Assembly constraints drive co-evolution among ribosomal constituents

Ribosome biogenesis, a central and essential cellular process, occurs through sequential association and mutual co-folding of protein–RNA constituents in a well-defined assembly pathway. Here, we construct a network of co-evolving nucleotide/amino acid residues within the ribosome and demonstrate that assembly constraints are strong predictors of co-evolutionary patterns. Predictors of co-evolution include a wide spectrum of structural reconstitution events, such as cooperativity phenomenon, protein-induced rRNA reconstitutions, molecular packing of different rRNA domains, protein–rRNA recognition, etc. A correlation between folding rate of small globular proteins and their topological features is known. We have introduced an analogous topological characteristic for co-evolutionary network of ribosome, which allows us to differentiate between rRNA regions subjected to rapid reconstitutions from those hindered by kinetic traps. Furthermore, co-evolutionary patterns provide a biological basis for deleterious mutation sites and further allow prediction of potential antibiotic targeting sites. Understanding assembly pathways of multicomponent macromolecules remains a key challenge in biophysics. Our study provides a ‘proof of concept’ that directly relates co-evolution to biophysical interactions during multicomponent assembly and suggests predictive power to identify candidates for critical functional interactions as well as for assembly-blocking antibiotic target sites.     

Comprehensive functional network analysis and screening of deleterious pathogenic variants in non-syndromic hearing loss causative genes

Hearing loss (HL) is a significant public health problem and causes the most frequent congenital disability in developed societies. The genetic analysis of non-syndromic hearing loss (NSHL) may be considered as a complement to the existent plethora of diagnostic modalities available. The present study focuses on exploring more target genes with respective non-synonymous single nucleotide polymorphisms (nsSNPs) involved in the development of NSHL. The functional network analysis and variant study have successfully been carried out from the gene pool retrieved from reported research articles of the last decade. The analyses have been done through STRING. According to predicted biological processes, various variant analysis tools have successfully classified the NSHL causative genes and identified the deleterious nsSNPs, respectively. Among the predicted pathogenic nsSNPs with rsIDs rs80356586 (I515T), rs80356596 (L1011P), rs80356606 (P1987R) in OTOF have been reported in NSHL earlier. The rs121909642 (P722S), rs267606805 (P722H) in FGFR1, rs121918506 (E565A) and rs121918509 (A628T, A629T) in FGFR2 have not been reported in NSHL yet, which should be clinically experimented in NSHL. This also indicates this variant’s novelty as its association in NSHL. The findings and the analyzed data have delivered some vibrant genetic pathogenesis of NSHL. These data might be used in the diagnostic and prognostic purposes in non-syndromic congenitally deaf children.     

Identification of two lipid phosphatases that regulate sphingosine-1-phosphate cellular uptake and recycling

Sphingosine-1-phosphate (S1P) is a sphingolipid metabolite that serves as a potent extracellular signaling molecule. Metabolic regulation of extracellular S1P levels impacts key cellular activities through altered S1P receptor signaling. Although the pathway through which S1P is degraded within the cell and thereby eliminated from reuse has been previously described, the mechanism used for S1P cellular uptake and the subsequent recycling of its sphingoid base into the sphingolipid synthesis pathway is not completely understood. To identify the genes within this S1P uptake and recycling pathway, we performed a genome-wide CRISPR/Cas9 KO screen using a positive-selection scheme with Shiga toxin, which binds a cell-surface glycosphingolipid receptor, globotriaosylceramide (Gb3), and causes lethality upon internalization. The screen was performed in HeLa cells with their sphingolipid de novo pathway disabled so that Gb3 cell-surface expression was dependent on salvage of the sphingoid base of S1P taken up from the medium. The screen identified a suite of genes necessary for S1P uptake and the recycling of its sphingoid base to synthesize Gb3, including two lipid phosphatases, PLPP3 (phospholipid phosphatase 3) and SGPP1 (S1P phosphatase 1). The results delineate a pathway in which plasma membrane–bound PLPP3 dephosphorylates extracellular S1P to sphingosine, which then enters cells and is rephosphorylated to S1P by the sphingosine kinases. This rephosphorylation step is important to regenerate intracellular S1P as a branch-point substrate that can be routed either for dephosphorylation to salvage sphingosine for recycling into complex sphingolipid synthesis or for degradation to remove it from the sphingolipid synthesis pathway.     

Migration defects by DISC1 knockdown in C57BL/6, 129X1/SvJ, and ICR strains via in utero gene transfer and virus-mediated RNAi

Disrupted-in-Schizophrenia 1 (DISC1) is a promising genetic risk factor for major mental disorders. Many groups repeatedly reported a role for DISC1 in brain development in various strains of mice and rats by using RNA interference (RNAi) approach. Nonetheless, due to the complexity of its molecular disposition, such as many splice variants and a spontaneous deletion in a coding exon of the DISC1 gene in some mouse strains, there have been debates on the interpretation on these published data. Thus, in this study, we address this question by DISC1 knockdown via short-hairpin RNAs (shRNAs) against several distinct target sequences with more than one delivery methodologies into several mouse strains, including C57BL/6, ICR, and 129X1/SvJ. Here, we show that DISC1 knockdown by in utero electroporation of shRNA against exons 2, 6, and 10 consistently results in neuronal migration defects in the developing cerebral cortex, which are successfully rescued by co-expression of full-length DISC1. Furthermore, lentivirus-mediated shRNA also led to migration defects, which is consistent with two other methodologies already published, such as plasmid-mediated and retrovirus-mediated ones. The previous study by Song’s group also reported that, in the adult hippocampus, the phenotype elicited by DISC1 knockdown with shRNA targeting exon 2 was consistently seen in both C57BL/6 and 129S6 mice. Taken together, we propose that some of DISC1 isoforms that are feasible to be knocked down by shRNAs to exon 2, 6, and 10 of the DISC1 gene play a key role for neuronal migration commonly in various mouse strains and rats.     

Stabilization and characterization of a heme-oxy reaction intermediate in inducible nitric-oxide synthase

Nitric-oxide synthases (NOS) are heme-thiolate enzymes that N-hydroxylate L-arginine (L-Arg) to make NO. NOS contain a unique Trp residue whose side chain stacks with the heme and hydrogen bonds with the heme thiolate. To understand its importance we substituted His for Trp188 in the inducible NOS oxygenase domain (iNOSoxy) and characterized enzyme spectral, thermodynamic, structural, kinetic, and catalytic properties. The W188H mutation had relatively small effects on l-Arg binding and on enzyme heme-CO and heme-NO absorbance spectra, but increased the heme midpoint potential by 88 mV relative to wild-type iNOSoxy, indicating it decreased heme-thiolate electronegativity. The protein crystal structure showed that the His188 imidazole still stacked with the heme and was positioned to hydrogen bond with the heme thiolate. Analysis of a single turnover L-Arg hydroxylation reaction revealed that a new heme species formed during the reaction. Its build up coincided kinetically with the disappearance of the enzyme heme-dioxy species and with the formation of a tetrahydrobiopterin (H4B) radical in the enzyme, whereas its subsequent disappearance coincided with the rate of l-Arg hydroxylation and formation of ferric enzyme. We conclude: (i) W188H iNOSoxy stabilizes a heme-oxy species that forms upon reduction of the heme-dioxy species by H4B. (ii) The W188H mutation hinders either the processing or reactivity of the heme-oxy species and makes these steps become rate-limiting for l-Arg hydroxylation. Thus, the conserved Trp residue in NOS may facilitate formation and/or reactivity of the ultimate hydroxylating species by tuning heme-thiolate electronegativity.     

A Comparison of Structural and Evolutionary Attributes of Escherichia coli and Thermus thermophilus Small Ribosomal Subunits: Signatures of Thermal Adaptation

Here we compare the structural and evolutionary attributes of Thermus thermophilus and Escherichia coli small ribosomal subunits (SSU). Our results indicate that with few exceptions, thermophilic 16S ribosomal RNA (16S rRNA) is densely packed compared to that of mesophilic at most of the analogous spatial regions. In addition, we have located species-specific cavity clusters (SSCCs) in both species. E. coli SSCCs are numerous and larger compared to T. thermophilus SSCCs, which again indicates densely packed thermophilic 16S rRNA. Thermophilic ribosomal proteins (r-proteins) have longer disordered regions than their mesophilic homologs and they experience larger disorder-to-order transitions during SSU-assembly. This is reflected in the predicted higher conformational changes of thermophilic r-proteins compared to their mesophilic homologs during SSU-assembly. This high conformational change of thermophilic r-proteins may help them to associate with the 16S ribosomal RNA with high complementary interfaces, larger interface areas, and denser molecular contacts, compared to those of mesophilic. Thus, thermophilic protein-rRNA interfaces are tightly associated with 16S rRNA than their mesophilic homologs. Densely packed 16S rRNA interior and tight protein-rRNA binding of T. thermophilus (compared to those of E. coli) are likely the signatures of its thermal adaptation. We have found a linear correlation between the free energy of protein-RNA interface formation, interface size, and square of conformational changes, which is followed in both prokaryotic and eukaryotic SSU. Disorder is associated with high protein-RNA interface polarity. We have found an evolutionary tendency to maintain high polarity (thereby disorder) at protein-rRNA interfaces, than that at rest of the protein structures. However, some proteins exhibit exceptions to this general trend.     

New Oral Anticoagulants Are Not Superior to Warfarin in Secondary Prevention of Stroke or Transient Ischemic Attacks,but Lower the Risk of Intracranial Bleeding: Insights from a Meta-Analysis and Indirect Treatment Comparisons

Purpose

Patients with Atrial Fibrillation (AF) and prior stroke are classified as high risk in all risk stratification schemes. A systematic review and meta-analysis was performed to compare the efficacy and safety of New Oral Anticoagulants (NOACs) to warfarin in patients with AF and previous stroke or transient ischemic attack (TIA).

Methods

Three randomized controlled trials (RCTs), including total 14527 patients, comparing NOACs (apixaban, dabigatran and rivaroxaban) with warfarin were included in the analysis. Primary efficacy endpoint was ischemic stroke, and primary safety endpoint was intracranial bleeding. Random-effects models were used to pool efficacy and safety data across RCTs. RevMan and Stata software were used for direct and indirect comparisons, respectively.

Results

In patients with AF and previous stroke or TIA, effects of NOACs were not statistically different from that of warfarin, in reduction of stroke (Odds Ratio [OR] 0.86, 95% confidence interval [CI] 0.73- 1.01), disabling and fatal stroke (OR 0.85, 95% CI 0.71-1.04), and all-cause mortality (OR 0.90, 95% CI 0.79 -1.02). Randomization to NOACs was associated with a significantly lower risk of intracranial bleeding (OR 0.42, 95% CI 0.25-0.70). There were no major differences in efficacy between apixaban, dabigatran (110 mg BID and 150 mg BID) and rivaroxaban. Major bleeding was significantly lower with apixaban and dabigatran (110 mg BID) compared with dabigatran (150 mg BID) and rivaroxaban.

Conclusion

NOACs may not be more effective than warfarin in the secondary prevention of ischemic stroke in patients with a prior history of cerebrovascular ischemia, but have a lower risk of intracranial bleeding.     

Effect of orally administered azadirachtin on non-specific immune parameters of goldfish Carassius auratus (Linn. 1758) and resistance against Aeromonas hydrophila

Modulation of the immune responses using active bio-ingredients as a possible prophylaxis measure has been novel prospect for aquaculture. The present study evaluated the effects of azadirachtin EC 25% on non-specific immune responses in goldfish Carassius auratus and resistance against pathogenic bacteria Aeromonas hydrophila. The experimental trial for effects of azadirachtin on immuno-haematoloical parameters in goldfish was conducted by feeding the various levels of azadirachtin as control T₀ (without azadirachtin), T₁ (0.1%), T₂ (0.2%), T₃ (0.4%), T₄ (0.8%) and T₅ (1.6%) for a period of 28 days. Fishes were challenged with A. hydrophila 28 days post feeding and relative percentage survival (%) was recorded over 14 days post infection. Immuno-haematoloical (total erythrocyte count, total leukocyte count, haemoglobin, packed cell volume, NBT activity, phagocytic activity, serum lysozyme activity, myeloperoxidase activity, total immunoglobulin) and serum biochemical parameters (serum glutamate oxaloacetate transaminase (SGOT), serum glutamate pyruvate transaminase (SGPT) and blood glucose) of fishes were examined at 14 and 28 days of feedings. Fish fed with azadirachtin, showed significantly (p < 0.05) enhanced TEC, TLC, Total Ig, total protein, NBT activity, serum lysozyme activity and myeloperoxidase level in different treatment groups in comparison with control group. Similarly, SGOT, SGPT and blood glucose level were found to be significantly (p < 0.05) high but PCV and Hb did not differ significantly (p > 0.05) in the treatment groups compared to control groups. Azadirachtin at the concentration of 4 g kg^?1 showed significantly (p < 0.05) higher relative percentage survival (42.60%) when compared with the control against A. hydrophila infection. This study indicated that azadirachtin EC 25% (4 g kg^?1) showed higher NBT activity, serum lysozyme, protein profiles, leukocyte counts and resistance against A. hydrophila infection and thus, can be used as a potential immunostimulant in aquaculture.     

Autophagy regulates sphingolipid levels in the liver

Sphingolipid levels are tightly regulated to maintain cellular homeostasis. During pathologic conditions such as in aging, inflammation, and metabolic and neurodegenerative diseases, levels of some sphingolipids, including the bioactive metabolite ceramide, are elevated. Sphingolipid metabolism has been linked to autophagy, a critical catabolic process in both normal cell function and disease; however, the in vivo relevance of the interaction is not well-understood. Here, we show that blocking autophagy in the liver by deletion of the Atg7 gene, which is essential for autophagosome formation, causes an increase in sphingolipid metabolites including ceramide. We also show that overexpression of serine palmitoyltransferase to elevate de novo sphingolipid biosynthesis induces autophagy in the liver. The results reveal autophagy as a process that limits excessive ceramide levels and that is induced by excessive elevation of de novo sphingolipid synthesis in the liver. Dysfunctional autophagy may be an underlying mechanism causing elevations in ceramide that may contribute to pathogenesis in diseases.     

The RAS subfamily Evolution – tracing evolution for its utmost exploitation

In the development of multicellularity, signaling proteins has played a very important role. Among them, RAS family is one of the most widely studied protein family. However, evolutionary analysis has been carried out mainly on super family level leaving sub family information in scanty. Thus, a subfamily evolutionary study on RAS evolutionary expansion is imperative as it will aid in better drug designing against dreadful diseases like Cancer and other developmental diseases. The present study was aimed to understand RAS evolution on both holistic as well as reductive level. All human RAS family genes and protein were subjected to BLAST tools to find orthologs and paralogs with different parameters followed by phylogenetic tree generation. Our results clearly showed that H-RAS is the most primitive RAS in higher eukaryotes and then diverged into other RAS family members due to different gene modification events. Furthermore, a site specific selection pressure analysis was carried out using SELECTON server which showed that H-RAS, M-RAS and N-RAS are evolving faster than K-RAS and R-RAS. Thus, the results ascertain a new ground to cancer biologists to exploit negatively selected K-RAS and R-RAS as potent drug targets in cancer therapeutics.