiting a diverse set of regulators. However, additional factors may be required for proper heterochromatin organization, but they are not fully known. Here, using several molecular and biochemical approaches, we report that Vgl1, a member of a large family of multiple KH-domain proteins, collectively known as vigilins, is indispensable for the heterochromatin-mediated gene silencing in S. pombe ChIP analysis revealed that Vgl1 binds to pericentromeric heterochromatin in an RNA-dependent manner and that Vgl1 deletion leads to loss of H3K9 methylation and Swi6 recruitment to centromeric and telomeric heterochromatic loci. Furthermore, we show that Vgl1 interacts with the H3K9 methyltransferase, Clr4, and that loss of Vgl1 impairs Clr4 recruitment to heterochromatic regions of the genome. These findings uncover a novel role for Vgl1 as a key regulator in heterochromatin-mediated gene silencing in S. pombe.

In silico approaches for investigating the binding propensity of apigenin and luteolin against class I HDAC isoforms.

AIM: Aberrant activity of class I histone deacetylases (HDACs) has strong implications for various cancers. Targeting these HDACs with synthetic HDAC inhibitors has shown significant side effects such as atrial fibrillation and QT prolongation emphasizing the need of natural inhibitors as substitutes to synthetic ones. RESULTS: The binding propensity of the two plant-derived inhibitors apigenin and luteolin towards class I HDAC isoforms was checked using extra-precision molecular docking and implicit solvation MMGBSA. Apigenin showed a superior binding affinity against these isoforms as compared to luteolin. Both inhibitors docked stable to the binding pocket of these HDACs as determined by molecular dynamics simulation study. CONCLUSION: Apigenin and luteolin may serve as substitutes to synthetic inhibitors for effective HDAC based anticancer therapy.

Posterior Reversible Encephalopathy Syndrome after Augmentation Cystoplasty in a Child with Neurogenic Bladder.

Posterior reversible encephalopathy syndrome (PRES) or leukoencephalopathy syndrome was introduced into clinical practice in 1996 by Hinchey et al., to describe unique syndrome, clinically expressed during hypertensive and uremic encephalopathy, eclampsia, and immunosuppressive therapy. Hyperperfusion with resultant disruption of the blood-brain barrier results in vasogenic edema, but not infarction, most commonly in the parieto-occipital regions. The severity of this clinical symptom varies. For example, the visual disturbance can manifest as blurred vision, homonymous hemianopsia, or even cortical blindness. Patients may be mildly confused or agitated but can become comatose. Other symptoms less commonly seen include nausea, vomiting, seizures, and brainstem deficits. Chronic kidney disease (CKD) and acute kidney injury are both commonly present in patients with PRES. We are presenting a rare case of neurogenic bladder who developed PRES after augmentation cystoplasty due to underlying CKD.

Modulating epigenetic HAT activity for reinstating acetylation homeostasis: A promising therapeutic strategy for neurological disorders.

Epigenetic mechanisms are emerging as a fundamental regulatory switch in neuronal function. Acetylation homeostasis governed by the antagonistic activities of HATs and HDACs plays a critical role in neuronal gene activity. It is now becoming increasingly clear that several neurodevelopmental, neurodegenerative, and neuropsychiatric disorders are caused by aberrant changes in chromatin acetylation. Several HATs have been shown to be vital for neuronal processes such as synaptic plasticity and memory formation. Thus not surprisingly, dysregulation of such HATs has been implicated in the pathogenesis of several neurodegenerative diseases including Huntington's disease (HD) and Alzheimer's disease (AD). The current therapeutic strategy involves the use of small-molecule histone deacetylase inhibitors to compensate the acetylation deficits arising due to loss of HAT activity. Despite the promising therapeutic effects, the lack of isoform (target) specificity of HDACi raises concerns regarding their applicability. Mounting evidences about the role of HATs in neuronal survival, learning and memory has triggered a new wave of modulating specific HATs as a novel therapeutic option to tackle neurodegenerative diseases. In this review we focus on different HAT families and the critical roles they play in neural development and how the altered acetylation homeostasis culminates in neurodegeneration. Further, we describe the HDACi-based therapeutic approach and its flip side in overcoming neurodegenerative diseases. Furthermore, we discuss the therapeutic potential of HAT modulators in reinstating acetylation homeostasis to ameliorate neurodegenerative disorders.

Role of Inner Nuclear Membrane Protein Complex Lem2-Nur1 in Heterochromatic Gene Silencing.

Heterochromatin in the fission yeast Schizosaccharomyces pombe is clustered at the nuclear periphery and interacts with a number of nuclear membrane proteins. However, the significance and the factors that sequester heterochromatin at the nuclear periphery are not fully known. Here, we report that an inner nuclear membrane protein complex Lem2-Nur1 is essential for heterochromatin-mediated gene silencing. We found that Lem2 is physically associated with another inner nuclear membrane protein, Nur1, and deletion of either lem2 or nur1 causes silencing defect at centromeres, telomeres, and rDNA loci. We analyzed the genome-wide association of Lem2 using ChIP sequencing and we found that it binds to the central core region of centromeres, in striking contrast to Chp1, a component of pericentromeric heterochromatin, which binds H3K9me-rich chromatin in neighboring sequences. The recruitment of Lem2 and Nur1 to silent regions of the genome is dependent on H3K9 methyltransferase, Clr4. Finally, we show that the Lem2-Nur1 complex regulates the local balance between the underln]Snf2/HDAC-containing repressor complex (SHREC) histone deacetylase complex and the anti-silencing protein Epe1. These findings uncover a novel role for Lem2-Nur1 as a key functional link between localization at the nuclear periphery and heterochromatin-mediated gene silencing.

The many faces of histone H3K79 methylation.

Dot1/DOT1L (disruptor of telomeric silencing-1) is an evolutionarily conserved histone methyltransferase that methylates lysine 79 located within the globular domain of histone H3. Dot1 was initially identified by a genetic screen as a disruptor of telomeric silencing in Saccharomyces cerevisiae; further, it is the only known non-SET domain containing histone methyltransferase. Methylation of H3K79 is involved in the regulation of telomeric silencing, cellular development, cell-cycle checkpoint, DNA repair, and regulation of transcription. hDot1L-mediated H3K79 methylation appears to have a crucial role in transformation as well as disease progression in leukemias involving several oncogenic fusion proteins. This review summarizes the multiple functions of Dot1/hDOT1L in a range of cellular processes.