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.     

Live Cell Fluorescence Resonance Energy Transfer Predicts an Altered Molecular Association of Heterologous PrPSc with PrPC

Prion diseases result from the accumulation of a misfolded isoform (PrP^Sc) of the normal host prion protein (PrP^C). PrP^Sc propagates by templating its conformation onto resident PrP^C to generate new PrP^Sc. Although the nature of the PrP^Sc-PrP^C complex is unresolved, certain segments or specific residues are thought to feature critically in its formation. The polymorphic residue 129 is one such site under considerable study. We combined transmission studies with a novel live cell yeast-based fluorescence resonance energy transfer (FRET) system that models the molecular association of PrP in a PrP^Sc-like state, as a way to explore the role of residue 129 in this process. We show that a reduction in efficiency of prion transmission between donor PrP^Sc and recipient PrP^C that are mismatched at residue 129 correlates with a reduction in FRET between PrP-129M and PrP-129V in our yeast model. We further show that this effect depends on the different secondary structure propensities of Met and Val, rather than the specific amino acids. Finally, introduction of the disease-associated P101L mutation (mouse- equivalent) abolished FRET with wild-type mouse PrP, whereas mutant PrP-P101L displayed high FRET with homologous PrP-P101L, as long as residue 129 matched. These studies provide the first evidence for a physical alteration in the molecular association of PrP molecules differing in one or more residues, and they further predict that the different secondary structure propensities of Met and Val define the impaired association observed between PrP^Sc and PrP^C mismatched at residue 129.     

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.     

Comparison of SYPRO Ruby and Flamingo fluorescent stains for application in proteomic research

Fluorescent dyes are widely used for the detection and quantitation of proteins separated by polyacrylamide gel electrophoresis. SYPRO Ruby is one such fluorescent dye widely used for this purpose. More recently, another fluorescent dye, Flamingo, is available for expression proteomic research. Using a standard ultraviolet (UV) transilluminator and a charge-coupled device (CCD)-based imaging system, the relative sensitivity of these two different fluorescent stains with regard to detection of protein spots separated by two-dimensional gel electrophoresis (2D-GE) and identification by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) were compared. Using mouse kidney and liver homogenates as well as Escherichia coli extract, we detected a greater number of protein spots using Flamingo compared with SYPRO Ruby. In addition, when we compared the number of matched peptides and the percentage of amino acid residues identified for 22 different protein spots of mouse kidney proteome, we observed a higher number of matched peptides and a higher percentage of amino acid residues for the majority of the proteins using Flamingo compared with SYPRO Ruby. Also, we were able to characterize a protein spot that can be detected by Flamingo only. Therefore, we recommend Flamingo over SYPRO Ruby to be used for studies on expression proteomics.     

Evidence of indigenous NAH plasmid of naphthalene degrading Pseudomonas putida PpG7 strain implicated in limonin degradation

A well characterized naphthalene-degrading strain, Pseudomonas putida PpG7 was observed to utilize limonin, a highly-oxygenated triterpenoid compound as a sole source of carbon and energy. Limonin concentrations evidenced a 64% reduction over 48 h of growth in batch cultures. Attempts were made to acquire a plasmid-less derivative via various methods (viz. Ethidium Bromide, SDS, elevated temperature & mitomycin C), among which the method involving mitomycin C (20 ug/ml) proved successful. Concomitant with the loss of plasmid in P. putida PpG7 strain, the cured derivative was identified as a lim- phenotype. The lim+ phenotype could be conjugally transferred to the cured derivative. Based on the results of curing with mitomycin C, conjugation studies and presence of ndo gene encoding naphthalene 1,2 dioxygenase, it was demonstrated that genes for the limonin utilization were encoded on an 83 kb indigenous transmissible Inc. P9 NAH plasmid in Pseudomonas putida PpG7 strain.     

Fine needle aspiration cytology of follicular variant of papillary carcinoma of the thyroid: Morphologic pointers to its diagnosis

OBJECTIVE: To study the cytomorphologic features of 59 cases of histologically proven follicular variant of papillary carcinoma (FVPC), compare them to those described in the literature and highlight cytologic features that may aid in the preoperative diagnosis. STUDY DESIGN: Aspiration smears from 59 histologically proven cases of FVPC were examined independently by 2 observers, and a detailed cytologic evaluation was done for architectural, cytologic and nuclear features. surgical RESULTS: On initial cytology of the 59 cases, 36 (61%) were diagnosed aspapillary carcinoma, and 17 of these were subtyped as FVPC. On reviewing the smears, 50 cases were diagnosed as papillary carcinoma, and 33 of them were typed as FVPC; however, 4 cases were diagnosed as benign lesions. Most smears showed moderate to high cellularity, with 55 cases (93%) showing syncytial clusters and 48 (81%) showing microfollicular architecture. Chromatin clearing and nuclear grooves were seen in 55 (93.2%) and 54 (91.52%) cases but were easily detected in only 36 (61%) and 44 (74%) cases, respectively. Thick colloid was identified in 28 cases, and 3 of these cases also showed thin colloid in the background. CONCLUSION: Our findings suggest that syncytial clusters, microfollicular architecture, chromatin clearing and nuclear grooves are strong morphologic pointers to the diagnosis of FVPC.     

Chromatographic analysis of carbamazepine binding to human serum albumin. II. Comparison of the Schiff base and N-hydroxysuccinimide immobilization methods

Recent studies with carbamazepine on human serum albumin (HSA) columns have noted an appreciable degree of non-specific binding on supports prepared by the Schiff base immobilization method. This work examines an alternative immobilization method for HSA based on N-hydroxysuccinimide (NHS)-activated silica. This support was prepared by reacting HPLC-grade silica directly with disuccinimidyl carbonate. The resulting material was compared to an HSA support prepared by the Schiff base method in terms of its activity for carbamazepine and non-specific interactions with this drug. When examined by frontal analysis, both supports gave comparable association equilibrium constants for carbamazepine interactions with HSA ((0.53-0.55) x 10(4)M(-1) at 37 degrees C). However, columns prepared by the Schiff base method gave greater non-specific binding. These columns, as well as control columns prepared using the carbonyldiimidazole (CDI) immobilization method, were also evaluated for their non-specific binding to a variety of other solutes known to interact with HSA. From these results it was concluded that the NHS method was an attractive alternative to the Schiff base technique in the preparation of immobilized HSA for HPLC-based binding studies for carbamazepine. However, it was also noted that non-specific binding varies from one drug to the next in these immobilization methods, indicating that such properties should be evaluated on a case-by-case basis in the use and development of HSA columns for binding studies.