High Serum Ferritin Levels among Blood Transfused Thalassemic Patients Admitted to the Department of Paediatrics in a Tertiary Care Centre: A Descriptive Cross-Sectional Study.

Introduction: Raised serum ferritin levels often indicate iron overload, but they are not specific as the levels are elevated in inflammatory disorders, liver diseases, alcohol excess, or malignancy. If regular transfusions are required for the patient with thalassemia, this doubles the rate of iron accumulation leading to earlier massive iron overload and iron-related damage. The aim of this study aimed to find out the prevalence of high serum ferritin levels among blood-transfused thalassemic patients admitted to the Department of Paediatrics in a tertiary care centre. Methods: A descriptive cross-sectional study was conducted at a tertiary care centre from 1 March 2022 to 31 December 2022. Ethical approval was taken from the Institutional Review Committee (Reference number: 078/79-017/HG). Children who were confirmed by haemoglobin electrophoresis on regular blood transfusion were included in the study. Those who did not gave consent were excluded from the study. Convenience sampling method was used. Point estimate and 90% Confidence Interval were calculated. Results: Out of 53 cases, the prevalence of high serum ferritin level was seen in 46 (88.79%) (80.30-97.28, 95% Confidence Interval). Among 46, 34 (73.91%) had serum ferritin levels of more than 1000 to 2500 ng/ml whereas 12 (26.09%) had more than 25000 ng/ml. Conclusions: The prevalence of high serum ferritin levels among blood transfused thalassemic patients admitted to the Department of Paediatrics in a tertiary care centre was found to be higher than in other studies done in similar settings. Keywords: blood transfusion; ferritin; thalassemia.

Genetic Screen for Identification of Multicopy Suppressors in Schizosaccharomyces pombe.

Identification of genetic interactions is a powerful tool to decipher the functions of gene(s) by providing insights into their functional relationships with other genes and organization into biological pathways and processes. Although the majority of the genetic screens were initially developed in Saccharomyces cerevisiae, a complementary platform for carrying out these genetic screens has been provided by Schizosaccharomyces pombe. One of the common approaches used to identify genetic interactions is by overexpression of clones from a genome-wide, high-copy-number plasmid library in a loss-of-function mutant, followed by selection of clones that suppress the mutant phenotype. This paper describes a protocol for carrying out this 'multicopy suppression'-based genetic screen in S. pombe. This screen has helped identify multicopy suppressor(s) of the genotoxic stress-sensitive phenotype associated with the absence of the Ell1 transcription elongation factor in S. pombe. The screen was initiated by transformation of the query ell1 null mutant strain with a high-copy-number S. pombe cDNA plasmid library and selecting the suppressors on EMM2 plates containing 4-nitroquinoline 1-oxide (4-NQO), a genotoxic stress-inducing compound. Subsequently, plasmid was isolated from two shortlisted suppressor colonies and digested by restriction enzymes to release the insert DNA. Plasmids releasing an insert DNA fragment were retransformed into the ell1 deletion strain to confirm the ability of these suppressor plasmid clones to restore growth of the ell1 deletion mutant in the presence of 4-NQO and other genotoxic compounds. Those plasmids showing a rescue of the deletion phenotype were sequenced to identify the gene(s) responsible for suppression of the ell1 deletion-associated genotoxic stress-sensitive phenotype.

Sequence, structural and functional conservation among the human and fission yeast ELL and EAF transcription elongation factors.

BACKGROUND: Transcription elongation is a dynamic and tightly regulated step of gene expression in eukaryotic cells. Eleven nineteen Lysine rich Leukemia (ELL) and ELL Associated Factors (EAF) family of conserved proteins are required for efficient RNA polymerase II-mediated transcription elongation. Orthologs of these proteins have been identified in different organisms, including fission yeast and humans. METHODS AND RESULTS: In the present study, we have examined the sequence, structural and functional conservation between the fission yeast and human ELL and EAF orthologs. Our computational analysis revealed that these proteins share some sequence characteristics, and were predominantly disordered in both organisms. Our functional complementation assays revealed that both human ELL and EAF proteins could complement the lack of ell1+ or eaf1+ in Schizosaccharomyces pombe respectively. Furthermore, our domain mapping experiments demonstrated that both the amino and carboxyl terminal domains of human EAF proteins could functionally complement the S. pombe eaf1 deletion phenotypes. However, only the carboxyl-terminus domain of human ELL was able to partially rescue the phenotypes associated with lack of ell1+ in S. pombe. CONCLUSIONS: Collectively, our work adds ELL-EAF to the increasing list of human-yeast complementation gene pairs, wherein the simpler fission yeast can be used to further enhance our understanding of the role of these proteins in transcription elongation and human disease.

Arabidopsis thaliana possesses two novel ELL associated factor homologs.

Transcription elongation is one of the key steps at which RNA polymerase II-directed expression of protein-coding genes is regulated in eukaryotic cells. Different proteins have been shown to control this process, including the ELL/EAF family. ELL Associated Factors (EAFs) were first discovered in a yeast two-hybrid screen as interaction partners of the human ELL (Eleven nineteen Lysine-rich Leukemia) transcription elongation factor. Subsequently, they have been identified in different organisms, including Schizosaccharomyces pombe. However, no homolog(s) of EAF has as yet been characterized from plants. In the present work, we identified EAF orthologous sequences in different plants and have characterized two novel Arabidopsis thaliana EAF homologs, AtEAF-1 (At1g71080) and AtEAF-2 (At5g38050). Sequence analysis showed that both AtEAF-1 and AtEAF-2 exhibit similarity with its S. pombe EAF counterpart. Moreover, both Arabidopsis thaliana and S. pombe EAF orthologs share conserved sequence characteristic features. Computational tools also predicted a high degree of disorder in regions towards the carboxyl terminus of these EAF proteins. We demonstrate that AtEAF-2, but not AtEAF-1 functionally complements growth deficiencies of Schizosaccharomyces pombe eaf mutant. We also show that only AtEAF-1 displays transactivation potential resembling the S. pombe EAF ortholog. Subsequent expression analysis in A. thaliana showed that both homologs were expressed at varying levels during different developmental stages and in different tissues tested in the study. Individual null-mutants of either AtEAF-1 or AtEAF-2 are developmentally normal implying their functional redundancy. Taken together, our results provide first evidence that A. thaliana also possesses functional EAF proteins, suggesting an evolutionary conservation of these proteins across organisms.

Functional interaction between ELL transcription elongation factor and Epe1 reveals the role of Epe1 in the regulation of transcription outside heterochromatin.

Eleven-nineteen lysine-rich leukemia (ELL) is a eukaryotic RNA polymerase II transcription elongation factor. In Schizosaccharomyces pombe, it is important for survival under genotoxic stress conditions. However, the molecular basis underlying this function of ELL in S. pombe is yet to be deciphered. Here, we carried out a genetic screen to identify multicopy suppressor(s) that could restore normal growth of ell1 deletion mutant in the presence of DNA damaging agent. Sequence analysis of the identified suppressors revealed the anti-silencing protein, Epe1, as one of the suppressors of ell1 deletion associated genotoxic stress sensitivity. Our results further demonstrate that the overexpression of Epe1 could suppress all other phenotypes associated with the absence of Ell1. Moreover, transcriptional defect of ell1Δ strain could also be alleviated by the overexpression of Epe1. Epe1 also showed a physical interaction with Ell1. Interestingly, we also observed that the region of Epe1 encompassing 403-948 amino acids was indispensable for all the above functions. Furthermore, our results show that the overexpression of Epe1 causes increased H3K9 acetylation and RNA polymerase II recruitment. Taken together, our results show a functional interaction between Epe1 and Ell1, and this function is independent of the well-known JmjC and N-terminal transcriptional activation domains of Epe1 in S. pombe.

Synthesis, In-Vitro and In-Silico Evaluation of Silver Nanoparticles with Root Extract of Withania somnifera for Antibacterial Activity via Binding of Penicillin-Binding Protein-4.

BACKGROUND: Metal Nanoparticles (NPs) have been widely used for various applications in biomedical sciences, including in drug delivery, and as therapeutic agents, but limited owing to their toxicity towards the healthy tissue. This warrants an alternative method, which can achieve the desired activity with much reduced or no toxicity. Being a biological product, Withania somnifera (W. somnifera) is environment friendly, besides being less toxic as compared to metal-based NPs. However, the exact mechanism of action of W. somnifera for its antibacterial activities has not been studied so far. OBJECTIVE: To develop "silver nanoparticles with root extract of W. somnifera (AgNPs-REWS)" for antimicrobial and anticancer activities. Furthermore, the analysis of their mechanism of action will be studied. METHODS: Using the in-silico approach, the molecular docking study was performed to evaluate the possible antibacterial mechanism of W. somnifera phytochemicals such as Anaferine, Somniferine, Stigmasterol, Withaferin A, Withanolide- A, G, M, and Withanone by the inhibition of Penicillin- Binding Protein 4 (PBP4). Next, we utilized a bottom-up approach for the green synthesis of AgNPs- REWS, performed an in-detail phytochemical analysis, confirmed the AgNPs-REWS by SEM, UVvisible spectroscopy, XRD, FT-IR, and HPLC. Eventually, we examined their antibacterial activity. RESULTS: The result of molecular docking suggests that WS phytochemicals (Somniferine, Withaferin A, Withanolide A, Withanolide G, Withanolide M, and Withanone) possess the higher binding affinity toward the active site of PBP4 as compared to the Ampicillin (-6.39 kcal/mol) reference molecule. These phytochemicals predicted as potent inhibitors of PBP4. Next, as a proof-of-concept, AgNPs- REWS showed significant antibacterial effect as compared to crude, and control; against Xanthomonas and Ralstonia species. CONCLUSION: The in-silico and molecular docking analysis showed that active constituents of W. somnifera such as Somniferine, Withaferin A, Withanolide A, Withanolide G, Withanolide M, and Withanone possess inhibition potential for PBP4 and are responsible for the anti-bacterial property of W. somnifera extract. This study also establishes that AgNPs via the green synthesis with REWS showed enhanced antibacterial activity towards pathogenic bacteria.

Detection of Disease-Specific Parent Cells Via Distinct Population of Nano-Vesicles by Machine Learning.

BACKGROUND: The diagnosis and prognosis of pathological conditions, such as age-related macular degeneration (AMD) and cancer still need improvement. AMD is primarily caused due to the dysfunction of retinal pigment epithelium (RPE), whereas endothelial cells (ECs) play one of the major roles in angiogenesis; an important process which occurs in malignant progression of cancer. Several reports suggested the augmented release of nano-vesicles under pathological conditions, including from RPE as well as cancer-associated ECs, which take part in various biological processes, including intercellular communication in disease progression. Importantly, these nano-vesicles are around 30-1000 nm and carry the fingerprint of their initiating parent cells (IPCs). Therefore, these nano-vesicles could be utilized as the diagnostic tool for AMD and cancer, respectively. However, the analysis of nano-vesicles for biomarker study is confounded by their extensive heterogeneous nature. METHODS: To confront this challenge, we utilized artificial intelligence (AI) based machine learning (ML) algorithms such as support vector machine (SVM) and decision tree model on the dataset of nano-vesicles from RPE and ECs cell lines with low dimensionality. RESULTS: Overall, Gaussian SVM demonstrated the highest prediction accuracy of the IPCs of nano-vesicles, among all the chosen SVM classifiers. Additionally, the bagged tree showed the highest prediction among the chosen decision tree-based classifiers. CONCLUSION: Therefore, the overall bagged tree showed the best performance for the prediction of IPCs of nanovesicles, suggesting the applicability of AI-based prediction approach in diagnosis and prognosis of pathological conditions, including non-invasive liquid biopsy via various biofluids-derived nano-vesicles.

Prevalence and causes of avoidable blindness and visual impairment, including the prevalence of diabetic retinopathy in Siwan district of Bihar, India: A population-based survey.

Purpose: The aim of this study was to estimate the prevalence and causes of visual impairment (VI) and blindness and diabetic retinopathy (DR) in Siwan district, Bihar. Methods: A population-based cross-sectional study was done from January to March 2016 using the Rapid Assessment of Avoidable Blindness 6 (RAAB 6, incorporating DR module) methodology. All individuals aged ≥50 years were examined in 57 randomly selected clusters within the district. Results: A total of 3476 individuals were enumerated and 3189 (92%) completed examination. The overall prevalence of blindness and severe VI was 2.2% (95% confidence interval (CI): 1.6-2.8) and 3.4% (95% CI: 2.6-4.3), respectively. Untreated cataract was the leading cause of blindness (73%) and severe VI (93%). The cataract surgical coverage (CSC) at <3/60 was 71.5% for eyes and 89.3% for persons in this sample and the CSC was similar between the genders. Refractive error (71%) was the primary cause of early VI. The overall prevalence of known and newly diagnosed diabetes was 6.3% (95% CI, 5.4-7.2%). Prevalence of any DR, maculopathy, and sight-threatening DR was 15, 12.4, and 6%, respectively. Conclusion: To conclude, as compared to previous reports, the prevalence of blindness and DR in Siwan district of Bihar was found to be lower and the CSC was higher. However, the problem of avoidable blindness remains a major problem in this region.

Structure function characterization of the ELL Associated Factor (EAF) from Schizosaccharomyces pombe.

EAF (ELL Associated Factor) proteins interact with the transcription elongation factor, ELL (Eleven nineteen Lysine rich Leukemia) and enhance its ability to stimulate RNA polymerase II-mediated transcriptional elongation in vitro. Schizosaccharomyces pombe contains a single homolog of EAF (SpEAF), which is not essential for survival of S. pombe in contrast to its essential higher eukaryotic homologs. The physiological role of SpEAF is not well understood. In this study, we show that S. pombe EAF is important in regulating growth of S. pombe cells during normal growth conditions. Moreover, SpEAF is also essential for survival under conditions of DNA damage, while its deletion does not affect growth under environmental stress conditions. Our in vivo structure-function studies further demonstrate that while both the amino and carboxyl terminal domains of SpEAF possess the potential to activate transcription, only the amino terminal domain of SpEAF is involved in interaction with the S. pombe ELL protein. The carboxyl-terminus of SpEAF is required for rescue of the growth defect under normal and DNA damaging conditions that is associated with the absence of SpEAF. Using bioinformatics and circular dichroism spectroscopy, we show that the carboxyl-terminus of SpEAF has a disordered conformation. Furthermore, addition of trifluoroethanol triggered its transition from a disordered to α-helical conformation. Taken together, the results presented here identify novel structural and functional features of SpEAF protein, providing insights into how EAF proteins may enforce transcriptional control of gene expression.

The amino-terminal domain of ELL transcription elongation factor is essential for ELL function in Schizosaccharomyces pombe.

Transcriptional elongation is a critical step for regulating expression of protein-coding genes. Multiple transcription elongation factors have been identified in vitro, but the physiological roles of many of them are still not clearly understood. The ELL (Eleven nineteen Lysine rich Leukemia) family of transcription elongation factors are conserved from fission yeast to humans. Schizosaccharomyces pombe contains a single ELL homolog (SpELL) that is not essential for its survival. Therefore to gain insights into the in vivo cellular functions of SpELL, we identified phenotypes associated with deletion of ell1 in S. pombe. Our results demonstrate that SpELL is required for normal growth of S. pombe cells. Furthermore, cells lacking ell1+ exhibit a decrease in survival when exposed to DNA-damaging conditions, but their growth is not affected under environmental stress conditions. ELL orthologs in different organisms contain three conserved domains, an amino-terminal domain, a middle domain and a carboxyl-terminal domain. We also carried out an in vivo functional mapping of these conserved domains within S. pombe ELL and uncovered a critical role for its amino-terminus in regulating all its cellular functions, including growth under different conditions, transcriptional elongation potential and interaction with S. pombe EAF. Taken together our results suggest that the domain organization of ELL proteins is conserved across species, but the in vivo functions as well as the relationship between the various domains and roles of ELL show species-specific differences.

Homotypic clustering of OsMYB4 binding site motifs in promoters of the rice genome and cellular-level implications on sheath blight disease resistance.

The promoter regions (1 kb upstream sequences) of 45,836 annotated genes of rice were analyzed for the presence of OsMYB4 binding sites using a Perl program algorithm. Based on the homotypic clustering concept, 113 promoters were found to have more than 4 binding site motifs. Among the downstream genes of these promoters, five genes which are known to have a role in disease resistance were selected and the binding capacity of OsMYB4 protein in the promoter regions was analyzed by docking studies. Expression level of these genes was analyzed by RT-PCR in Rhizoctonia solani infected rice seedlings. Upon pathogen challenge, higher expression of aminotransferase, ankyrin and WRKY 12 genes was observed corresponding to higher expression of Osmyb4. Over-expression of Osmyb4 cDNA in rice leaf tissues by agro-infection failed to result in similar over-expression of aminotransferase, ankyrin and WRKY 12 as expected. Although the role of OsMYB4 in sheath blight resistance was found to be definitive based on our initial results, artificial over-expression of this TF was observed to be insufficient in regulating the disease resistance related genes.

A comparison of glomerular filtration rate by creatinine based equations and DTPA-renogram in healthy adult kidney donors.

INTRODUCTION: Accurate determination of donor kidney function has important long-term implications for both donor health and recipient outcome. Many centers use 24 hour urinary creatinine clearance or creatinine-based GFR estimations to assess kidney function but their performance when compared with GFR measurements by isotope clearance remains inconclusive. We assessed the performance of creatinine based equations against DTPA GFR for evaluating Nepalese kidney donors. METHODS: All kidney donors who had undergone both DTPA GFR estimation and 24 hour urine CrCl were included. The performance of the urine-CrCl, CG-CrCl, modified MDRD GFR against DTPA GFR was evaluated by analyzing global bias, precision (R2),Pearson correlation and accuracy percentage within 30% and 15%. The sensitivity and specificity of each predictive equation in selecting donor with GFR of ≥80 mL/min/1.73 m2 was also calculated. RESULTS: Of 51 donors analysed, only 18 (35.29%) were male. The mean measured GFR was 102.752±16.71 mL/min/1.73 m2. Of all prediction equations, urine-CrCL has most precision (R2=0.207) with the highest pearson correlation (0.455) and highest accuracy percentage within 30% and 15%. However, predictive performance was poor for all the equations. The urine CrCl had highest sensitivity of 100% for detecting donor with measured GFR>80 mL/min/1.73 m2 with positive predictive value of 92.1%. CONCLUSIONS: The performance of all equations was disappointing and even the best performing equation urine-CrCl was suboptimal for donor selection. So considering the potential risk of living kidney donation, other more accurate methods of GFR estimation should be used.

The SbASR-1 gene cloned from an extreme halophyte Salicornia brachiata enhances salt tolerance in transgenic tobacco.

Salinity severely affects plant growth and development. Plants evolved various mechanisms to cope up stress both at molecular and cellular levels. Halophytes have developed better mechanism to alleviate the salt stress than glycophytes, and therefore, it is advantageous to study the role of different genes from halophytes. Salicornia brachiata is an extreme halophyte, which grows luxuriantly in the salty marshes in the coastal areas. Earlier, we have isolated SbASR-1 (abscisic acid stress ripening-1) gene from S. brachiata using cDNA subtractive hybridisation library. ASR-1 genes are abscisic acid (ABA) responsive, whose expression level increases under abiotic stresses, injury, during fruit ripening and in pollen grains. The SbASR-1 transcript showed up-regulation under salt stress conditions. The SbASR-1 protein contains 202 amino acids of 21.01-kDa molecular mass and has 79 amino acid long signatures of ABA/WDS gene family. It has a maximum identity (73 %) with Solanum chilense ASR-1 protein. The SbASR-1 has a large number of disorder-promoting amino acids, which make it an intrinsically disordered protein. The SbASR-1 gene was over-expressed under CaMV 35S promoter in tobacco plant to study its physiological functions under salt stress. T(0) transgenic tobacco seeds showed better germination and seedling growth as compared to wild type (Wt) in a salt stress condition. In the leaf tissues of transgenic lines, Na(+) and proline contents were significantly lower, as compared to Wt plant, under salt treatment, suggesting that transgenic plants are better adapted to salt stress.