Prevalence and clinicopathological features of driver gene mutations profile in BCR: ABL1 negative classical myeloproliferative neoplasm-A single-center study from North India.

BACKGROUND: Recurrent somatic mutations in the JAK2, CALR, and the MPL genes are noted in BCR: ABL1 negative classic myeloproliferative neoplasms (MPN) that includes polycythemia vera (PV), essential thrombocytosis (ET), and primary myelofibrosis (PMF). MATERIALS AND METHODS: Mutation profile and clinical features of MPN cases diagnosed at a tertiary care center in North India are being described. JAK2V617F mutation was screened using ARMS PCR, and CALR mutation was screened using allele-specific PCR followed by fragment analysis. MPL and JAK2 Exon 12 mutations were screened by Sanger sequencing. Some of the samples were also screened using commercial kits based on single-plex RT PCR. RESULTS: A total of 378 cases (including 124 PV, 121 ET, and 133 PMF cases) were screened over 6.5 years. JAK2V617F mutation was noted in 90.3%, 61.1%, and 69.2% of cases of PV, ET, and PMF, respectively. In PV, JAK2V617F wild-type cases were associated with a significantly lower age (44 yrs vs 54 yrs; P = 0.001), lower TLC (6.3 vs 16.9; P = 0.001), and a lower platelet count (188 × 109/L vs 435 × 109/L; P = 0.009) as compared to the JAK2V617F mutated cases. CALR and MPL mutations were noted in 17.4% and 12% and 0.8% and 5.3% of ET and PMF cases, respectively. Type 1 CALR mutations were commoner in both ET and PMF. The triple negative cases constituted 20.7% and 13.5% cases of ET and PMF, respectively. In ET, the triple negative cases were found to have a significantly lower median age of presentation (42 yrs vs 52 yrs; P = 0.001), lower median TLC (10.2 × 109/L vs 13.2 × 109/L; P = 0.024), and a higher median platelet count (1238 × 109/L vs 906 × 109/L; P = 0.001) as compared to driver genes mutated cases. In PMF, the triple negative cases were found to have a significantly lower hemoglobin level (7.9 g/dl vs 11.0 gl/dl; P = 0.001) and a significant female preponderance (P = 0.05) as compared to the mutated cases. CALR mutations were found to have a significantly lower median age (43 yrs vs 56 yrs; P = 0.001) and lower hemoglobin (9.6 g/dl vs 11.3 g/dl) as compared to the JAK2 mutations. CONCLUSION: Our data on the driver gene mutational profile of BCR: ABL1 negative MPN is one of the largest patient cohorts. The prevalence and clinicopathological features corroborate with that of other Asian studies.

AAV mediated genome engineering with a bypass coagulation factor alleviates the bleeding phenotype in a murine model of hemophilia B.

It is crucial to develop a long-term therapy that targets hemophilia A and B, including inhibitor-positive patients. We have developed an Adeno-associated virus (AAV) based strategy to integrate the bypass coagulation factor, activated FVII (murine, mFVIIa) gene into the Rosa26 locus using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 mediated gene-editing. AAV vectors designed for expression of guide RNA (AAV8-gRNA), Cas9 (AAV2 neddylation mutant-Cas9), and mFVIIa (AAV8-mFVIIa) flanked by homology arms of the target locus were validated in vitro. Hemophilia B mice were administered with AAV carrying gRNA, Cas9 (1 × 1011 vgs/mouse), and mFVIIa with homology arms (2 × 1011 vgs/mouse) with appropriate controls. Functional rescue was documented with suitable coagulation assays at various time points. The data from the T7 endonuclease assay revealed a cleavage efficiency of 20-42 %. Further, DNA sequencing confirmed the targeted integration of mFVIIa into the safe-harbor Rosa26 locus. The prothrombin time (PT) assay revealed a significant reduction in PT in mice that received the gene-editing vectors (22 %), and a 13 % decline in mice that received only the AAV-FVIIa when compared to mock treated mice, 8 weeks after vector administration. Furthermore, FVIIa activity in mice that received triple gene-editing vectors was higher (122.5mIU/mL vs 28.8mIU/mL) than the mock group up to 15 weeks post vector administration. A hemostatic challenge by tail clip assay revealed that hemophilia B mice injected with only FVIIa or the gene-editing vectors had significant reduction in blood loss. In conclusion, AAV based gene-editing facilitates sustained expression of coagulation FVIIa and phenotypic rescue in hemophilia B mice.

Peripheral blood quantitation of CD26 positive leukemic stem cells as a predictor of tyrosine kinase inhibitor response in chronic myeloid leukemia.

INTRODUCTION: Leukemic stem cells (LSCs) are the transcriptionally low/silent cells which are resistant to the tyrosine kinase inhibitor. These have been found to play a pivotal role in disease relapse in chronic myeloid leukemia (CML) cases. The present study evaluated the correlation of absolute CML-LSC count in the peripheral blood (PB) at diagnosis and achievement of major molecular response (MMR) at 12 months in patients of CML-CP. METHODS: This was a prospective, observational, non-interventional single center study including newly diagnosed adult (>18 yrs) CML-CP patients. Absolute CD26 + CML-LSC quantification was done by multiparametric flow cytometry. Patients were treated with Imatinib treatment and subsequently monitored at 3-month intervals for BCR::ABL transcript levels. MMR was defined as a BCR::ABL1 transcript level of less than 0.1% on international scale. RESULTS: A total of 89 patients were enrolled in the study out of which 40.5% achieved MMR at 12 months. There was a significant difference in the median absolute CML-LSC count of the patients who achieved MMR at 12 months as compared to those who did not (58.5 vs 368.1 cells/µL; p value <0.001). Using a ROC analysis, a count of <165.69 CML LSC/µL was identified to have a sensitivity of 83.8% and specificity of 72.4%, in predicting the MMR at 12 months. CONCLUSION: Absolute CML-LSC count at diagnosis in the PB predicts the MMR achievement at 12 months. An absolute count of less than 165 cells/µL is highly predictive of achieving MMR at 12 months.

Role of miRNAs in T-cell activation and Th17/Treg-cell imbalance in acquired aplastic anemia.

BACKGROUND: Altered T-cell repertoire with an aberrant T-cell activation and imbalance of the Th17/Treg cells has been reported in acquired aplastic anemia (aAA). miRNAs are well known to orchestrate T-cell activation and differentiation, however, their role in aAA is poorly characterized. The study aimed at identifying the profile of miRNAs likely to be involved in T-cell activation and the Th17/Treg-cell imbalance in aAA, to explore newer therapeutic targets. METHODS: Five milliliters peripheral blood samples from 30 patients of aAA and 15 healthy controls were subjected to flow cytometry for evaluating Th17- and Treg-cell subsets. The differential expression of 7 selected miRNAs viz; hsa-miR-126-3p, miR-146b-5p, miR-155-5p, miR-16, miR-17, miR-326, and miR-181c was evaluated in the PB-MNCs. Expression analysis of the miRNAs was performed using qRT-PCR and fold change was calculated by 2-ΔΔCt method. The alterations in the target genes of deregulated miRNAs were assessed by qRT-PCR. The targets studied included various transcription factors, cytokines, and downstream proteins. RESULTS: The absolute CD3+ lymphocytes were significantly elevated in the PB of aAA patients when compared with healthy controls (p < 0.0035), however, the CD4:CD8 ratio was unperturbed. Th17: Treg-cell ratio was altered in aAA patients (9.1 vs. 3.7%, p value <0.05), which correlated positively with disease severity and the PNH positive aAA. Across all severities of aAA, altered expression of the 07 miRNAs was noted in comparison to controls; upregulation of miR-155 (FC-2.174, p-value-0.0001), miR-146 (FC-2.006, p-value-0.0001), and miR-17 (FC-3.1, p-value-0.0001), and downregulation of miR-126 (FC-0.329, p-value-0.0001), miR-181c (FC-0.317, p-value-0.0001), miR-16 (FC-0.348, p-value-0.0001), and miR-326 (FC-0.334, p-value-0.0001). Target study for these miRNAs revealed an increased expression of transcription factors responsible for Th1 and Th17 differentiation (T-bet, RORϒt, IL-17, IL-6, and IFN-ϒ), T-cell activation (NFκB, MYC, and PIK3R2), downregulation of FOX-P3, and other regulatory downstream molecules like SHIP-1, ETS-1, IRAK-1, TRAF-6, and PTEN. CONCLUSION: The study for the first time highlights the plausible role of different miRNAs in deregulating the Th17/Treg-cell imbalance in aAA, and comprehensively suggest the role of altered NF-kB and mTOR pathways in aAA. The axis may be actively explored for development of newer therapeutic targets in aAA.

Targeting caspase pathway by novel N-Me aziridine derivatives for hepatocellular carcinoma drug discovery.

Azaheterocycles are three-membered rings, known as aziridines, that occur naturally and have pharmaceutical applications.These compounds are present as several secondary metabolites produced by plants and microorganisms.Recent studies have demonstrated the effectiveness of aziridine derivatives (N-H/N-Me) as anticancer agents.We synthesized 18 compounds containing an N-Me enone aziridine group, the chemistry of which has been previously published. However, these compounds have drug-likeness properties; therefore, we aimed to demonstrate their drug-like properties using in silico and in vitro investigations.The molecular structures of the compounds were optimized using density functional theory (DFT). The ADMET parameters of the derivatives were calculated using SwissADME and PreADMET. Additionally, these derivatives were evaluated for their ability to bind to caspase-3 and caspase-9 and then subjected to molecular docking. The lead chemical AY128 maintained stable complexes with target proteins during molecular dynamics simulations, as evidenced by the root mean square deviation (RMSD) and root mean square fluctuation (RMSF) parameters. In vitro cytotoxicity and ELISA tests showed that the novel aziridine derivatives, especially AY128, had strong anticancer activity against HepG2 hepatocellular carcinoma cells.Our study suggests that AY128 may be a potential drug candidate for hepatocellular carcinoma through the caspase-3 and caspase-9-dependent apoptotic pathways.Communicated by Ramaswamy H. Sarma.

Oriental theileriosis associated with a new genotype of Theileria orientalis in buffalo (Bubalus bubalis) calves in Uttar Pradesh, India.

Theileria orientalis is known to cause a benign infection in cattle and buffalo (Bubalus bubalis). However, the Ikeda and Chitose genotypes of the parasite cause lethal disease in beef and dairy cattle. Recently an outbreak of clinical oriental theileriosis occurred in buffalo calves in a Government Animal Husbandry and Agricultural Farm located in Uttar Pradesh, India. Examination of Giemsa stained thin blood smears revealed typical rod-shaped T. orientalis piroplasms in the erythrocytes. The clinical signs included pyrexia, nasal discharge, lacrimation, lethargy, inappetence and anaemia with varying degrees of paleness of the visible mucous membranes. Vascular congestion in internal organs, pulmonary emphysema and consolidation of lungs, focal areas of necrosis in the heart with mononuclear cell infiltration, focal mononuclear cell aggregation in the cortex and tubular degeneration of the kidney were significant necropsy findings. The T. orientalis major piroplasm surface protein (MPSP) gene was amplified by polymerase chain reaction (PCR) using specific primers. The nucleotide sequence analysis of the PCR product revealed 84.8% identity between the T. orientalis Uttar Pradesh isolate and other reference genotypes available in the public domain. Furthermore, the phylogenetic analysis of the MPSP gene sequence ratified that this is a new genotype of T. orientalis. This is the first report of a clinical outbreak of oriental theileriosis in Indian buffalo calves caused by a novel genotype of T. orientalis.

Role of T Lymphocyte Activation Profile in Predicting SARS-CoV-2 Severity: Experience from Tertiary Care Centre of North India.

Background: Immune dysregulation plays a key role in determining COVID-19 disease severity. We aimed to analyze the T cell activation profile in COVID - 19 cases and its predictive role in disease severity and outcome. Material & methods: This was a prospective observational pilot study from a tertiary care COVID-19 hospital. Peripheral blood samples obtained between the fifth and seventh day of COVID-19 illness, were subjected to lymphocyte subset analysis using multicolor flowcytometry using a single tube, 8 antibodies (CD45, CD19, CD3, CD4, CD8, CD38, HLADR, and CD56) analysis. Correlation between lymphocyte subset analysis and clinical profile was determined. Results: 26 patients including 11 with mild disease and 15 with severe disease were enrolled. The median age was 58 years (range: 33-81), with a male: female ratio of 1.36:1. Significant lymphopenia was observed in the severe group compared to the mild group (p < 0.02). The absolute numbers of CD3+, CD4+, CD8 + T cells, B cells, and NK cells were significantly reduced in the severe group as compared to the mild group (p < 0.05). In patients with severe disease, the proportion of CD8 + and CD4 + T cells were significantly higher than those in patients with mild disease (p = 0.0372). Using ROC analysis, a CD4:8 T cell ratio of ≥ 2.63 and an activated (CD38 + HLA-DR+) CD8 T cell proportion of > 15.85% of the total CD8 T cell population, significantly determined the severe disease category. Conclusions: Severe COVID-19 is associated with severe lymphopenia, altered CD4/CD8 ratio and markedly increased CD8 T cell activation profile. Supplementary Information: The online version contains supplementary material available at 10.1007/s12288-022-01558-6.

An Analysis of M-protein in Plasma cell Dyscrasia Patients Identifies that IgG Lambda Subtype is More Commonly Associated with Normal Serum Free Light Chain (SFLC) Ratio.

The determination of monoclonal protein (M-protein) by SPE, IFE and SFLC assay is fundamental in the diagnosis of Plasma cell proliferative disorder (PCPD). In the present study, we seek to assess the diagnostic performance and concordance of these three techniques in un-treated PCPD patients. All new patients with dysproteinemia and/or suspected PCPD were included in this retrospective observational study. The baseline parameters were retrieved from electronic medical records. SPE was performed on gel electrophoresis system; monoclonal component was identified by IFE. SFLC assays were performed by nephelometry using a latex-enhanced immunoassay. Total 402 patients of PCPD were included (10.9% of MGUS/SMM and 89.1% of multiple myeloma). The combination of SPE + rSFLC (ratio of kappa/lambda light chain) and SPE + IFE + rSFLC was able to detect M-protein across all subgroups of patients. In 61 patients, rSFLC values were within normal range (54.5% of MGUS/SMM and 10.3% of MM) and was more commonly seen with IgG lambda M-protein (57.4% vs. all-others). The median dFLC value, among these patients, was higher for MM than MGUS/SMM patients (23.8 vs. 14.4 mg/L, respectively). The combination of SPE and rSFLC can be reliably used to detect M-protein in PCPD patients. In a small subgroup of MM patients, despite the presence of an intact immunoglobulin (M-protein), the rSFLC is not abnormal. Historically, these patients should respond better to treatment. However, a further follow-up analysis with more number of such patients would be advantageous for better understanding.

Hepatosplenic T-cell lymphoma diagnosed using flow cytometry. A single-center study of 12 cases from North India.

Background: Hepatosplenic T-cell lymphoma (HSTCL) is a rare fatal T-cell neoplasm with unique clinical and laboratory features. There is, however, significant morphological and immunophenotypic heterogeneity which may lead to diagnostic dilemma. Aims and Objectives: The study was aimed to study the prevalence and clinic-pathological spectrum of this rare variant of T cell lymphoma in the Indian subcontinent. Material and Methods: A retrospective analysis of all consecutive cases of HSTCL diagnosed over a period of 6 years was carried out. The clinical and laboratory parameters of all these patient were reviewed and analysed. Results: A total of 12 cases of HSTCL were diagnosed during this period which accounted for 1.76% of all non-Hodgkin's lymphomas (NHLs) and 9.1% of all T-cell NHLs. The median (range) age of presentation was 23 (16-30) years.Leukocytosis, peripheral blood (PB) involvement, and a blastic morphology were noted in 41%, 67%, and 58% of the cases, respectively. FCI proved these cells to have a mature, dual-negative (CD4-/CD8-) T-cell phenotype with a gamma-delta T-cell receptor restriction. Frequent loss of CD5 expression (84%) was also noted. These patients invariably had a fatal outcome and majority died within a year of diagnosis. Conclusion: The incidence of leukocytosis and a blastoid morphology is quite frequent in HSTCL. Hence, a differential diagnosis of HSTCL should always be considered in young patients presenting with splenomegaly and exhibiting atypical lymphoid/blastoid cells in the PB or a marrow. An FCI can readily diagnose and differentiate them from an acute lymphoblastic leukemia/lymphoma.

Simultaneous determination of additive concentration in rubber using ATR-FTIR spectroscopy.

Using attenuated total reflection (ATR) Fourier transform infrared (FTIR) spectroscopy for direct quantitative analysis is highly desirable for many sample systems due to advantages such as rapid spectra collection and being completely non-destructive. However, for many complex sample matrices the feasibility of direct quantitative analysis using ATR-FTIR is uncertain. The commonly used Beer-Lambert law may not be applicable for many systems in general, besides sample related complexities such as inhomogeneity, variable optical properties, or heavily overlapping absorption bands. In this study, we consider fully formulated vulcanized rubber with carbon black or silica as the primary filler as our system of interest. We developed a method to simultaneously quantify the concentration of three different antidegradents of similar chemical structure directly on rubber samples using ATR-FTIR spectra. Results show that absorbance follows the Beer-Lambert law well for the range of antidegradent concentrations considered. Despite this, a direct application of the Beer-Lambert law to deconvolute overlapping peaks between antidegradents proved insufficient. Through the application of partial least squares (PLS) multivariate analysis, remarkable prediction accuracy of within about 0.15 wt% error for all three antidegradents was achieved for both types of rubber formulations, even with high levels of carbon black. These results show the value this method has for quantitative analysis of additives in rubber. Our investigation highlights the potential usefulness of FTIR spectroscopy in general for rapid quantitative analysis directly on samples of interest without any prior chemical separation.

Mutation rate evaluation at 21 autosomal STR loci: Paternity testing experience.

The short tandem repeats (STRs) or microsatellites are used for paternity testing and these sequences mutate more rapidlythanbulkDNAsequences. A total of 746 paternity cases were analysed to understand the mutation rate of 21 autosomal STR loci. We identified 41 mutations in 11 STR Loci with a maximum at SE33. No mutations occurred in the remaining 10 STR loci. The overall average mutation rate was estimated as 0.004523 and the estimated locus-specific mutation rate varied between 0.001214 and 0.016990. Among these 90.24% was accounted for single-step mutation, 2.44% for two steps, and 7.32 % for three or muti steps. The obtained data is crucial and could be helpful for ensuring the accuracy of DNA testing and interpretation.

CD157 Can Replace CD24 and CD14 in a Single-Tube Flow-Cytometric Assay to Detect Paroxysmal Nocturnal Hemoglobinuria (PNH) Clones on Both Neutrophils and Monocytes: A Prospective Study From North India.

Introduction As per current guidelines, detection of paroxysmal nocturnal hematuria (PNH) clones on leucocytes requires the demonstration of the loss of at least two glycosyl-phosphatidyl-inositol (GPI)-linked molecules on both neutrophils and monocytes by flow cytometry. CD24 and CD14 are GPI-linked molecules expressed on neutrophils and monocytes respectively, whereas another GPI-linked molecule, CD157, is expressed on both neutrophils and monocytes. This prospective study evaluated the ability of CD157 to replace both CD24 and CD14 in a single-tube flow-cytometric assay to detect PNH clones on both neutrophils and monocytes. Materials and methods PNH clones were newly detected in 52 patients by an existing "standard" single-tube six-color flow-cytometric method, which was routinely performed in our laboratory at the time of undertaking this study. Six antibodies (CD45/CD15/CD64/CD24/CD14/FLAER) were used in this "standard" technique. Subjects were divided into two groups: (i) PNH disease (n=10), and (ii) aplastic anemia/myelodysplastic syndrome (AA/MDS) (n=42). Diagnosis of PNH disease and AA/MDS were made as per standard literature and guidelines. Results were compared with a single-tube five-color "test" assay using the antibodies CD45/CD15/CD64/CD157/FLAER by flow cytometry. Samples from 20 healthy control subjects were used to calculate cut-off values for the "test" assay. Results By the "test" method, cut-off values for detecting PNH clones obtained from receiver operating-characteristic curve analysis were >0.4% for neutrophils (sensitivity=96.15%, specificity=95%), and >0.9% for monocytes (sensitivity=98.08%, specificity=95%). There was significant correlation between PNH clone sizes measured by both the "standard" and "test" assays in neutrophils (PNH disease: r=0.976, p<0.001; AA/MDS: r=0.980, p<0.001) as well as monocytes (PNH disease: r=0.806, p=0.005; AA/MDS: r=0.915, p<0.001). Bland-Altman analysis showed agreement between both assays in all the 52 patients and in individuals with AA/MDS. The cost of the test to the patients was about 15% less in the "test" method than the "standard" technique, with improved technical efficiency. Conclusion CD157 can replace both CD24 and CD14 in a single-tube flow-cytometric assay to detect PNH clones on both neutrophils and monocytes, with reduced cost to the patients and improved technical efficiency.

Real-world challenges in the management of acute myeloid leukemia: a single-center experience from North India.

BACKGROUND: The outcome of acute myeloid leukemia (AML) in low-middle-income countries (LMIC) is dismal due to delayed clinical presentation and infection-related complications. We aimed to analyze the outcome of patients with AML and the factors associated with its prognosis. METHODS: A retrospective observational study was conducted at a tertiary care university hospital in North India from January 2015 to December 2019. RESULTS: A total of 137 AML patients (median age 32 year (3-66 years) received intensive chemotherapy during study period. The median delay from diagnosis to treatment was 45 days (6-177 days). Among the 352 febrile neutropenia (FN) episodes analyzed, 175 (49.7%) were culture positive; Gram-negative multi-drug resistant organism (MDRO) sepsis during induction being 57.4% with 34.5% infections due to carbapenem-resistant Enterobacteriaceae (CRE) leading to a mortality rate of 14.6%. The median EFS and OS were 12.0 ± 1.57 (95% CI 8.91-15.08) and 15.0 ± 2.44 (95% CI 10.21-19.78) months respectively. Multivariable analysis revealed significant difference in median OS between favorable vs high risk AML groups (20.0 (95% CI: 12.50-27.49) vs 9.0 (95% CI: 2.99-15.01) months; p = 0.002); time from diagnosis to treatment (< 30 days vs ≥ 30 days; not reached vs 9.0 (95% CI: 6.81-11.18) months; p = 0.001), performance status (1 vs 2 vs 3; not reached vs 12.0 (95% CI: 10.32-13.67) vs 4.0 (95% CI:2.77-5.22); p = 0.001), and attainment of complete remission vs induction failure (not reached vs 6.0 (95% CI: 3.78-8.21); p = 0.002). CONCLUSION: Patient-related factors like delayed treatment initiation and high incidence of MDRO-associated sepsis are critical determinants of AML outcome in LMIC.

Listeria delivers tetanus toxoid protein to pancreatic tumors and induces cancer cell death in mice.

Pancreatic ductal adenocarcinoma (PDAC) is a highly metastatic disease. Tumors are poorly immunogenic and immunosuppressive, preventing T cell activation in the tumor microenvironment. Here, we present a microbial-based immunotherapeutic treatment for selective delivery of an immunogenic tetanus toxoid protein (TT856-1313) into PDAC tumor cells by attenuated Listeria monocytogenes. This treatment reactivated preexisting TT-specific memory T cells to kill infected tumor cells in mice. Treatment of KrasG12D,p53R172H, Pdx1-Cre (KPC) mice with Listeria-TT resulted in TT accumulation inside tumor cells, attraction of TT-specific memory CD4 T cells to the tumor microenvironment, and production of perforin and granzyme B in tumors. Low doses of gemcitabine (GEM) increased immune effects of Listeria-TT, turning immunologically cold into hot tumors in mice. In vivo depletion of T cells from Listeria-TT + GEM-treated mice demonstrated a CD4 T cell-mediated reduction in tumor burden. CD4 T cells from TT-vaccinated mice were able to kill TT-expressing Panc-02 tumor cells in vitro. In addition, peritumoral lymph node-like structures were observed in close contact with pancreatic tumors in KPC mice treated with Listeria-TT or Listeria-TT + GEM. These structures displayed CD4 and CD8 T cells producing perforin and granzyme B. Whereas CD4 T cells efficiently infiltrated the KPC tumors, CD8 T cells did not. Listeria-TT + GEM treatment of KPC mice with advanced PDAC reduced tumor burden by 80% and metastases by 87% after treatment and increased survival by 40% compared to nontreated mice. These results suggest that Listeria-delivered recall antigens could be an alternative to neoantigen-mediated cancer immunotherapy.

CD26 expression on circulating CD34+/CD38- progenitor population is a specific and reliable tool for the rapid flow cytometric diagnosis of chronic myeloid leukemia-A single-center validation study.

BACKGROUND: Recently, CD26 have been identified as one of the promising and specific marker for the identification of leukemic stem cells (LSCs) in chronic myeloid leukemia (CML). METHODS: This was a prospective, observational validation study. Peripheral blood (PB) samples from suspected cases of CML and other hematolymphoid neoplasm were evaluated for the expression of CD26 on stem cells (SC) (CD45 dim/CD34+/CD38-) fraction by flow cytometry (FCM) using a single tube four-color antibodies cocktail: CD45-V500 /CD26-PE/CD34-PerCPcy5.5/CD38-APC-H7. The diagnosis of CML was confirmed using cytogenetics and/or molecular studies. Additionally, 12 paired PB and bone marrow (BM) samples of CML cases were compared for the proportion of CD26+ LSCs. RESULTS: Expression of CD26 on the SC fraction was invariably noted in all cases (116/116) of CML, irrespective of the disease phase and transcript type. None of other neoplasm (0/26), including the Ph + ALLs expressed CD26. Proportion of SCs expressing CD26 was variable with a median (range) proportion being 61.3% (7.6%-98.6%). Evaluation of paired PB and BM samples showed similar proportion of CD26 + LSCs (R2 : 0.969). CONCLUSION: We confirmed that FCM evaluation of CD26 expression in the PB LSCs is a rapid and specific tool for CML diagnosis. Its utility as a marker for residual disease evaluation can also be explored in the future.

Direct N-Me Aziridination of Enones.

The first direct general method for N-Me aziridination of electron-deficient olefins, enones, is described using N-methyl-O-tosylhydroxylamine as the aminating agent in the presence of a Cu(OTf)2 catalyst. The aziridination of vinyl ketones, hitherto unknown for N-Me as well as N-H, has been achieved efficiently. The open-flask reaction is stereospecific, operationally simple, and additive-free. It also efficiently affords N-H aziridinated products under a similar reaction condition.

A Multi-copy Nucleic Acid-Based Diagnostic Test for Bovine Tropical Theileriosis.

BACKGROUND: Bovine tropical theileriosis (BTT) is a haemoprotozoan tick-borne disease that implicates huge losses to livestock in terms of considerable mortality and morbidity in tropical and subtropical regions of the globe. Currently available diagnostic methods have less specificity and sensitivity towards the detection of Theileria species. Therefore, an attempt was made to diagnose Theileria annulata by targeting a multi-copy gene, viz. mitochondrially encoded cytochrome b (MT-CYB) gene via polymerase chain reaction (PCR) in different agro-zones of India. METHODS AND RESULTS: 129 cattle blood samples were collected from major livestock rearing regions of India and processed for both molecular and microscopic techniques. Screening of Giemsa-stained thin blood smears was able to detect 14 samples (10.85%) as positive for T. annulata. However, the MT-CYB gene-based PCR assay detected 107 samples (82.94%) positive for T. annulata out of 129 samples. Furthermore, the MT-CYB gene-based PCR assay was standardized in terms of its sensitivity and specificity. Specificity of PCR assay was evaluated against other common haemoprotozoan parasites of tropical countries viz. Babesia bigemina, Anaplasma marginale and Trypanosoma evansi. The multi-copy MT-CYB gene-based PCR assay provided an optimum level of sensitivity (up to the level of 10 femtogram) and high specificity. Haematological examination (Hb, PCV and TLC) of 113 samples revealed significantly (p < 0.05) decreased Hb and PCV levels in positive animals in comparison with the control group of healthy animals. However, the control group had significantly higher (p < 0.001) TLC levels than the positive group. CONCLUSION: The MT-CYB gene-based PCR assay was found to be highly sensitive that can accurately detect the occurrence of T. annulata infection in carrier animals which are potential infection sources to healthier populations in naive demographic locations through infected ticks.

Development and validation of novel kit for quantification of SARS-CoV-2 antibodies on clinical samples.

Since the pandemic occurred due to the emergence of SARS-CoV-2, there has always been a demand for a simple and sensitive diagnostic kit for detection of SARS-Cov-2 infection. In January 2020, WHO approved the Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) for detecting the presence of Covid-19 genetic material in individuals. Till date many diagnostic kits have arrived in the market for quantification of SARS-CoV-2 antibodies. In spite of being the gold standard method of Covid-19 detection, there are some drawbacks associated with RT-PCR which leads to false-negative results. Hence, in order to fulfil the need for an antibody testing kit for evaluating seroconversion and immunity acquisition in the population, an efficient, highly specific and sensitive assay, Chimera Soochak, an enzyme-linked immunoassay (ELISA) Kit has been developed. It works on the principle of detecting IgG antibodies developed specifically against the S1-RBD by employing a recombinant strain of S1-RBD produced in the HEK293 cell line. The developed kit was validated using different modes and methods to attain the utmost confidence on the samples collected from patients. The validation methodology included, validation with known samples, blind study, third-party validation, validation using WHO Reference Panel and comparison with FDA approved Surrogate virus neutralization kit. The kit was found successful in detecting IgG against the S1-RBD of SARS-CoV-2. The kit had been validated on multiple parameters. A total of 900 samples had been tested by using this kit and it has exhibited the sensitivity, specificity and accuracy for all the above-mentioned parameters.