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.

Functional characterization of two WD40 family proteins, Alr0671 and All2352, from Anabaena PCC 7120 and deciphering their role in abiotic stress management.

WD40 domain-containing proteins are one of the eukaryotes' most ancient and ubiquitous protein families. Little is known about the presence and function of these proteins in cyanobacteria in general and Anabaena in particular. In silico analysis confirmed the presence of WD40 repeats. Gene expression analysis indicated that the transcript levels of both the target proteins were up-regulated up to 4 fold in Cd and drought and 2-3 fold in heat, salt, and UV-B stress. Using a fluorescent oxidative stress indicator, we showed that the recombinant proteins were scavenging reactive oxygen species (ROS) (4-5 fold) more efficiently than empty vectors. Chromatin immunoprecipitation analysis (ChIP) and electrophoretic mobility shift assay (EMSA) revealed that the target proteins function as transcription factors after binding to the promoter sequences. The presence of kinase activity (2-4 fold) in the selected proteins indicated that these proteins could modulate the functions of other cellular proteins under stress conditions by inducing phosphorylation of specific amino acids. The chosen proteins also demonstrated interaction with Zn, Cd, and Cu (1.4-2.5 fold), which might stabilize the proteins' structure and biophysical functions under multiple abiotic stresses. The functionally characterized Alr0671 and All2352 proteins act as transcription factors and offer tolerance to agriculturally relevant abiotic stresses.

Alr0671 and All2352 are novel WD40 proteins of Anabaena capable of regulating biochemical functions and abiotic stress tolerance by acting as a transcription factor and mediating DNA-protein interaction.

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.

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

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.

Hits and misses in treatment of ESCPM gram negative infections.

Treatment of Enterobacter infections is complex and often associated with development of resistance when wrong antibiotics are chosen for treatment despite in vitro susceptibility. This infectious diseases grand round highlights two cases, how antimicrobial and diagnostic stewardship approach could detect and prevent development of such resistance in - vivo.

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.

Regulation of antioxidant defense and glyoxalase systems in cyanobacteria.

Oxidative stress is common consequence of abiotic stress in plants as well as cyanobacteria caused by generation of reactive oxygen species (ROS), an inevitable product of respiration and photosynthetic electron transport. ROS act as signalling molecule at low concentration however, when its production exceeds the endurance capacity of antioxidative defence system, the organisms suffer oxidative stress. A highly toxic metabolite, methylglyoxal (MG) is also produced in cyanobacteria in response to various abiotic stresses which consequently augment the ensuing oxidative damage. Taking recourse to the common lineage of eukaryotic plants and cyanobacteria, it would be worthwhile to explore the regulatory role of glyoxalase system and antioxidative defense mechanism in combating abiotic stress in cyanobacteria. This review provides comprehensive information on the complete glyoxalase system (GlyI, GlyII and GlyIII) in cyanobacteria. Furthermore, it elucidates the recent understanding regarding the production of ROS and MG, noteworthy link between intracellular MG and ROS and its detoxification via synchronization of antioxidants (enzymatic and non-enzymatic) and glyoxalase systems using glutathione (GSH) as common co-factor.

Does Malaria Co-Infection Alter the Clinical Course in Children Infected with Dengue? Analysis from 623 Indian Children Admitted with Dengue Infection.

BACKGROUND: Dengue and malaria co-infection has been reported in several case reports. We aim to study effect of malaria co-infection on clinical outcomes of dengue infection. METHODS: Records of 623 children with dengue infection, based on NS-1 antigen and IgM ELISA testing, were collected. Malaria co-infection was identified in 20 cases, based on peripheral blood smear examination. Clinical and hematological parameters were compared in two groups (malaria co-infection vs. dengue mono-infection). RESULTS: Duration of hospitalization was significantly higher in co-infected group. Significantly higher proportion of malaria co-infection cases had hepatosplenomegaly, hemoglobin ≤8 g/dl, serum albumin ≤3 g/dl, serum bilirubin ≥1 mg/dl, serum aspartate aminotransferase ≥500 U/l and serum alanine aminotransferase ≥300 U/l. Number of transfusions (PRBC and platelets) required in malaria co-infection group was higher. CONCLUSION: Malaria co-infection in dengue impacts clinical presentation, hematological parameters, requirement of blood transfusion and morbidity. High index of suspicion is warranted while evaluating febrile patients.

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

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. 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.

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.

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.

Abacavir-based Regimen for HIV-infected Children and Adolescents.

We studied 48 children receiving abacavir-based HAART regimen, over a period of one-year for side effects and failure rates. None of the children developed hypersensitivity reaction. The CD4 count significantly improved from the time of enrolment till 12 months of therapy while the failure rate was 14.5%.

Alr2321, a multiple stress inducible glyoxalase I of Anabaena sp. PCC7120 detoxifies methylglyoxal and reactive species oxygen.

Abiotic stresses enhance the cellular level of reactive oxygen species (ROS) which consequently leads to toxic methylglyoxal (MG) production. Glyoxalases (GlyI & GlyII) catalyze the conversion of toxic MG into non-toxic lactic acid but their properties and functions have been overlooked in cyanobacteria. This is the first attempt to conduct a genome-wide analysis of GlyI protein (PF00903) from Anabaena sp. PCC7120. Out of total nine GlyI domain possessing proteins, only three (Alr2321, Alr4469, All1022) harbour conserve His/Glu/His/Glu metal binding site at their homologous position and are deficient in conserved region specific for Zn2+ dependent members. Their biochemical, structural and functional characterization revealed that only Alr2321 is a homodimeric Ni2+ dependent active GlyI with catalytic efficiency 11.7 × 106 M-1 s-1. It has also been found that Alr2321 is activated by various divalent metal ions and has maximum GlyI activity with Ni2+ followed by Co2+ > Mn2+ > Cu2+ and no activity with Zn2+. Moreover, the expression of alr2321 was found to be maximally up-regulated under heat (19 fold) followed by cadmium, desiccation, arsenic, salinity and UV-B stresses. BL21/pGEX-5X2-alr2321 showed improved growth under various abiotic stresses as compared to BL21/pGEX-5X2 by increased scavenging of intracellular MG and ROS levels. Taken together, these results suggest noteworthy links between intracellular MG and ROS, its detoxification by Alr2321, a member of GlyI family of Anabaena sp. PCC7120, in relation to abiotic stress.

Molecular and biochemical characterization of All0580 as a methylglyoxal detoxifying glyoxalase II of Anabaena sp. PCC7120 that confers abiotic stress tolerance in E. coli.

Abiotic stresses enhance cellular reactive oxygen species (ROS) level which results in toxic methylglyoxal (MG) production. Glyoxalases catalyze the conversion of toxic MG into non-toxic lactic acid whose properties and function are still unknown in cyanobacteria. This is the first attempt to characterize All0580 from Anabaena sp. PCC7120 as GlyII using in silico and wet lab approaches. Data of functional complementation of E. coli GlyII mutant (ΔgloB), enzyme kinetics and ESI-MS analysis suggested that All0580 harbors distinctive GlyII activity. The catalytic efficiency of All0580 (3 × 106 M-1 s-1) is higher than Arabidopsis GlyII. AAS analysis revealed the presence of a binuclear Zn/Fe centre in All0580 active site. The qRT-PCR of the target gene revealed maximum up-regulation in salinity followed by drought, arsenic, heat, and UV-B stresses. BL21/pET-21a-all0580 showed 1.5 to 10 fold increased growth and up to 4 fold decreased intracellular MG level as compared to BL21/pET-21a cells under various abiotic stresses and MG. A 39% drop in ROS generation by BL21/pET-21a-all0580 under MG stress suggested its potential to manage MG toxicity. Above attributes suggest that the hypothetical protein All0580 is a novel GlyII of cyanobacteria which heterologously confers tolerance to multiple abiotic stresses in E. coli.

Molecular characterization of two novel proteins All1122 and Alr0750 of Anabaena PCC 7120 conferring tolerance to multiple abiotic stresses in Escherichia coli.

In- silico and functional genomics approaches have been used to determine cellular functions of two hypothetical proteins All1122 and Alr0750 of Anabaena sp. PCC 7120. Motif analysis and multiple sequence alignment predicted them as typical α/ß ATP binding universal stress family protein-A (UspA) with G-(2×)-G-(9×)-G(S/T) as conserved motif. qRT-PCR data under UV-B, NaCl, heat, As, CdCl2, mannitol and methyl viologen registered approximately 1.4 to 4.3 fold induction of all1122 and alr0750 thus confirming their multiple abiotic stress tolerance potential. The recombinant E. coli (BL21) cells harboring All1122 and Alr0750 showed 12-41% and 23-41% better growth respectively over wild type control under said abiotic stresses thus revalidating their stress coping ability. Functional complementation on heterologous expression in UspA mutant E. coli strain LN29MG1655 (ΔuspA::Kan) attested their UspA family membership. This study tempted us to suggest that recombinant Anabaena PCC 7120 over expressing all1122 and alr0750 might contribute to the nitrogen economy in paddy fields experiencing array of abiotic stresses including drought and nutrient limitation.