All3048, a DnaJ III homolog of Anabaena sp. PCC7120 mediates heat shock response in E. coli and its N-terminus J-domain stimulates DnaK ATPase activity.

Cyanobacterial DnaJ offers thermo-tolerance and effectively prevents aggregation of denatured protein in coordination with DnaK. The hypothetical protein All3048 of Anabaena sp. PCC7120 was found to be a 24 kDa DnaJ III protein with a putative J-domain at the extreme N-terminus. This paper decodes the role of All3048 in thermo-tolerance and as a co-chaperon of DnaK. Semi-quantitative and RT-PCR results showed up-accumulation of all3048 in heat, UV-B, cadmium, arsenic and salt. BL21/pET-28a-all3048, all3048(1-95) and all3048(31-128) reduced the heat stress-induced ROS generation by 40 %, 21 % and 24 % as compared to BL21/pET-28-a. Conformational properties of All3048 and its truncated variants were assessed using bis ANS, guanidine hydrochloride and acrylamide quenching. All3048(1-95), All3048 and All3048(31-128) increased DnaK ATPase activity by 8.6, 8.2, and 2.5 fold, respectively. The thermostability investigated using DSC and DSF methods affirmed the relative stability of All3048 and All3048 (31-128), whereas All3048 (1-95) was the least stable. All3048 is a novel cyanobacterial DnaJ III that imparts heat stress tolerance in E. coli; however, only the J-domain present at N-terminus was sufficient for stimulating DnaK's ATPase activity.

Hypothyroxinemia in sick term neonates and its risk factors in an extramural neonatal intensive care unit: a prospective cohort study

ABSTRACT Objective: Thyroid functions in the sick newborns may be altered in the first week of life. Transient hypothyroxinemia has been reported in these babies, which could be due to the immaturity of the hypothalamic-pituitary-thyroid axis or to acute illness. We conducted this study to estimate the incidence of hypothyroxinemia and determine its risk factors in sick term newborns. Materials and methods: We analyzed free T4 (FT4) and thyroid-stimulating hormone (TSH) levels in sick term neonates (≤7 days of life) admitted to the neonatal intensive care unit. FT4 and TSH levels were estimated in the first week of life in all the enrolled neonates (N = 98) and then repeated at 14-21 days of life in 46 babies. Risk analysis was conducted using univariate and multivariate logistic regression, and numerical data was compared using the Mann-Whitney U test and t-test. Results: Hypothyroxinemia was seen in 10 (10.2%) of the admitted term babies. Male gender, vaginal delivery, presence of hypoxic ischemic encephalopathy, and need for mechanical ventilation (>24 hours) were identified as risk factors. There was a significant negative linear correlation between FT4 level in the first week of life and duration of hospital stay. Conclusion: Hypothyroxinemia is common in sick term neonates.

Transient hypothyroxinemia of prematurity and its risk factors in an extramural neonatal intensive care unit

ABSTRACT Objective: Thyroid functions in preterm newborns may be altered in the first week of life. Hypothyroxinemia has been commonly reported in these babies, which could be due to the immaturity of the hypothalamic pituitary thyroid axis or acute illness. It could have a long-term impact on the developing brain of these babies. We conducted this study to estimate the incidence of transient hypothyroxinemia of prematurity (THOP) and to determine its risk factors. Materials and methods: We analyzed thyroid stimulating hormone (TSH) and free T4 levels of 64 preterm neonates admitted in the neonatal intensive care unit. TSH and free T4 levels were measured in the first week and then at 14-21 days of life to estimate the incidence of THOP and determine its risk factors. We also estimated the incidence of congenital hypothyroidism (CH) and delayed TSH elevation in CH. Risk analysis was conducted using simple and multiple logistic regression, and numerical data was compared using the Mann Whitney U test and t test. Results: THOP was seen in 25% of the preterm babies. Caesarean delivery, presence of one or more morbidities, mechanical ventilation, birth weight ≥ 1,500 g, and gestational age ≥ 32 weeks were identified as risk factors for THOP based on simple logistic regression. In multiple regression, mechanical ventilation and gestational age ≥ 32 weeks were significantly associated with THOP. CH was seen in 2 (3.1%) babies, and 1 of these cases had delayed TSH elevation. Conclusion: Thyroid abnormalities are common in preterm admitted neonates. Mechanical ventilation is an independent risk factor for development of THOP.

Early hematological parameters as predictors for outcomes in children with dengue in northern India: A retrospective analysis

Abstract INTRODUCTION: Dengue presents with a variable clinical course, ranging from mild illness to potentially fatal hemorrhage and shock. We aimed to evaluate the capabilities of various hematological parameters observed early in the course of illness for predicting the clinical outcomes of illness. METHODS: We retrospectively analyzed the records of children admitted in the pediatric inpatient services of the institute with dengue between 2017 and 2019. We determined the relationships between the hematological parameters observed during the first evaluation and the various clinical outcomes. RESULTS: We evaluated data from 613 patients (age range, 26 days to 17 years). Of these, 29.85% exhibited fever with warning signs, and 8.97% had severe dengue. Lower values of hemoglobin, platelet count, mean corpuscular volume, mean corpuscular hemoglobin concentration, and mean platelet volume, and higher values of total leukocyte count (TLC), hematocrit, and red cell distribution width variably correlated with numerous clinical outcomes-duration of hospital stay, development of complications, requirement of blood component transfusion, inotropic support, and mortality. Among the parameters, TLC ≥20,000/mL and initial platelet count ≤20,000/mL significantly associated with mortality, with odds ratios (95% confidence interval) of 11.81 (4.21-33.80) and 5.53 (1.90-16.09), respectively. CONCLUSIONS: Hematological parameters observed early during dengue infection may predict its clinical outcomes in infected children. Initial high TLC and low platelet count are potential predictors of fatal outcomes in the course of disease.

Functional Characterization of Alr0765, A Hypothetical Protein from Anabaena PCC 7120 Involved in Cellular Energy Status Sensing, Iron Acquisition and Abiotic Stress Management in E. coli Using Molecular, Biochemical and Computational Approaches.

BACKGROUND: Cyanobacteria are excellent model to understand the basic metabolic processes taking place in response to abiotic stress. The present study involves the characterization of a hypothetical protein Alr0765 of Anabaena PCC7120 comprising the CBS-CP12 domain and deciphering its role in abiotic stress tolerance. METHODS: Molecular cloning, heterologous expression and protein purification using affinity chromatography were performed to obtain native purified protein Alr0765. The energy sensing property of Alr0765 was inferred from its binding affinity with different ligand molecules as analyzed by FTIR and TNP-ATP binding assay. AAS and real time-PCR were applied to evaluate the iron acquisition property and cyclic voltammetry was employed to check the redox sensitivity of the target protein. Transcript levels under different abiotic stresses, as well as spot assay, CFU count, ROS level and cellular H2O2 level, were used to show the potential role of Alr0765 in abiotic stress tolerance. In-silico analysis of Alr0765 included molecular function probability analysis, multiple sequence analysis, protein domain and motif finding, secondary structure analysis, protein-ligand interaction, homologous modeling, model refinement and verification and molecular docking was performed with COFACTOR, PROMALS-3D, InterProScan, MEME, TheaDomEx, COACH, Swiss modeller, Modrefiner, PROCHECK, ERRAT, MolProbity, ProSA, TM-align, and Discovery studio, respectively. RESULTS: Transcript levels of alr0765 significantly increased by 20, 13, 15, 14.8, 12, 7, 6 and 2.5 fold when Anabaena PCC7120 treated with LC50 dose of heat, arsenic, cadmium, butachlor, salt, mannitol (drought), UV-B, and methyl viologen respectively, with respect to control (untreated). Heterologous expression resulted in 23KDa protein observed on the SDS-PAGE. Immunoblotting and MALDI-TOF-MS/MS, followed by MASCOT search analysis, confirmed the identity of the protein and ESI/MS revealed that the purified protein was a dimer. Binding possibility of Alr0765 with ATP was observed with an almost 6-fold increment in relative fluorescence during TNP-ATP binding assay with a λ max of 538 nm. FTIR spectra revealed modification in protein confirmation upon binding of Alr0765 with ATP, ADP, AMP and NADH. A 10-fold higher accumulation of iron was observed in digests of E. coli with recombinant vector after induction as compared to control, which affirms the iron acquisition property of the protein. Moreover, the generation of the redox potential of 146 mV by Alr0765 suggested its probable role in maintaining the redox status of the cell under environmental constraints. As per CFU count recombinant, E. coli BL21 cells showed about 14.7, 7.3, 6.9, 1.9, 3 and 4.9 fold higher number of colonies under heat, cadmium (CdCl2), arsenic (Na3AsO4), salt (NaCl), UV-B and drought (mannitol) respectively compared to pET21a harboring E. coli BL21 cells. Deterioration in the cellular ROS level and total cellular H2O2 concentration validated the stress tolerance ability of Alr0765. In-silico analysis unraveled novel findings and attested experimental findings in determining the role of Alr0765. CONCLUSION: Alr0765 is a novel CBS-CP12 domain protein that maintains cellular energy level and iron homeostasis which provides tolerance against multiple abiotic stresses.

Alr2954 of Anabaena sp. PCC 7120 with ADP-ribose pyrophosphatase activity bestows abiotic stress tolerance in Escherichia coli.

In silico derived properties on experimental validation revealed that hypothetical protein Alr2954 of Anabaena sp. PCC7120 is ADP-ribose pyrophosphatase, which belongs to nudix hydrolase superfamily. Presence of ADP-ribose binding site was attested by ADP-ribose pyrophosphatase activity (K m 44.71 ± 8.043 mM, V max 7.128 ± 0.417 µmol min-1 mg protein-1, and K cat/K m 9.438 × 104 µM-1 min-1). Besides ADP-ribose, the enzyme efficiently hydrolyzed various nucleoside phosphatases such as 8-oxo-dGDP, 8-oxo-dADP, 8-oxo-dGTP, 8-oxo-dATP, GDP-mannose, ADP-glucose, and NADH. qRT-PCR analysis of alr2954 showed significant expression under different abiotic stresses reconfirming its role in stress tolerance. Thus, Alr2954 qualifies to be a member of nudix hydrolase superfamily, which serves as ADP-ribose pyrophosphatase and assists in multiple abiotic stress tolerance.

A randomized, double-blind, placebo-controlled trial of ormeloxifene in breast pain and nodularity.

BACKGROUND: Breast pain and non-discrete breast nodularity are common in women. METHODS: We did a randomized, double-blind, placebocontrolled trial of oral ormeloxifene 30 mg, a selective oestrogen receptor modulator (SERM) or placebo twice a week for 3 months in 20-50-year-old women with breast pain with or without lumpiness. Women with a discrete benign lump or cancer were excluded from the study. Serial assessments of pain on a visual analogue scale and nodularity grade on a 5-point ordinal Lucknow-Cardiff scale were done. A total of 151 patients were randomly allocated to two interventions using a block size of 4. RESULTS: Of the 151 patients, 121 (active 57, placebo 64) were available for efficacy analysis. The mean pain level showed a systematic downward trend over five visits (F=105.23, p<0.0001) that significantly reduced in the active group compared to that in the placebo group (F=18.66, p<0.0001). The patterns of variation in pain over time for the individual groups differ from the overall mean pattern for the two groups and thus from one another (F=44.43, p<0.0001). Cumulative frequencies of breast nodularity grades during successive visits showed significant improvement (p=0.001) compared to placebo at the end of the third month. The effect of the active drug persisted till the completion (6 months) of treatment (p<0.001). At the last visit, 93.3% of women in the active group had grade 2 or lower nodularity as compared to 71.1% in the placebo group. Oligomenorrhoea alone was reported by 12 patients. CONCLUSION: Ormeloxifene showed significant efficacy for treating breast pain and nodularity.

Human sodium iodide symporter (hNIS) in fibroadenoma breast--a immunohistochemical study.

Human sodium iodide symporter (hNIS), responsible for the active transport of iodine is an integral plasma membrane glycoprotein present in the thyroid cells and extrathyroid tissues like breast and salivary glands. If its functional form is unequivocally shown in benign or malignant breast tissues, then it may serve as a basis for diagnosis and treatment using radioactive iodine. With an aim to analyze the hNIS expression in a distinct benign breast condition of fibroadenoma, biopsy proven fibroadenoma tissues, normal non-lactating breast tissue and biopsy proven infiltrating duct carcinoma tissues were examined for hNIS expression using immunohistochemistry. Out of 20 biopsy proven fibroadenoma tissues, 19 (95%) showed positivity for hNIS protein and only one was negative. Of these 10% were mildly positive, 50% cases were moderately positive and 35% showed intense positivity. None of the control tissue obtained from reduction mammoplasty specimens or normal breast tissues samples (5 cms away from the tumor) were positive, hNIS was also intensely positive in 9 out of 10 (90%) infiltrating duct carcinoma tissues and moderately positive in one case. These preliminary results show that hNIS was present in high frequency as demonstrated by immunohistochemistry in fibroadenoma breast.

Double fetus in fetu.

Fetus in fetu (FIF) is an extremely rare cause of infantile abdominal mass where a rudimentary, malformed monozygotic-diamniotic twin grows inside the other twin. We describe a male infant with double or twin fetuses in fetu. The diagnosis was made on a computerized tomography (CT) scan of the abdomen and confirmed on surgery. Surgical excision was done and the baby did well post operatively.