T Cell Receptor Gamma and Delta Gene Rearrangements in T-cell Acute Lymphoblastic Leukemia in South India and Quantitation of Minimal Residual Disease

Background: T cell-Acute Lymphoblastic Leukemia (T-ALL) arises by clonal proliferation of lymphoid precursors arrested at particular stage of differentiation. The incidence of T-ALL in India is 37-43% of ALL. In this study, TCR gamma (TCRG) and TCR delta (TCRD) gene rearrangements were detected in diagnostic samples of ALL. Those clonal rearrangements detected at diagnosis are used as clonal markers for the quantitation of minimal residual disease (MRD) in follow up samples. 

Patients and Methods: BM/PB from 54 T-ALL patients (34 pediatrics and 20 adults) at diagnosis, treated by MCP 841 protocol were studied. Median age of the patients was 13. The frequency of clonal TCRG and TCRD gene rearrangements were studied by Polymerase Chain Reaction (PCR) coupled with Heteroduplex analysis (HD). Allele Specific Oligos (ASO) was designed by sequencing the junctional region sequence of clonal TCRG and TCRD gene rearrangements. MRD was studied in 4 patients with follow up samples. Diagnostic DNA with almost 100% tumor involvement as standard was serially diluted (50ng to 5ng) in 500ng control DNA to give a final concentration of one leukemic cell in 101 (1 in 101) cells to one leuekemic cell in 105 (1 in 105) cells. The serially diluted diagnostic standard in triplicates was amplified with TaqMan probe for respective TCRG/TCRD gene rearrangements in Real-time PCR along with 500ng of follow up DNA samples at different time points (Unknown) in triplicates. Analysis was done to calculate the amount of leukemic cells in follow up samples. 

Results: Using PCR-HD analysis, TCRG gene rearrangements were detected in 37 of 54 cases (68.5%) and TCRD gene rearrangements in 16 of 54 cases (29.6%). VgI-Jg1.3/2.3 was more commonly rearranged in 29 cases (53.7%) of T-ALL; VgII-Jg1.3/2.3 in 14 cases (26%). Both VgIII-Jg1.3/2.3 and VgIV-Jg1.3/2.3 rearrangements were detected in 4 cases respectively and VgI-Jg1.1/2.1 was detected in 3 cases. Vd1-Jd1 rearrangement was detected in 9 cases (16.6%); Vd2-Dd3 in 5 cases and Dd2-Dd3 in 4 cases of T-ALL. Both TCRG and TCRD gene rearrangements were detected in 8 cases (14.8%). The junctional region in TCRG rearrangements ranged from 1nucleotide to 11nucleotide (average 7.6 nt) and in TCRD rearrangements ranged from 14 nucleotide to 42 nucleotide (average 27 nt). In patient 1, the initial leukeimia load of 1 (before treatment) was reduced to 3 leukemic cell in 104 cells at the lost follow up. In patient 2, the initial leukemia load was reduced to 3.7 in 104 cells. In Patient 3, the initial leukemia load 1 was reduced to 6 in 102 cells at end of M2 and disease relapsed at M5. In patient 4, the initial leukemia load was reduced to 1.6 leukemic cell in 104 cells. 

Conclusion: Real-time PCR experiments reached a reproducible sensitivity of detecting one leukemic cell in 104 normal cells. Real-time PCR analysis showed that Patient 3 was not all responded to treatment and it was detectable by Real-time PCR at RI1 stage itself though the disease was clinically evident only at M5 stage of treatment. Thus, monitoring the MRD using Real-time PCR will help to quantitate the accurate amount of residual leukemic load, predate relapse and assess the response to treatment. 
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Assessment of sustainable vermiconversion of water hyacinth by Eudrilus eugeniae and Eisenia fetida

The present work has assessed sustainable vermiconversion of aquatic weed water hyacinth (Eichornia crassipes). The garden soil, water hyacinth and cow dung were taken in the following the combinations of 1: 2 : 1, 2: 1: 1 and 1 : 1: 2. Two species of earthworms Eudrilus eugeniae and Eisenia fetida was used for the experiment. The total nitrogen (0.18% in control and 1.68% in earthworm treated) and phosphate (0.63 % in control and 1.64 % in earthworm treated) levels were increased and toxic heavy metals zinc (7.66 ppm in control and 2.58 ppm in earthworm treated) and copper (6.68 ppm in control and 1.15 ppm in earthworm treated) were significantly decreased. The earthworm enriches the compost with various nutrients for plant and microbial growth. Plant growth studies were conducted in all the combination of water hyacinth, maximum growth of root length (8.9cm and 7.2 in control) and shoot length (21.6cm and 16.2 in control) observed compare to control. Gut microbial analysis revealed that Bacillus cereus, Micrococcus luteus were predominantly present in the earthworm. The study recommended that the aquatic weed compost was suitable of agricultural usage

Potential of distillery effluents for safe water through vermifiltration

Vermifiltration of wastewater using waste eater earthworms is a newly conceived novel technology. The present study evaluated BOD, COD and TS showing significant variation in decrease by 95%, 90% and 80% respectively through vermifiltration of distillery effluents. The nutrient contents (TN, TP, TK, TCa and TMg) in the vermicasts had increase (1.82 % in TN, 0.24% in TP, 2.15% in TK, 2.07% in TCa and 2.86 % in TMg) in the range of fold than the control level. The morphology of the control and experimental vermicast samples were analyzed with SEM and the image showed significant variation. The FT-IR spectrum analysis showed reduction of aliphatic/aromatic (C=C and OH) compounds in the vermicompost. Thus, the present study significantly highlights the vermifiltration technology in treating distillery effluent

Strategies to modulate charge variants of a Biosmilar monoclonal antibody through cell culture conditions

Monoclonal antibodies (mAb) are the most successful and rapidly growing class of biopharmaceuticals used in treating several diseases. Biosimilar mAb, an approved version of an original biological medicinal product (reference product) with demonstrated similarity to the reference in terms of critical quality attributes, safety and efficacy, is an increasingly accepted solution to provide greater access at affordable cost to the patients across the world. But, given the complex nature of mAbs, developing a biosimilar using a new cell line and a process is challenging, especially with regards to matching the glycosylation and charge profiles to the appropriate level. It is reported that culture environment during the production of monoclonal antibody affects its various quality attributes including charge variant profiles1. The charge variants are usually formed due to chemical modification of amino acids by deamidation, oxidation, glycation and methylglyoxal adducts2, and may lead to increase in acidic charge variants. These unintended changes in the protein are mainly due to it being exposed during the long duration of the cell culture to an environment, like elevated temperature, nutrients from media and feed, metabolites from live and lysed cells, culture pH, which favours certain chemical modifications. Understanding and controlling cell culture process parameters are vital in developing a protein biologic to ensure process consistency and product quality. In the present study, we discuss a case study of development of a cell culture process to produce a proposed biosimilar mAb using a CHO cell line, and ways to modulate its charge variants in the cell culture. The initial screening experiments were performed in an ambr® 15 cell culture micro bioreactor system, from which an optimal 12-day process was chosen and subsequently tested in 3L and 10L bioreactors. Significant time-dependent increase in acidic charge variants was observed from day 10 to 12 at both bioreactor scales, while all other quality parameters remained largely unchanged during the last days of the culture. Further various strategies such as use of different basal media, feed, and additives (amino acids/metal ions and insulin), and changes in culture temperature and pH, were applied during the cell culture process to control the charge variants, in particular the acidic charge variants. The impact of various additives, cell culture pH, temperature on the charge profiles, as well as on productivity and glycosylation, during the development of this biosimilar mAb using a CHO cell line is discussed in detail. References: Liu, H., Nowak, C., Shao, M., Ponniah, G. and Neill, A. (2016), Impact of cell culture on recombinant monoclonal antibody product heterogeneity. Biotechnol Progress, 32: 1103–1112. Chumsae, C., Gifford, K., Lian, W., Liu, H., Radziejewski, C. H., & Zhou, Z. S. (2013). Arginine modifications by methylglyoxal: discovery in a recombinant monoclonal antibody and contribution to acidic species. Analytical chemistry, 85(23), 11401-11409

Anemia due to a rare anomaly - Case Report

Anemia due to gastrointestinal blood loss can occur due to many conditions and rarely to bowel structural anomalies. We report a 12 years old girl with anemia due to small bowel duplication cyst, posing diagnostic challenge intra operatively. Surgery offered cure without recurrence of bleeding. Common symptoms can be due to a rare surgical condition in practice

A study to evaluate association of nuclear grooving in benign thyroid lesions with RET/PTC1 and RET/PTC3 gene translocation

Abstract Introduction Papillary thyroid carcinoma (PTC) is the most common malignant lesion of the thyroid characterized by unique histological features like nuclear grooving, nuclear clearing, and intra-nuclear inclusions. However, nuclear grooves are observed even in benign thyroid lesions (BTL) like nodular goiter (NG), Hashimoto's thyroiditis (HT), and follicular adenoma (FA) resulting in diagnostic dilemma of the presence of PTC in such BTL. RET/PTC gene translocation is one of the most common oncogenic rearrangements seen in PTC, known to be associated with nuclear grooving. Among different types of RET/PTC translocations, RET/PTC1 and RET/PTC3 gene translocations are the most common types. These translocations have also been identified in many BTL like hyperplastic nodules and HT. Our study aimed to determine the frequency of nuclear grooving in BTL and evaluate their association with RET/PTC1 and RET/PTC3 gene translocation. Methods Formalin-fixed, paraffin-embedded (FFPE) tissue blocks of NG, HT, and FA were included in the study. The hematoxylin and eosin (H&E) stained sections were evaluated for the presence of nuclear grooving/high power field (hpf) and a scoring of 0 to 3 was used for the number of grooves. Sections of 10 μ thickness were cut and the cells containing the nuclear grooves were picked using Laser-Capture microdissection. About 20 to 50 such cells were microdissected in each of the cases followed by RNA extraction, cDNA conversion, realtime-polymerase chain reaction (RQ-PCR) for RET/PTC1 and RET/PTC3 gene translocation, and the findings were analyzed for statistical significance. Results Out of 87 BTL included in the study, 67 (77.0%) were NG, 12 (13.7%) were HT, and 8 (9.2%) were FA. Thirty-two cases (36.8%) had nuclear grooving with 18 out of 67 NG, 6 out of 12 HT, and all 8 cases of FA showing a varying number of nuclear grooves. A significant association between the number of nuclear grooves with RET/PTC gene translocation (p-value of 0.001) was obtained. A significant association of HT with RET/PTC gene translocation (p-value of 0.038) was observed. RET/PTC1 and RET/PTC3 translocation were seen in 5 out of 87 cases, with HT showing positivity in 2 and FA in 1 case for RET/PTC1 and HT in 1 and FA in 2 cases for RET/PTC3 gene translocation with 1 case of FA being positive for both RET/PTC1 and RET/PTC3 gene translocation. Conclusions The frequency of nuclear grooving among BTLs in our study was 36.8%. Our study shows, that when BTLs, show nuclear grooves, with an increase in the nuclear size, oval and elongated shape, favors the possibility of an underlying genetic aberration like RET/PTC gene translocation, which in turn supports the reporting pathologist to suggest a close follow up of the patients on seeing such nuclear features on cytology or histopathology sample, particularly in HT

Phosphorene, antimonene, silicene and siloxene based novel 2D electrode materials for supercapacitors-A brief review

In the past decade, 2D materials such as graphitic carbon nitride, transition metal dichalcogenides, layered metal oxides and hydroxides, hexagonal boron nitride and MXenes have garnered a great attention with the discovery of graphene. Very recently, novel 2D materials analogous to graphene such as phosphorene, antimonene, silicene, siloxene, germanene etc., were discovered and are emerging as strong competitors to the existing 2D materials. These materials pose explicit properties making them suitable for various applications. This article reviews the properties, synthesis techniques and the supercapacitive nature of phosphorene, antimonene, silicene and siloxene while briefing the properties of other 2D materials viz. germanene, stanene, arsenene and bismuthene. With the successful implementation of phosphorene as supercapacitor electrode, research is progressing in exploring the supercapacitive nature of other novel 2D materials. The investigations on these materials are still in its infancy and most of the properties lack experimental evidence. The current research trends on these novel materials are discussed in this review