A Functional Loop Spanning Distant Domains of Glutaminyl-tRNA Synthetase Also Stabilizes a Molten Globule State

Molten globule and other disordered states of proteins are now known to play important roles in many cellular processes. From equilibrium unfolding studies of two paralogous proteins and their variants, glutaminyl-tRNA synthetase (GlnRS) and two of its variants [glutamyl-tRNA synthetase (GluRS) and its isolated domains, and a GluRS-GlnRS chimera], we demonstrate that only GlnRS forms a molten globule-like intermediate at low urea concentrations. We demonstrated that a loop in the GlnRS C-terminal anticodon binding domain that promotes communication with the N-terminal domain and indirectly modulates amino acid binding is also responsible for stabilization of the molten globule state. This loop was inserted into GluRS in the eukaryotic branch after the archaea-eukarya split, right around the time when GlnRS evolved. Because of the structural and functional importance of the loop, it is proposed that the insertion of the loop into a putative ancestral GluRS in eukaryotes produced a catalytically active molten globule state. Because of their enhanced dynamic nature, catalytically active molten globules are likely to possess broad substrate specificity. It is further proposed that the putative broader substrate specificity allowed the catalytically active molten globule to accept glutamine in addition to glutamic acid, leading to the evolution of GlnRS.     

Hepatotoxic effects of tert-butyl hydroperoxide (t-BHP) and protection by antioxidants

t-BHP induced oxidative stress and Ca2+ function impairment in fresh hepatocytes was studied in order to understand its role in cytotoxicity. Viability of hepatocytes by the release of lactate dehydrogenase and methyl thiazoletetrazolium reduction method alongwith malondialdehyde formation indicated oxidative stress in the hepatotoxic action of t-BHP.     

Genetic and substrate-level modulation of Bacillus subtilis physiology for enhanced extracellular human interferon gamma production

Human interferon-gamma (hIFNG) production is limited by various gene-level bottlenecks including translation, protein folding, and secretion which depends upon the physiological state of the organism. In this study gene-level and substrate-level modulations have been used to control Bacillus subtilis physiology for >15 fold extracellular soluble hIFNG production. Two variants of the native human interferon-gamma gene (hifng) were designed and synthesized, namely, cohifng_his and cohifng having codon adaptation index 25.33 and 26.89% higher than the native gene, respectively. BScoIFNG and BScoIFNGhis with ΔG of ?100.0 and ?113.7?kcal?mol^?1 resulted in 30 and 6.5% higher hIFNG compared to the native gene in complex medium. BScoIFNG produced 1.53 fold higher hIFNG using glucose-based defined medium as compared to the complex medium by modulating the physiological parameter growth rate from 0.35 to 0.26?hr^?1. Further modulatory effect of various phosphotransferase transport system (PTS) and no-PTS sugars, sugar alcohols, and organic acids was quantified on the physiology of B. subtilis WB800N for extracellular hIFNG production. Sorbitol and glycerol emerged as the best hIFNG producers with lowest growth and substrate consumption rates. BScoIFNG produced maximum 3.15?mg?L^?1 hIFNG at 50?g?L^?1 glycerol with highest hIFNG yield (Y_p/x?=?0.136) and lowest substrate uptake rate (q_s?=?0.26).     

Women’s Awareness and Knowledge of Abortion Laws: A Systematic Review

Background

Incorrect knowledge of laws may affect how women enter the health system or seek services, and it likely contributes to the disconnect between official laws and practical applications of the laws that influence women’s access to safe, legal abortion services.

Objective

To provide a synthesis of evidence of women’s awareness and knowledge of the legal status of abortion in their country, and the accuracy of women’s knowledge on specific legal grounds and restrictions outlined in a country’s abortion law.

Methods

A systematic search was carried for articles published between 1980–2015. Quantitative, mixed-method data collection, and objectives related to women’s awareness or knowledge of the abortion law was included. Full texts were assessed, and data extraction done by a single reviewer. Final inclusion for analysis was assessed by two reviewers. The results were synthesised into tables, using narrative synthesis.

Results

Of the original 3,126 articles, and 16 hand searched citations, 24 studies were included for analysis. Women’s correct general awareness and knowledge of the legal status was less than 50% in nine studies. In six studies, knowledge of legalization/liberalisation ranged between 32.3% - 68.2%. Correct knowledge of abortion on the grounds of rape ranged from 12.8% – 98%, while in the case of incest, ranged from 9.8% - 64.5%. Abortion on the grounds of fetal impairment and gestational limits, varied widely from 7% - 94% and 0% - 89.5% respectively.

Conclusion

This systematic review synthesizes literature on women’s awareness and knowledge of the abortion law in their own context. The findings show that correct general awareness and knowledge of the abortion law and legal grounds and restrictions amongst women was limited, even in countries where the laws were liberal. Thus, interventions to disseminate accurate information on the legal context are necessary.     

Mitochondrial DNA mutations contribute to high altitude pulmonary edema via increased oxidative stress and metabolic reprogramming during hypobaric hypoxia

High altitude pulmonary edema (HAPE) is experienced by non-acclimatized sea level individuals on exposure to high altitude hypoxic conditions. Available evidence suggests that genetic factors and perturbed mitochondrial redox status may play an important role in HAPE pathophysiology. However, the precise mechanism has not been fully understood. In the present study, sequencing of mitochondrial DNA (mtDNA) from HAPE subjects and acclimatized controls was performed to identify pathogenic mutations and to determine their role in HAPE. Hypobaric hypoxia induced oxidative stress and metabolic alterations were also assessed in HAPE subjects. mtDNA copy number, mitochondrial oxidative phosphorylation (mtOXPHOS) activity, mitochondrial biogenesis were measured to determine mitochondrial functions. The data revealed that the mutations in Complex I genes affects the secondary structure of protein in HAPE subjects. Further, increased oxidative stress during hypobaric hypoxia, reduced mitochondrial biogenesis and mtOXPHOS activity induced metabolic reprogramming appears to contribute to mitochondrial dysfunctions in HAPE individuals. Haplogroup analysis suggests that mtDNA haplogroup H2a2a1 has potential contribution in the pathobiology of HAPE in lowlanders. This study also suggests contribution of altered mitochondrial functions in HAPE susceptibility.     

Mechanisms of resistance to an amino acid antibiotic that targets translation.

Structural and functional diversity among the aminoacyl-tRNA synthetases prevent infiltration of the genetic code by noncognate amino acids. To explore whether these same features distinguish the synthetases as potential sources of resistance against antibiotic amino acid analogues, we investigated bacterial growth inhibition by S-(2-aminoethyl)-L-cysteine (AEC). Wild-type lysyl-tRNA synthetase (LysRS) and a series of active site variants were screened for their ability to restore growth of an Escherichia coli LysRS null strain at increasing concentrations of AEC. While wild-type E. coli growth is completely inhibited at 5 microM AEC, two LysRS variants, Y280F and F426W, provided substantial resistance and allowed E. coli to grow in the presence of up to 1 mM AEC. Elevated resistance did not reflect changes in the kinetics of amino acid activation or tRNA (Lys) aminoacylation, which showed at best 4-6-fold improvements, but instead correlated with the binding affinity for AEC, which was decreased approximately 50-fold in the LysRS variants. In addition to changes in LysRS, AEC resistance has also been attributed to mutations in the L box riboswitch, which regulates expression of the lysC gene, encoding aspartokinase. The Y280F and F426W LysRS mutants contained wild-type L box riboswitches that responded normally to AEC in vitro, indicating that LysRS is the primary cellular target of this antibiotic. These findings suggest that the AEC resistance conferred by L box mutations is an indirect effect resulting from derepression of lysC expression and increased cellular pools of lysine, which results in more effective competition with AEC for binding to LysRS.     

hnRNPA1 couples nuclear export and translation of specific mRNAs downstream of FGF-2/S6K2 signalling

The increased cap-independent translation of anti-apoptotic proteins is involved in the development of drug resistance in lung cancer but signalling events regulating this are poorly understood. Fibroblast growth factor 2 (FGF-2) signalling-induced S6 kinase 2 (S6K2) activation is necessary, but the downstream mediator(s) coupling this kinase to the translational response is unknown. Here, we show that S6K2 binds and phosphorylates hnRNPA1 on novel Ser4/6 sites, increasing its association with BCL-XL and XIAP mRNAs to promote their nuclear export. In the cytoplasm, phosphoS4/6-hnRNPA1 dissociates from these mRNAs de-repressing their IRES-mediated translation. This correlates with the phosphorylation-dependent association of hnRNPA1 with 14-3-3 leading to hnRNPA1 sumoylation on K183 and its re-import into the nucleus. A non-phosphorylatible, S4/6A mutant prevented these processes, hindering the pro-survival activity of FGF-2/S6K2 signalling. Interestingly, immunohistochemical staining of lung and breast cancer tissue samples demonstrated that increased S6K2 expression correlates with decreased cytoplasmic hnRNPA1 and increased BCL-XL expression. In short, phosphorylation on novel N-term sites of hnRNPA1 promotes translation of anti-apoptotic proteins and is indispensable for the pro-survival effects of FGF-2.     

Locomotion with flexible propulsors: II. Computational modeling of pectoral fin swimming in sunfish

This paper describes a computational fluid dynamics (CFD) based investigation of the pectoral fin hydrodynamics of a bluegill sunfish. The pectoral fin of this fish undergoes significant shape-change during its abduction-adduction cycle and the effect of this deformation on the thrust performance remains far from understood. The current study is part of a combined experimental-numerical approach wherein the numerical simulations are being used to examine features and issues that are not easily amenable to the experiments. These numerical simulations are highly challenging and we briefly describe the computational methodology that has been developed to handle such flows. Finally, we describe some of the key computational results including wake vortex topologies and hydrodynamics forces.