Targeting NF-κB signaling cascades of glioblastoma by a natural benzophenone, garcinol, via in vitro and molecular docking approaches.

Glioblastoma multiforme (GBM) is regarded as the most aggressive form of brain tumor delineated by high cellular heterogeneity; it is resistant to conventional therapeutic regimens. In this study, the anti-cancer potential of garcinol, a naturally derived benzophenone, was assessed against GBM. During the analysis, we observed a reduction in the viability of rat glioblastoma C6 cells at a concentration of 30 µM of the extract (p < 0.001). Exposure to garcinol also induced nuclear fragmentation and condensation, as evidenced by DAPI-stained photomicrographs of C6 cells. The dissipation of mitochondrial membrane potential in a dose-dependent fashion was linked to the activation of caspases. Furthermore, it was observed that garcinol mediated the inhibition of NF-κB (p < 0.001) and decreased the expression of genes associated with cell survival (Bcl-XL, Bcl-2, and survivin) and proliferation (cyclin D1). Moreover, garcinol showed interaction with NF-κB through some important amino acid residues, such as Pro275, Trp258, Glu225, and Gly259 during molecular docking analysis. Comparative analysis with positive control (temozolomide) was also performed. We found that garcinol induced apoptotic cell death via inhibiting NF-κB activity in C6 cells, thus implicating it as a plausible therapeutic agent for GBM.

Phytochemicals-based ß-amyloid cleaving enzyme-1 and MAO-B inhibitors for the treatment of Alzheimer's disease: molecular simulations-based predictions.

Alzheimer's disease (AD) is among the highly prevalent neurodegenerative disorder of the aging brain and is allied with cognitive and behavioral abnormalities. Unfortunately, there is very limited drug discovery for the effective management of AD, and the clinically approved drugs have limited efficacy. Consequently, there is an immediate demand for the development of new compounds that have the ability to act as multitarget-directed ligands (MTDLs). As major pathological targets of the disease, the current study aimed to investigate lead natural bioactive compounds including apigenin, epigallocatechin-3-gallate, berberine, curcumin, genistein, luteolin, quercetin, resveratrol for their inhibitory potentials against ß-amyloid cleaving enzyme-1 (BACE1) and monoamine oxidase-B (MAO-B) enzymes. The study compounds were docked against the target enzymes (MAO-B and BACE1) using MOE software and subsequent molecular dynamics simulations (MDS) studies. The molecular docking analysis revealed that these phytochemicals (MTDLs) showed good interactions with the target enzymes as compared to the reference inhibitors. Among these eight phytocompounds, the epigallocatechin-3-gallate compound was an active inhibitor against both drug targets, with the highest docking scores and good interactions with the active residues of the enzymes. Furthermore, the docking result of the active one inhibitor in complex with the target enzymes (epigallocatechin-3-gallate/BACE1, epigallocatechin-3-gallate/MAO-B, reference/BACE1 and reference/MAO-B) were further validated by MDS. According to the findings of our study, epigallocatechin-3-gallate has the potential to be a candidate for use in the treatment of neurological illnesses like AD. This compound has MTDL potential and may be exploited to create new compounds with disease-modifying features.Communicated by Ramaswamy H. Sarma.

Application of CRISPR-Cas System to Mitigate Superbug Infections.

Multidrug resistance in bacterial strains known as superbugs is estimated to cause fatal infections worldwide. Migration and urbanization have resulted in overcrowding and inadequate sanitation, contributing to a high risk of superbug infections within and between different communities. The CRISPR-Cas system, mainly type II, has been projected as a robust tool to precisely edit drug-resistant bacterial genomes to combat antibiotic-resistant bacterial strains effectively. To entirely opt for its potential, advanced development in the CRISPR-Cas system is needed to reduce toxicity and promote efficacy in gene-editing applications. This might involve base-editing techniques used to produce point mutations. These methods employ designed Cas9 variations, such as the adenine base editor (ABE) and the cytidine base editor (CBE), to directly edit single base pairs without causing DSBs. The CBE and ABE could change a target base pair into a different one (for example, G-C to A-T or C-G to A-T). In this review, we addressed the limitations of the CRISPR/Cas system and explored strategies for circumventing these limitations by applying diverse base-editing techniques. Furthermore, we also discussed recent research showcasing the ability of base editors to eliminate drug-resistant microbes.

Pharmacological evaluations of amide carboxylates as potential anti-Alzheimer agents: anti-radicals, enzyme inhibition, simulation and behavioral studies in animal models.

Alzheimer's disease (AD) is a neurological disorder that progresses gradually but irreversibly leading to dementia and is difficult to prevent and treat. There is a considerable time window in which the progression of the disease can be intervened. Scientific advances were required to help the researchers to identify the effective methods for the prevention and treatment of disease. This research was designed to investigate potential mediators for the remedy of AD, five new carboxylate amide zinc complexes (AAZ9-AAZ13) were synthesized and characterized by spectroscopic and physicochemical techniques. The biological evaluation was carried out based on the cholinesterase inhibitory mechanism. The preparation methodology provided the effective synthesis of targeted moieties. The in vitro pharmacological activities were evaluated involving AChE/BChE inhibition and antioxidant potential. All synthesized compounds displayed activity against both enzymes in higher or comparable to the standard drug Galantamine, a reversible inhibitor but the results displayed by compound AAZ10 indicated IC50 of 0.0013 µM (AChE) and 0.061 µM (BChE) as high values for dual AChE/BChE inhibition with potent anti-oxidant results. Structure activity relationship (SAR) indicated that the potent activity of compound AAZ10 appeared due to the presence of nitro clusters at the ortho position of an aromatic ring. The potent synthesized compound AAZ10 was also explored for the in-vivo Anti-Alzheimer activity and anti-oxidant activity. Binding approaches of all synthesized compounds were revealed through molecular docking studies concerning binding pockets of enzymes that analyzed the best posture interaction with amino acid (AA) residues providing an appreciable understanding of enzyme inhibitory mechanisms. Results indicate that synthesized zinc (II) amide carboxylates can behave as an effective remedy in the treatment of Alzheimer's disease.Communicated by Ramaswamy H. Sarma.

COVID-19 Vaccine Acceptance and Hesitancy among Migrants, Refugees, and Foreign Workers: A Systematic Review and Meta-Analysis.

Despite the effectiveness of current vaccines in reducing the spread and severity of SARS-CoV-2 infections, many people, including migrants, refugees, and foreign workers, are hesitant to be vaccinated. This systematic review and meta-analysis (SRMA) was conducted to determine the pooled prevalence estimate of the acceptance and hesitancy rates of the COVID-19 vaccine among these populations. A comprehensive search of the peer-reviewed literature indexed in PubMed, Scopus, Science Direct, and Web of Science databases was conducted. Initially, 797 potential records were identified, of which 19 articles met the inclusion criteria. A meta-analysis of proportions using data from 14 studies revealed that the overall acceptance rate of COVID vaccination among 29,152 subjects was 56.7% (95% CI: 44.9-68.5%), while the prevalence of vaccine hesitancy among 26,154 migrants reported in 12 studies was estimated to be 31.7% (95% CI: 44.9-68.5%). The acceptance rate for the COVID-19 vaccination first declined from 77.3% in 2020 to 52.9% in 2021 and then slightly increased to 56.1% in 2022. The most frequent factors influencing vaccine hesitancy were worries about vaccine efficacy and safety. Intensive vaccination campaigns should be implemented to raise vaccination awareness among migrants, which will increase the acceptance rate for the COVID-19 vaccine and result in herd immunity.

Homozygous Missense Variant in the N-Terminal Region of ANK3 Gene Is Associated with Developmental Delay, Seizures, Speech Abnormality, and Aggressive Behavior.

Introduction: Intellectual disability (ID) is a lifelong disability that affects an individual‧s learning capacity and adaptive behavior. Such individuals depend on their families for day-to-day survival and pose a significant challenge to the healthcare system, especially in developing countries. ID is a heterogeneous condition, and genetic studies are essential to unravel the underlying cellular pathway for brain development and functioning. Methods: Here we studied a female index patient, born to a consanguineous Pakistani couple, showing clinical symptoms of ID, ataxia, hypotonia, developmental delay, seizures, speech abnormality, and aggressive behavior. Whole exome sequencing (WES) coupled with Sanger sequencing was performed for molecular diagnosis. Further, 3D protein modeling was performed to see the effect of variant on protein structure. Results: WES identified a novel homozygous missense variant (c.178T>C; p.Tyr60His) in the ANK3 gene. In silico analysis and 3-dimensional (3D) protein modeling supports the deleterious impact of this variant on the encoding protein, which compromises the protein‧s overall structure and function. Conclusion: Our finding supports the clinical and genetic diversity of the ANK3 gene as a plausible candidate gene for ID syndrome. Intelligence is a complex polygenic human trait, and understanding molecular and biological pathways involved in learning and memory can solve the complex puzzle of how cognition develops. Intellectual disability (ID) is defined as a deficit in an individual‧s learning and adaptive behavior at an early age of onset [American Psychiatric Association, 2013]. It is one of the major medical, and cognitive disorders with a prevalence of 1-3% in the population worldwide [Leonard and Wen, 2002]. ID often exists with other disabling mental conditions such as autism, attention deficit hyperactivity disorder, epilepsy, schizophrenia, bipolar disorder, or depression. Almost half of the cases appear to have a genetic explanation that ranges from cytogenetically visible abnormalities to monogenic defects [Flint, 2001; Ropers, 2010; Tucker-Drob et al., 2013]. Intellectual disability is a genetically heterogeneous condition, and more than 700 genes have been identified to cause ID alone or as a part of the syndrome. Research in X-linked ID has identified more than 100 disease-causing genes on the X chromosome that play a role in cognition; however, research into autosomal causes of ID is still ongoing [Vissers et al., 2016].

Efficient megakaryopoiesis and platelet production require phospholipid remodeling and PUFA uptake through CD36.

Lipids contribute to hematopoiesis and membrane properties and dynamics, however, little is known about the role of lipids in megakaryopoiesis. Here, a lipidomic analysis of megakaryocyte progenitors, megakaryocytes, and platelets revealed a unique lipidome progressively enriched in polyunsaturated fatty acid (PUFA)-containing phospholipids. In vitro, inhibition of both exogenous fatty acid functionalization and uptake and de novo lipogenesis impaired megakaryocyte differentiation and proplatelet production. In vivo, mice on a high saturated fatty acid diet had significantly lower platelet counts, which was prevented by eating a PUFA-enriched diet. Fatty acid uptake was largely dependent on CD36, and its deletion in mice resulted in thrombocytopenia. Moreover, patients with a CD36 loss-of-function mutation exhibited thrombocytopenia and increased bleeding. Our results suggest that fatty acid uptake and regulation is essential for megakaryocyte maturation and platelet production, and that changes in dietary fatty acids may be a novel and viable target to modulate platelet counts.

Aloe-emodin exhibits growth-suppressive effects on androgen-independent human prostate cancer DU145 cells via inhibiting the Wnt/ß-catenin signaling pathway: an in vitro and in silico study.

At the molecular level, several developmental signaling pathways, such as Wnt/ß-catenin, have been associated with the initiation and subsequent progression of prostate carcinomas. The present report elucidated the anti-cancerous attributes of an anthraquinone, aloe-emodin (AE), against androgen-independent human prostate cancer DU145 cells. The cytotoxicity profiling of AE showed that it exerted significant cytotoxic effects and increased lactose dehydrogenase levels in DU145 cells (p < 0.01 and p < 0.001). AE also induced considerable reactive oxygen species (ROS)-mediated oxidative stress, which escalated at higher AE concentrations of 20 and 25 µM. AE also efficiently instigated nuclear fragmentation and condensation concomitantly, followed by the activation of caspase-3 and -9 within DU145 cells. AE further reduced the viability of mitochondria with increased cytosolic cytochrome-c levels (p < 0.01 and p < 0.001) in DU145 cells. Importantly, AE exposure was also correlated with reduced Wnt2 and ß-catenin mRNA levels along with their target genes, including cyclin D1 and c-myc. Furthermore, the molecular mechanism of AE was evaluated by performing molecular docking studies with Wnt2 and ß-catenin. Evidently, AE exhibited good binding energy scores toward Wnt2 and ß-catenin comparable with their respective standards, CCT036477 (Wnt2 inhibitor) and FH535 (ß-catenin inhibitor). Thus, it may be considered that AE was competent in exerting anti-growth effects against DU145 androgen-independent prostate cancer cells plausibly by modulating the expression of Wnt/ß-catenin signaling.

Pectin: A Bioactive Food Polysaccharide with Cancer Preventive Potential.

Pectin is an acidic heteropolysaccharide found in the cell walls and the primary and middle lamella of land plants. To be authorized as a food additive, industrial pectins must meet strict guidelines set forth by the Food and Agricultural Organization and must contain at least 65% polygalacturonic acid to achieve the E440 level. Fruit pectin derived from oranges or apples is commonly used in the food industry to gel or thicken foods and to stabilize acid-based milk beverages. It is a naturally occurring component and can be ingested by dietary consumption of fruit and vegetables. Preventing long-term chronic diseases like diabetes and heart disease is an important role of dietary carbohydrates. Colon and breast cancer are among the diseases for which data suggest that modified pectin (MP), specifically modified citrus pectin (MCP), has beneficial effects on the development and spread of malignancies, in addition to its benefits as a soluble dietary fiber. Cellular and animal studies and human clinical trials have provided corroborating data. Although pectin has many diverse functional qualities, this review focuses on various modifications used to develop MP and its benefits for cancer prevention, bioavailability, clinical trials, and toxicity studies. This review concludes that pectin has anti-cancer characteristics that have been found to inhibit tumor development and proliferation in a wide variety of cancer cells. Nevertheless, further clinical and basic research is required to confirm the chemopreventive or therapeutic role of specific dietary carbohydrate molecules.

Curcumin-Encapsulated Nanomicelles Improve Cellular Uptake and Cytotoxicity in Cisplatin-Resistant Human Oral Cancer Cells.

Oral cancer has a high mortality rate, which is mostly determined by the stage of the disease at the time of admission. Around half of all patients with oral cancer report with advanced illness. Hitherto, chemotherapy is preferred to treat oral cancer, but the emergence of resistance to anti-cancer drugs is likely to occur after a sequence of treatments. Curcumin is renowned for its anticancer potential but its marred water solubility and poor bioavailability limit its use in treating multidrug-resistant cancers. As part of this investigation, we prepared and characterized Curcumin nanomicelles (CUR-NMs) using DSPE-PEG-2000 and evaluated the anticancer properties of cisplatin-resistant cancer cell lines. The prepared CUR-NMs were sphere-shaped and unilamellar in structure, with a size of 32.60 ± 4.2 nm. CUR-NMs exhibited high entrapment efficiency (82.2%), entrapment content (147.96 µg/mL), and a mean zeta potential of -17.5ζ which is considered moderately stable. The cellular uptake and cytotoxicity studies revealed that CUR-NMs had significantly higher cytotoxicity and cellular uptake in cisplatin drug-resistant oral cancer cell lines and parental oral cancer cells compared to plain curcumin (CUR). The DAPI and FACS analysis corroborated a high percentage of apoptotic cells with CUR-NMs (31.14%) compared to neat CUR (19.72%) treatment. Conclusively, CUR-NMs can potentially be used as an alternative carrier system to improve the therapeutic effects of curcumin in the treatment of cisplatin-resistant human oral cancer.

Cardioprotective Potential of Aqueous Extract of Fumaria indica on Isoproterenol-Induced Myocardial Infarction in SD Rats.

Ischemic heart disease (IHD) treatments and preventions by using plant extract and its phytochemical constituents have achieved considerable attention globally due to its cardioprotective effects. This study is aimed at investigating the cardioprotective and vascular effects of Fumaria indica (F. indica) crude extract on isoproterenol- (ISO-) induced myocardial infarction (MI) in Sprague-Dawley (SD) rats. Rats treated with isoproterenol (85 mg/kg, s.c), administered. Twice at an interval of 24 h showed a significant ST-segment elevation in ECG, edema, and necrosis in histopathology and also in troponin I (cTnI), creatine phosphokinase (CPK), lactate dehydrogenase (LDH), and aspartate aminotransferase (AST). Pretreatment with F. indica (10, 30, and 100 mg/kg, p.o) for 21 days significantly reversed the effects of isoproterenol-induced ischemic changes in the ECG, levels of cTnI, CPK, LDH, and AST, and histopathological changes. In isolated rat atrial strips, F. indica induced negative chronotropic and inotropic effects which were not affected by pretreatment with atropine, excluding role of cardiac muscarinic receptors. Cumulative addition of the extract induced a vasorelaxant effect on phenylephrine-evoked contractions in isolated rat aortic rings, which remained unchanged when challenged with L-NAME, excluding role of endothelial NO. However, extract of F. indica concentration dependently reversed contractions evoked with high K+, indicating calcium entry blocking effect. In conclusion, the F. indica extract is a cardioprotective remedy that ameliorates the isoproterenol-induced cardiotoxic effects and reverses cardiac ischemia, and the calcium antagonistic effect might be of useful in the treatment of MI.

Insights from the molecular docking analysis of SGLT2 and FIMH to combat uropathogenicity.

SGLT2 inhibitors are a novel class of FDA approved anti-diabetes drugs. They act by blocking the SGLT2 protein, which prevents glucose reabsorption, leading in enhance glucose excretion and lower blood glucose levels. In diabetic patients, SGLT2 inhibitors have been linked to urinary tract infections (UTIs). Therefore, the development of novel SGLT2 inhibitors with no adverse effects is a need of time. With this purpose, in this study, 48164natural compounds from ZINC database were screened targeting both the SGLT2 and FimH protein using insilico approaches. FimH has been discovered as a promising target for preventing and treating UTIs. The hit compounds ZINC69481892, ZINC1612996, and ZINC4039265 exhibited strong binding with both SGLT2 and FimH with binding energies values of -9.88, -8.96, and -10.57 kcal/mol for SGLT2, and -7.86, -7.01, and -8.92 kcal/mol for FimH, which is higher than that of controls (-6.78 kcal/mol (Empaglifozolin for SGLT2) and -5.14 kcal/mol (Heptyl α-d-mannopyranoside for FimH)). Hits were found to bind with key residues of both SGLT2 and FimH protein. In addition, physiochemical properties showed that these compounds have good drug-likeness properties. Therefore, we anticipate that if these compounds are investigated further, might be potential SGLT2 inhibitors with less uropathogenic adverse effects.

A novel RUNX1 exon 3 - 7 deletion causing a familial platelet disorder.

Familial Platelet Disorder with associated Myeloid Malignancy (FPDMM) is a rare inherited disorder confirmed with the presence of a pathogenic germline RUNX1 variant and is thought to be heavily underdiagnosed. RUNX1 has also been found to be mutated in up to 10% of adult AML cases and other cell malignancies. We performed targeted next-generation sequencing and subsequent MLPA analysis in a kindred with multiple affected individuals with low platelet counts and a bleeding history. We detected a novel heterozygous exon 3-7 large deletion in the RUNX1 gene in all affected family members which is predicted to remove all of the Runt-homology DNA-binding domain and a portion of the Activation domain. Our results show that the combination of targeted NGS and MLPA analysis is an effective way to detect copy number variants (CNVs) which would be missed by conventional sequencing methods. This precise diagnosis offers the possibility of accurate counseling and clinical management in such patients who could go onto develop other cell malignancies.

Evaluation of the Antiasthmatic Activity of Carissa opaca in Animal Models.

Carissa opaca Stapf ex Haines (C. opaca) fruit is used traditionally in the treatment of respiratory illnesses including asthma. However, there is no scientific evidence supporting its antiasthmatic activity. The current study was conducted to evaluate its antiasthmatic effects using in vivo and in vitro approaches. The methanolic crude extract of C. opaca fruit (Co.Cr.) was used and in vivo antiasthmatic activity was carried out using ovalbumin- (OVA-) sensitized and OVA-challenged BALB/c mice. In in vitro bronchorelaxant activity of crude extract, aqueous and n-hexane fractions of C. opaca were carried out on isolated rat tracheal strips. Co.Cr. (200 and 400 mg/kg) attenuated ovalbumin-induced changes in lung histochemistry with % decrease in peribronchial inflammation of 14.1 ± 0.21 and 65.8 ± 0.22 and % decrease in total inflammatory cell count of 35.7 ± 2.80 and 53.3 ± 2.30 in bronchoalveolar lavage fluid. Co.Cr., aqueous, and n-hexane fraction of C. opaca attenuated the precontractions induced by high K+ (80 mM) and carbachol (1 µM), respectively. In conclusion, the results showed that C. opaca possesses antiasthmatic activity via relaxant effect on bronchial smooth muscle which is mediated through calcium channel blockade and antimuscarinic activity. This study provides scientific evidence of the traditional use of C. opaca in the management of allergic asthma.

Novel gene variants in patients with platelet-based bleeding using combined exome sequencing and RNAseq murine expression data.

Essentials Identifying genetic variants in platelet disorders is challenging due to its heterogenous nature. We combine WES, RNAseq, and python-based bioinformatics to identify novel gene variants. We find novel candidates in patient data by cross-referencing against a murine RNAseq model of thrombopoiesis. This innovative combined bioinformatic approach provides novel data for future research in the field. ABSTRACT: Background The UK Genotyping and Phenotyping of Platelets study has recruited and analyzed 129 patients with suspected heritable bleeding. Previously, 55 individuals had a definitive genetic diagnosis based on whole exome sequencing (WES) and platelet morphological and functional testing. A significant challenge in this field is defining filtering criteria to identify the most likely candidate mutations for diagnosis and further study. Objective Identify candidate gene mutations for the remaining 74 patients with platelet-based bleeding with unknown genetic cause, forming the basis of future re-recruitment and further functional testing and assessment. Methods Using python-based data frame indexing, we first identify and filter all novel and rare variants using a panel of 116 genes known to cause bleeding across the full cohort of WES data. This identified new variants not previously reported in this cohort. We then index the remaining patients, with rare or novel variants in known bleeding genes against a murine RNA sequencing dataset that models proplatelet-forming megakaryocytes. Results Filtering against known genes identified candidate variants in 59 individuals, including novel variants in several known genes. In the remaining cohort of "unknown" patients, indexing against differentially expressed genes revealed candidate gene variants in several novel unreported genes, focusing on 14 patients with a severe clinical presentation. Conclusions We identified candidate mutations in a cohort of patients with no previous genetic diagnosis. This work involves innovative coupling of RNA sequencing and WES to identify candidate variants forming the basis of future study in a significant number of undiagnosed patients.

A comprehensive bioinformatic analysis of 126 patients with an inherited platelet disorder to identify both sequence and copy number genetic variants.

Inherited bleeding disorders (IBDs) comprise an extremely heterogeneous group of diseases that reflect abnormalities of blood vessels, coagulation proteins, and platelets. Previously the UK-GAPP study has used whole-exome sequencing in combination with deep platelet phenotyping to identify pathogenic genetic variants in both known and novel genes in approximately 40% of the patients. To interrogate the remaining "unknown" cohort and improve this detection rate, we employed an IBD-specific gene panel of 119 genes using the Congenica Clinical Interpretation Platform to detect both single-nucleotide variants and copy number variants in 126 patients. In total, 135 different heterozygous variants in genes implicated in bleeding disorders were identified. Of which, 22 were classified pathogenic, 26 likely pathogenic, and the remaining were of uncertain significance. There were marked differences in the number of reported variants in individuals between the four patient groups: platelet count (35), platelet function (43), combined platelet count and function (59), and normal count (17). Additionally, we report three novel copy number variations (CNVs) not previously detected. We show that a combined single-nucleotide variation (SNV)/CNV analysis using the Congenica platform not only improves detection rates for IBDs, suggesting that such an approach can be applied to other genetic disorders where there is a high degree of heterogeneity.

Inherited Thrombocytopenia: Update on Genes and Genetic Variants Which may be Associated With Bleeding.

Inherited thrombocytopenia (IT) is comprised of a group of hereditary disorders characterized by a reduced platelet count as the main feature, and often with abnormal platelet function, which can subsequently lead to impaired haemostasis. Inherited thrombocytopenia results from genetic mutations in genes implicated in megakaryocyte differentiation and/or platelet formation and clearance. The identification of the underlying causative gene of IT is challenging given the high degree of heterogeneity, but important due to the presence of various clinical presentations and prognosis, where some defects can lead to hematological malignancies. Traditional platelet function tests, clinical manifestations, and hematological parameters allow for an initial diagnosis. However, employing Next-Generation Sequencing (NGS), such as Whole Genome and Whole Exome Sequencing (WES) can be an efficient method for discovering causal genetic variants in both known and novel genes not previously implicated in IT. To date, 40 genes and their mutations have been implicated to cause many different forms of inherited thrombocytopenia. Nevertheless, despite this advancement in the diagnosis of IT, the molecular mechanism underlying IT in some patients remains unexplained. In this review, we will discuss the genetics of thrombocytopenia summarizing the recent advancement in investigation and diagnosis of IT using phenotypic approaches, high-throughput sequencing, targeted gene panels, and bioinformatics tools.