Insights into the evolution of mutations in SARS-CoV-2 non-spike proteins.

The COVID-19 pandemic has been driven by the emergence of SARS-CoV-2 variants with mutations across all the viral proteins. Although mutations in the spike protein have received significant attention, understanding the prevalence and potential impact of mutations in other viral proteins is essential for comprehending the evolution of SARS-CoV-2. Here, we conducted a comprehensive analysis of approximately 14 million sequences of SARS-CoV-2 deposited in the GISAID database until December 2022 to identify prevalent mutations in the non-spike proteins at the global and country levels. Additionally, we evaluated the energetics of each mutation to better understand their impact on protein stability. While the consequences of many mutations remain unclear, we discuss potential structural and functional significance of some mutations. Our study highlights the ongoing evolutionary process of SARS-CoV-2 and underscores the importance of understanding changes in non-spike proteins.

Synchronous presentation of ETV6::RUNX1 fusion positive concordant B-acute lymphoblastic leukaemia in identical twin toddlers.

Concordant leukaemia in identical twins is rare. The likelihood of concordance of leukaemia in twins is near 100% in infancy, around 10% from 1 to 6 years of age, and rare at a later age with variable latency. Reporting of new cases of concordant leukaemia in twins is encouraged to contribute to data pool of this infrequent but exceptional condition; especially when the theories with respect to evolution, natural history and molecular evidence explaining concordant leukaemia in identical twins are still evolving.We discuss identical pair of monochorionic twin toddlers who were detected to have pallor and blood investigations revealed pancytopenia. Further work up including bone marrow studies revealed synchronous diagnosis of B-acute lymphoblastic leukaemia (B-ALL) with ETV6::RUNX1 fusion. Synchronous presentation of concordant leukaemia in identical twins is extremely rare. Index twins are the only second set of twins and first one beyond infantile age with synchronous presentation of B-ALL.

Complete resolution of primary myelofibrosis in an infant with steroids and hydroxyurea.

Paediatric primary myelofibrosis (PMF) is exceedingly rare and distinct compared with adult PMF. It is characterised by peripheral blood cytopenias, leucoerythroblastosis, reticulin fibrosis, extramedullary haematopoiesis and hepatosplenomegaly. In the absence of laid down diagnostic criteria, the diagnosis is largely of exclusion. Though early haematological stem cell transplant (HSCT) remains the treatment of choice, spontaneous remission or remission with steroids and/or cytoreductive agents is described in around 20% of cases of paediatric PMF. Moreover, HSCT in paediatric PMF is associated with high mortality (30%-45%). Therefore, it may be prudent to consider a trial of steroids and/or cytoreductive agents in all transfusion-dependent paediatric PMF while considering HSCT and ongoing bone marrow donor search. We describe one such infant with PMF who had complete remission of clinical and haematological parameters with a combination therapy of steroids and hydroxyurea.

Clinico-Hematological Profile of Paroxysmal Nocturnal Hemoglobinuria in Indian Patients: FLAER Flow Cytometry Based Experience from an Indian Tertiary Care Centre.

PNH is a rare disease with wide spectrum of intra-vascular hemolysis and thrombosis to sub-clinical PNH clones. We aimed to study the clinico-hematological profile and clone size on granulocytes and monocytes of PNH patients classified as per International PNH Interest Group recommendations. A retrospective analysis of clinico-hematological profile of 112 PNH clone positive patients by FLAER based flow cytometry between January and September 2017 done and classified into classical PNH, PNH with aplastic anemia or myelodysplastic syndrome (PNH-AA/MDS) and sub-clinical PNH clones (PNH-sc). Of 112 patients, majority were PNH-sc (62) followed by PNH-AA/MDS (34) and classical PNH (16). The commonest clinical feature was anemia in all 3 groups followed by jaundice (87.5%) in classical PNH and fever in PNH-AA/MDS (64.7%) and PNH-sc (48.4%). Thrombosis was present in 25% (4/16) classical PNH and 2.9% (1/34) of PNH-AA/MDS. The mean hemoglobin, reticulocyte count and LDH was higher in classical PNH. Bone marrow was predominantly hypercellular in classical PNH (11/16) and hypocellular in PNH-AA/MDS (31/34) and PNH-sc (50/62) with dyserythropoiesis predominantly in PNH-AA/MDS (83.8%) and PNH-sc (74.1%). Marrow iron was reduced in 62.2% classical PNH contrary to increased in PNH-BMF (58%) and PNH-sc (91%). The mean clone size in PNH-sc was significantly lower with > 50% in 16.2% patients. Three patients with MDS-MLD and MDS-MLD-RS in PNH-sc had > 80% clone on granulocytes and monocytes. Most PNH patients in Indian setting are PNH-sc with significantly lower clone, however, a clone size > 50% is not uncommon in Indian PNH-sc.

Sigma Metrics: A Valuable Tool for Evaluating the Performance of Internal Quality Control in Laboratory.

Background Six Sigma is a widely accepted quality management system that provides an objective assessment of analytical methods and instrumentation. Six Sigma scale typically runs from 0 to 6, with sigma value above 6 being considered adequate and 3 sigma being considered the minimal acceptable performance for a process. Methodology Sigma metrics of 10 biochemistry parameters, namely glucose, triglycerides, high-density lipoprotein (HDL), albumin, direct bilirubin, alanine transaminase, aspartate transaminase, urea nitrogen, creatinine and uric acid, and hematology parameters such as hemoglobin (Hb), total leucocyte count (TLC), packed cell volume (PCV), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), and platelet were calculated by analyzing internal quality control (IQC) data of 3 months (June-August 2019). Results Sigma value was found to be > 6 for triglyceride, HDL, Hb, TLC, and MCH, signifying excellent results and no further modification with respect to IQC. Sigma value was between 3 and 6 for glucose, albumin, creatinine, uric acid, PCV, and MCHC, implying the requirement of improvement in quality control (QC) processes. Sigma value of < 3 was seen in AST, ALT, direct bilirubin, urea nitrogen, platelet, and MCV, signifying suboptimal performance. Discussion Six Sigma provides a more quantitative framework for evaluating process performance with evidence for process improvement and describes how many sigmas fit within the tolerance limits. Thus, for parameters with sigma value < 3, duplicate testing of the sample along with three QCs three times a day may be used along with stringent Westgard rules for rejecting a run. Conclusion Sigma metrics help assess analytical methodologies and augment laboratory performance.

Utility of Lupus Anticoagulant Assays (APTT-LA, KCT, DPT and DRVVT) in Detection of Antiphospholipid Syndrome (APS) in High Risk Pregnancy Cases.

Routine investigation for recurrent pregnancy loss includes measurement of antiphospholipid antibodies. The lupus anticoagulant has long been associated with increased risks for thrombosis and adverse obstetric outcomes. But there are some disadvantages with lupus anticoagulant (LAC) tests which includes varied sensitivity of different clot based assays. ISTH recommends only 2 assays (preferably DRVVT and APTT-LA) for the identification of lupus anticoagulant but there are some studies which don't support this contention. Our study analyzed 526 samples from high risk pregnancy cases for APLA by all four LAC tests from tertiary centre of northern India. Among all the cases studies 65 cases were positive for lupus anticoagulant 25 of this became negative after 12 weeks. Among the 40 repeated positive assays, dRVVT could able to diagnose 36 cases followed by APTT-LA which could able to diagnose 28 cases, while KCT could able to diagnose 23 cases and dPT could able to diagnose only 14 cases. There were 12 cases in whom all lupus assays were positive. Our study thus concluded that DRVVT was the most sensitive followed by APPT-LA, KCT, dPT. The combination of dRVVT with APTT-LA or KCT appeared to be superior to other combinations. No individual test per se is 100% sensitive for the diagnosis of APLA in high risk pregnancy cases. Further results confirmed that repeated LAC result is required even in a high-risk setting. Positive LAC assay in majority were not associated with exclusively recurrent pregnancy loss but were associated with sporadic stillbirth and thrombosis.

Utility of Labile Plasma Iron Assay in Thalassemia Major Patients.

Labile plasma iron (LPI) levels are proposed as marker of iron overload in thalassemia patients and are also known to be the earliest parameter to indicate efficacy of chelation therapy. It was a prospective study in 35 patients of thalassemia major. Patients were recruited in two groups-group A (n = 13) patients not on chelation therapy and group B (n = 22) patients who were on regular oral chelation therapy. Ten age and gender matched healthy controls were also studied. For all patients, ferritin levels and LPI levels were measured at baseline, 6 months and 12 months. For group B patients paired samples for LPI were taken (before and 2 h after chelator). LPI levels were found to be significantly higher in group B patients versus group A patients versus normal healthy controls at all time-points. (P value-< 0.0001, 0.001) In group A, both LPI levels and ferritin levels follow an upward trend and correlated well with each other (P value-< 0.0001). In group B, the serum ferritin trend was not significant over follow up period of 1 year (P value 0.16), however LPI levels showed a significant decreasing trend on continued chelation (P value 0.0347) In patients on chelation therapy, the immediate change (2 h) in LPI levels on administration of chelators was not found to be significant (P value 0.22). LPI assay appears potentially attractive alternate to serum ferritin and can serve to monitor the trend of iron overload during long-term follow up.

Factor VII deficiency - an enigma; clinicohematological profile in 12 cases.

OBJECTIVE: Factor VII deficiency is the commonest of the rare bleeding disorders with limited knowledge on clinical profile. The objective of this study was to study the prevalence and clinico-hematological profile of factor VII-deficient patients. METHODS: It is a retrospective observational study of probable inherited factor VII deficiency covering 18 months. Their clinical profile, family history, investigation and treatment records were studied in detail. RESULTS: The study group comprised of total 12 factor VII deficiency cases with mean age of 17.5 years of onset of symptoms. The commonest symptom was menorrhagia (41.6%) followed by epistaxis (25%) and easy bruisability (16.6%). These 12 patients when categorized according to bleeding severity: severe bleeding - 2, moderate bleeding - 3, mild bleeding - 6 and asymptomatic - 1. All cases had prolonged prothrombin time (PT) with mean PT of 35.4 seconds (range 18-50 seconds) and mean prolongation of PT from upper limit of normal - 19.4 seconds (range 2-34 seconds). Factor VII levels ranged from < 1-40% in these patients. Clinical symptoms were not in concordance with factor levels. Of 12 patients, required treatment other than local measures. DISCUSSION AND CONCLUSION: Inherited factor VII deficiency is the commonest autosomally inherited factor deficiency with marked variation in the age of presentation and clinical symptoms. The laboratory results in form of PT and factor VII levels do not correlate with the severity of clinical presentation. A comprehensive evaluation to exclude acquired causes of factor VII deficiency, e.g. obesity, liver diseases, vitamin K deficiency and acquired inhibitors is required before labeling it as inherited in the absence of family history and molecular studies.

Syndecan-1 (sCD138) levels in chronic lymphocytic leukemia: clinical and hematological correlations.

BACKGROUND: Syndecan-1 (sCD138) has recently been suggested to predict the clinical course of early-stage chronic lymphocytic leukemia (CLL), but few studies have been reported. This study assessed the role of syndecan-1 in the prognosis of patients with CLL and its correlation with other prognostic markers. METHODS: This prospective study was performed in the hematology department of an Indian tertiary care center, over nineteen months (Jun. 2009-Jan. 2011). Forty-nine new patients with CLL presented during this period and were included. Twenty age- and gender-matched healthy patients served as controls, and six patients with multiple myeloma were included as positive controls. Baseline serum syndecan-1 concentrations were measured for all patients at presentation using ELISA (Diaclone, Besancon, France). At baseline, patients were divided into low (N=10), intermediate (N=18) and high (N=21) risk cohorts. Serum syndecan-1 levels in these patient subgroups were compared with clinical and laboratory parameters. RESULTS: The median syndecan-1 level in patients with CLL (73.32 ng/mL, range, 28.71-268.0 ng/mL) was marginally higher than that in healthy patients (63.10 ng/mL, range, 55.0-75.11 ng/mL). At presentation, syndecan-1 levels in patients with CLL correlated strongly with symptomatic disease (cytopenias, P=0.004) and higher clinical stage (Rai stage III and IV, P=0.001) markers and poorly with ß2-microglobulin level (P=0.270), diffuse BM infiltration (P=0.882), and surrogate mutation status markers (CD 38, P=0.174 and ZAP-70, P=0.459). Syndecan-1 levels dichotomized by the median value were higher with progressive disease markers, e.g. shorter lymphocyte doubling time (LDT, P=0.015) and increased treatment (P=0.099). CONCLUSION: In CLL, serum syndecan-1 (sCD138) levels at presentation correlate with disease burden, and higher baseline levels may predict early treatment.

Comparison of Immunohistochemistry, Cytochemistry, and Flow Cytometry in AML for Myeloperoxidase Detection.

Acute Myeloid Leukemia (AML) as per World Health Organization (WHO 2008) classification is on the basis of the antigenic characterization, enzymes restriction in the neoplastic myeloid cells and the specific translocations/mutations. AML can be assessed and differentiated by flowcytometry (FCM)/immunohistochemistry (IHC)/cytochemistry techniques. Myeloperoxidase (MPO) is an unequivocal marker to differentiate AML from the acute lymphoblastic leukemia. Despite FCM popularity, it has its limitations, in form of 'dry-tap', cost, and inability of being performed by retrospective analysis. IHC, though an old technique has overcome these disadvantages of FCM. Cytochemistry, on the other hand has its own advantages in being cost-effective; technically easy to do while its disadvantages are its inability to be carried out in the old samples, 'dry-tap' conditions in aleukemic leukemia. There has been non-uniformity in the literature among these techniques especially concerning their sensitivity for MPO. A prospective study was done at All India Institute of Medical Sciences New Delhi from 01 July 2014 to 30 Nov 2015 to include 120 diagnosed acute myeloid leukemia cases. Myeloperoxidase stain was done by cytochemistry, immunohistochemistry and flow cytometry and results were compared. There were 28 cases which showed discrepancies. Out of these 28 cases immunohistochemistry showed positivity in majority (22 cases) followed by flow cytometry (14 cases). Therefore it is important to employ more than one technique and IHC must be included for detection of MPO in all suspected cases of AML especially when negative with FCM .

Phytase from Citrobacter koseri PM-7: Enhanced production using statistical method and application in ameliorating mineral bioaccessibility and protein digestibility of high-phytate food.

The present study was aimed at enhancing phytase (Phy-Ck) production from Citrobacter koseri PM-7 using response surface methodology (RSM) and improving the bioaccessibility of minerals (Fe and Zn) and protein digestibility in high-phytate food using Phy-Ck. A five-variable and three-level central composite design of RSM using wheat bran (6.681%, w/v), inoculum level (2.5%, v/v), and triton X-100 (0.2%, v/v) resulted in up to 5.57-fold (1.047 U/ml) improvement in Phy-Ck yield from C. koseri PM-7 when compared with fermentation media I and II. The model was successfully validated in the design space by taking a random set of variable combinations. Treatment of high-phytate food with partially purified Phy-Ck showed improvement in mineral bioaccessibility maximally for defatted sesame flour (DSF) (Fe 45.5%; Zn 50.7%) followed by wheat flour (WF) (Fe 13.5%; Zn 14.4%), green gram flour (GGF) (Fe 0.7%; Zn 3.8%) and defatted groundnut flour (DGF) (Zn 5.6%). The in vitro protein digestibility (IVPD) of WF increased from 48.83 to 65.04%, GGF from 45.04 to 57.12%, and DSF from 47.34 to 55.7% after Phy-Ck treatment.

Arsenic tolerant Trichoderma sp. reduces arsenic induced stress in chickpea (Cicer arietinum).

Toxic metalloids including arsenic (As) can neither be eliminated nor destroyed from environment; however, they can be converted from toxic to less/non-toxic forms. The form of As species and their concentration determines its toxicity in plants. Therefore, the microbe mediated biotransformation of As is crucial for its plant uptake and toxicity. In the present study the role of As tolerant Trichoderma in modulating As toxicity in chickpea plants was explored. Chickpea plants grown in arsenate spiked soil under green house conditions were inoculated with two plant growth promoting Trichoderma strains, M-35 (As tolerant) and PPLF-28 (As sensitive). Total As concentration in chickpea tissue was comparable in both the Trichoderma treatments, however, differences in levels of organic and inorganic As (iAs) species were observed. The shift in iAs to organic As species ratio in tolerant Trichoderma treatment correlated with enhanced plant growth and nutrient content. Arsenic stress amelioration in tolerant Trichoderma treatment was also evident through rhizospheric microbial community and anatomical studies of the stem morphology. Down regulation of abiotic stress responsive genes (MIPS, PGIP, CGG) in tolerant Trichoderma + As treatment as compared to As alone and sensitive Trichoderma + As treatment also revealed that tolerant strain enhanced the plant's potential to cope with As stress as compared to sensitive one. Considering the bioremediation and plant growth promotion potential, the tolerant Trichoderma may appear promising for its utilization in As affected fields for enhancing agricultural productivity.

Selenite modulates the level of phenolics and nutrient element to alleviate the toxicity of arsenite in rice (Oryza sativa L.).

Arsenic (As) contamination of paddy rice is a serious threat all over the world particularly in South East Asia. Selenium (Se) plays important role in protection of plants against various abiotic stresses including heavy metals. Moreover, arsenite (AsIII) and selenite (SeIV) can be biologically antagonistic due to similar electronic configuration and sharing the common transporter for their uptake in plant. In the present study, the response of oxidative stress, phenolic compounds and nutrient elements was analyzed to investigate Se mediated As tolerance in rice seedlings during AsIII and SeIV exposure in hydroponics. Selenite (25µM) significantly decreased As accumulation in plant than As (25µM) alone treated plants. Level of oxidative stress related parameters viz., reactive oxygen species (ROS), lipid peroxidation, electrical conductivity, nitric oxide and pro-oxidant enzyme (NADPH oxidase), were in the order of As>As+Se>control>Se. Selenium ameliorated As phytotoxicity by increased level of phenolic compounds particularly gallic acid, protocatechuic acid, ferulic acid and rutin and thiol metabolism related enzymes viz., serine acetyl transferase (SAT) and cysteine synthase (CS). Selenium supplementation enhanced the uptake of nutrient elements viz., Fe, Mn, Co, Cu, Zn, Mo, and improved plant growth. The results concluded that Se addition in As contaminated environment might be an important strategy to reduce As uptake and associated phytotoxicity in rice plant by modulation of phenolic compounds and increased uptake of nutrient elements.

Effects of Local Heart Irradiation in a Glutathione S-Transferase Alpha 4-Null Mouse Model.

Glutathione S-transferase alpha 4 (GSTA4-4) is one of the enzymes responsible for the removal of 4-hydroxynonenal (4-HNE), an electrophilic product of lipid peroxidation in cellular membranes during oxidative stress. 4-HNE is a direct activator of nuclear factor (erythroid-derived 2)-like 2 (Nrf2), a transcription factor with many target genes encoding antioxidant and anti-electrophile enzymes. We have previously shown that Gsta4-null mice on a 129/Sv background exhibited increased activity of Nrf2 in the heart. Here we examined the sensitivity of this Gsta4-null mouse model towards cardiac function and structure loss due to local heart irradiation. Male Gsta4-null and wild-type mice were exposed to a single X-ray dose of 18 Gy to the heart. Six months after irradiation, immunohistochemical staining for respiratory complexes 2 and 5 indicated that radiation exposure had caused most pronounced alterations in mitochondrial morphology in Gsta4-null mice. On the other hand, wild-type mice showed a decline in cardiac function and an increase in plasma levels of troponin-I, while no such changes were observed in Gsta4-null mice. Radiation-induced Nrf2-target gene expression only in Gsta4-null mice. In conclusion, although loss of GSTA4-4 led to enhanced susceptibility of cardiac mitochondria to radiation-induced loss of morphology, cardiac function was preserved in Gsta4-null mice. We propose that this protection against cardiac function loss may occur, at least in part, by upregulation of the Nrf2 pathway.

Arsenite stress variably stimulates pro-oxidant enzymes, anatomical deformities, photosynthetic pigment reduction, and antioxidants in arsenic-tolerant and sensitive rice seedlings.

Contamination of arsenic (As) in rice (Oryza sativa L.) paddies and subsequent uptake by rice plants is a serious concern, because rice is a staple crop for millions of people. Identification of As toxicity and detoxification mechanisms in paddy rice cultivars would help to reduce As-associated risk. Arsenic tolerance and susceptibility mechanisms were investigated in 2 differential As-accumulating rice genotypes, Triguna and IET-4786, selected from initial screening of 52 rice cultivars as an As-tolerant and an As-sensitive cultivar, respectively, on the basis of root and shoot length during various arsenite (AsIII) exposures (0-50 µM). Indicators of oxidative stress, such as pro-oxidant enzymes (reduced nicotinamide adenine dinucleotide phosphate [NADPH] oxidase and ascorbate oxidase) and nitric oxide, were more numerous in the sensitive cultivar than in the tolerant cultivar. Arsenic-induced anatomical deformities were frequent in the sensitive cultivar, showing more distorted and flaccid root cells than the tolerant cultivar. Chlorophyll and carotenoid synthesis were inhibited in both cultivars, although the decline was more prominent in the sensitive cultivar at higher doses of As. Furthermore, the tolerant cultivar tolerated As stress by producing more antioxidants, such as proline, sustaining the ratio of ascorbate, dehydroascorbate, and glutathione peroxidase (GPX) activity as well as As detoxifying enzymes arsenate reductase, whereas these respective metabolic activities declined in sensitive cultivar, resulting in greater susceptibility to As toxicity.

A tocotrienol-enriched formulation protects against radiation-induced changes in cardiac mitochondria without modifying late cardiac function or structure.

Radiation-induced heart disease (RIHD) is a common and sometimes severe late side effect of radiation therapy for intrathoracic and chest wall tumors. We have previously shown that local heart irradiation in a rat model caused prolonged changes in mitochondrial respiration and increased susceptibility to mitochondrial permeability transition pore (mPTP) opening. Because tocotrienols are known to protect against oxidative stress-induced mitochondrial dysfunction, in this study, we examined the effects of tocotrienols on radiation-induced alterations in mitochondria, and structural and functional manifestations of RIHD. Male Sprague-Dawley rats received image-guided localized X irradiation to the heart to a total dose of 21 Gy. Twenty-four hours before irradiation, rats received a tocotrienol-enriched formulation or vehicle by oral gavage. Mitochondrial function and mitochondrial membrane parameters were studied at 2 weeks and 28 weeks after irradiation. In addition, cardiac function and histology were examined at 28 weeks. A single oral dose of the tocotrienol-enriched formulation preserved Bax/Bcl2 ratios and prevented mPTP opening and radiation-induced alterations in succinate-driven mitochondrial respiration. Nevertheless, the late effects of local heart irradiation pertaining to myocardial function and structure were not modified. Our studies suggest that a single dose of tocotrienols protects against radiation-induced mitochondrial changes, but these effects are not sufficient against long-term alterations in cardiac function or remodeling.