Systematic Proteomic Analysis of Protein Methylation in Prokaryotes and Eukaryotes Revealed Distinct Substrate Specificity.
Proteomics. 2017 Nov 18;:
Authors: Zhang M, Xu JY, Hu H, Ye BC, Tan M
The studies of protein methylation mainly focus on lysine and arginine residues due to their diverse roles in essential cellular processes from gene expression to signal transduction. Nevertheless, atypical protein methylation occurring on amino acid residues, such as glutamine and glutamic acid, was largely neglected until recently. In addition, the systematic analysis for the distribution of methylation on different amino acids in various species is still lacking, which hinders our understanding of its functional roles. In this study, we deeply explored the methylated sites in three species E. coli, S. cerevisiae, and HeLa cells by employing mass spectrometry-based proteomic approach coupled with heavy methyl SILAC method. We identified a total of 234 methylated sites on 187 proteins with high localization confidence, including 94 unreported methylated sites on 9 different amino acid residues. KEGG and GO analysis showed the pathways enriched with methylated proteins were mainly involved in central metabolism for E. coli and S. cerevisiae, but related to spliceosome for HeLa cells. The analysis of methylation preference on different amino acids were conducted in three species. Protein N-terminal methylation was dominant in E. coli while lysine and arginine methylation were widely identified in S. cerevisiae and HeLa cells, respectively. To study whether some atypical protein methylation have biological relevance in the pathological process in mammalian cells, we focused on histone methylation in diet-induced obese (DIO) mouse. Two glutamate methylation sites showed statistical significance in DIO mice compared with chow-fed mice, suggesting their potential roles in diabetes and obesity. Together, these findings expanded the methylome database from microbes to mammals, which will benefit our further appreciation for the protein methylation as well as its possible functions on disease. This article is protected by copyright. All rights reserved.
PMID: 29150981 [PubMed - as supplied by publisher]
Genotype and diet affect resistance, survival, and fecundity but not fecundity tolerance.
J Evol Biol. 2017 Nov 18;:
Authors: Kutzer MAM, Kurtz J, Armitage SAO
Insects are exposed to a variety of potential pathogens in their environment, many of which can severely impact fitness and health. Consequently, hosts have evolved resistance and tolerance strategies to suppress or cope with infections. Hosts utilising resistance improve fitness by clearing or reducing pathogen loads and hosts utilising tolerance reduce harmful fitness effects per pathogen load. To understand variation in, and selective pressures on resistance and tolerance we asked to what degree they are shaped by host genetic background, whether plasticity in these responses depends upon dietary environment, and whether there are interactions between these two factors. Females from ten wild-type Drosophila melanogaster genotypes were kept on high or low protein (yeast) diets, and infected with one of two opportunistic bacterial pathogens, Lactococcus lactis or Pseudomonas entomophila. We measured host resistance as the inverse of bacterial load in the early infection phase. The slope linking fly fecundity and individual-level bacteria load provided our fecundity tolerance measure. Genotype and dietary yeast determined host fecundity and strongly affected survival after infection with pathogenic P. entomophila. There was considerable genetic variation in host resistance, a commonly found phenomenon resulting from e.g. varying resistance costs or frequency-dependent selection. Despite this variation and the reproductive cost of higher P. entomophila loads, the slopes linking bacteria load and fecundity did not vary across genotypes. Absence of genetic variation in tolerance may suggest that at this early infection stage fecundity tolerance is fixed or that any evolved tolerance mechanisms are not expressed under these infection conditions. This article is protected by copyright. All rights reserved.
PMID: 29150962 [PubMed - as supplied by publisher]
Regulation of Calcification in Human Aortic Smooth Muscle Cells Infected with High-glucose-treated Porphyromonas Gingivalis.
J Cell Physiol. 2017 Nov 18;:
Authors: Chen TC, Lin CT, Chien SJ, Chang SF, Chen CN
Porphyromonas (P.) gingivalis infection leading to the periodontitis has been associated with the development of systemic diseases, including cardiovascular diseases and diabetes. However, the effect of a high concentration of glucose (HG) on the invasion efficiency of P. gingivalis and the consequent modulation of pathogenesis in vascular cells, especially in the vascular smooth muscle cells (VSMCs), remains unclear. Hence, the aim of this study was to investigate whether treating P. gingivalis with HG could change its invasion capability and result in VSMC calcification and the underlying mechanism. Human aortic SMCs (HASMCs) and P. gingivalis strain CCUG25226 were used in this study. We found that HGPg infection of HASMCs could initiate the HASMC calcification by stimulating the autocrine regulation of bone morphogenetic protein (BMP) 4 in HASMCs. The upregulation of BMP4 expression in HASMCs was mediated by toll-like receptor 4 and ERK1/2-p38 signaling after P. gingivalis infection. Moreover, the autocrine action of BMP4 in HGPg infection-initiated HASMC calcification upregulated BMP4-specific downstream smad1/5/8-runx2 signaling to increase the expressions of bone-related matrix proteins, i.e. osteopontin, osteocalcin, and alkaline phosphatase. This study elucidates the detailed mechanism of HGPg infection-initiated calcification of HASMCs and indicates a possible therapeutic role of BMP4 in P. gingivalis infection-associated vascular calcification. This article is protected by copyright. All rights reserved.
PMID: 29150938 [PubMed - as supplied by publisher]
High fat diet-induced oxidative stress blocks hepatocyte nuclear factor 4α and leads to hepatic steatosis in mice.
J Cell Physiol. 2017 Nov 18;:
Authors: Yu D, Chen G, Pan M, Zhang J, He W, Liu Y, Nian X, Sheng L, Xu B
Nonalcoholic fatty liver disease (NAFLD) is the most common form of chronic liver disease with manifestation of over-accumulation of fat in liver. Increasing evidences indicate that NAFLD may be in part caused by malfunction of very low density lipoprotein (VLDL) secretion. Hepatocyte nuclear factor 4α (HNF4α), a nuclear receptor protein, plays an important role in sustain hepatic lipid homeostasis via transcriptional regulation of genes involved in secretion of VLDL, such as apolipoprotein B (ApoB). However, the exact functional change of HNF4α in NAFLD remains to be elucidated. In the present study, we found that high fat diet (HFD) induced cytoplasmic retention of HNF4α in hepatocytes, which led to down-regulation of hepatic ApoB expression and its protein level in serum, as well as reduced secretion of VLDL. We further revealed that oxidative stress, elevated in fatty liver, was the key factor inducing the cytoplasmic retention of HNF4α in hepatocytes by activating protein kinase C (PKC)-mediated phosphorylation in HNF4α. Thus, our findings reveal a novel mechanism underlying HFD-induced fatty liver that oxidative stress impairs function of HNF4α on ApoB expression and VLDL secretion via PKC activation, eventually promoting fat accumulation in the liver. Therefore, oxidative stress/PKC/HNF4α pathway may be a novel target to treat diet-induced fatty liver. This article is protected by copyright. All rights reserved.
PMID: 29150932 [PubMed - as supplied by publisher]
Sustaining an efficient and effective CHO cell line development platform by incorporation of 24-deep well plate screening and multivariate analysis.
Biotechnol Prog. 2017 Nov 18;:
Authors: Mora A, Zhang SS, Carson G, Nabiswa B, Hossler P, Yoon S
Efficient and effective cell line screening is paramount towards a successful biomanufacturing program. Here we describe the implementation of 24-deep well plate screening of CHO lines as part of the cell line development platform at AbbVie. Incorporation of this approach accelerated the identification of the best candidate lines for process development. In an effort to quantify and predict process performance comparability, we compared cell culture performance in 24-deep well plates (24-DWP) and in shake flasks, for a panel of Chinese Hamster Ovary cell lines expressing a monoclonal antibody. The results in 24-DWP screening showed reduced growth profiles, but comparable viability profiles. Slow growers in 24-DWP achieved the highest productivity improvement upon scaling-up to shake flasks. Product quality of the protein purified from shake flasks and 24-DWP were also compared. The 24-DWP culture conditions were found to influence the levels of acidic species, reduce the G0 N-glycan species, and increase the high-mannose N-glycan species. Nevertheless, the identification of undesirable profiles is executed consistently with the scaled-up culture. We further employed multivariate data analysis to capture differences depending on the two scales and we could demonstrate that cell line profiles were adequately clustered, regardless of the vessel used for the development. In conclusion, the 24-DWP platform was reasonably predictive of the parameters crucial for upstream process development activities, and has been adapted as part of the AbbVie cell line development platform. This article is protected by copyright. All rights reserved.
PMID: 29150912 [PubMed - as supplied by publisher]
CK4, CK6, cyclin D1, p16(INK4a) and EGFR expression in glioblastoma with a primitive neuronal component.
J Neurooncol. 2017 Nov 17;:
Authors: Xu G, Li JY
Glioblastoma with primitive neuroectodermal tumor-like component (GBM-PNET) is a rare variant of glioblastoma, which was renamed as glioblastoma with a primitive neuronal component (GBM-PN) in new WHO classification of tumours of the central nervous system in 2016. There are few publications on the investigation of GBM-PN. In this study, PCR mRNA arrays on 6 cases of conventional GBM and 10 cases of GBM-PN showed high mRNA level of CDK4 in GBM-PN and low mRNA level of EGFR in GBM-PN. Immunohistochemical stains on tissue microarrays with 28 cases of conventional GBM and 13 cases of GBM-PN demonstrated that CDK4 was selectively expressed in the primitive neuronal component of all GBM-PN cases while EGFR was positive in conventional GBM and glial component of GBM-PN, but was negative in the primitive neuronal component of all GBM-PN cases. Immunohistochemical stains with antibodies against proteins that interact with CDK4 in cell cycle regulation, such as CDK6, cyclin D1 and p16(INK4a), were performed on these GBM-PN and GBM cases. CDK6 was patchily positive in rare cases of GBM-PN and cyclin D1 was negative in GBM-PN cases. p16(INK4a) is traditionally known as an inhibitor of CDK4 and CDK6. p16(INK4a) might not be the inhibitor of CDK4 in GBM-PN cases because seven GBM-PN cases were positive for both CDK4 and p16(INK4a). It indicates that CDK4 and p16(INK4a) might play a crucial role in GBM-PN pathogenesis. Since CDK4 and EGFR are highly expressed in the primitive neuronal component and in the glial component of GBM-PN respectively, the combination of CDK4/6 inhibitor and targeted therapy against EGFR might be potential effective therapeutic regimen for GBM-PN. CDK4 and EGFR immuohistochemical stain patterns make the diagnosis of GBM-PN much easier.
PMID: 29150788 [PubMed - as supplied by publisher]
TSPO imaging using the novel PET ligand [(18)F]GE-180: quantification approaches in patients with multiple sclerosis.
EJNMMI Res. 2017 Oct 26;7(1):89
Authors: Vomacka L, Albert NL, Lindner S, Unterrainer M, Mahler C, Brendel M, Ermoschkin L, Gosewisch A, Brunegraf A, Buckley C, Kümpfel T, Rupprecht R, Ziegler S, Kerschensteiner M, Bartenstein P, Böning G
BACKGROUND: PET ligands targeting the translocator protein (TSPO) represent promising tools to visualise neuroinflammation. Here, we analysed parameters obtained in dynamic and static PET images using the novel TSPO ligand [(18)F]GE-180 in patients with relapsing remitting multiple sclerosis (RRMS) and an approach for semi-quantitative assessment of this disease in clinical routine. Seventeen dynamic [(18)F]GE-180 PET scans of RRMS patients were evaluated (90 min). A pseudo-reference region (PRR) was defined after identification of the least disease-affected brain area by voxel-based comparison with six healthy controls (HC) and upon exclusion of voxels suspected of being affected in static 60-90 min p.i. images. Standardised uptake value ratios (SUVR) obtained from static images normalised to PRR were correlated to the distribution volume ratios (DVR) derived from dynamic data with Logan reference tissue model.
RESULTS: Group comparison with HC revealed white matter and thalamus as most affected regions. Fewest differences were found in grey matter, and normalisation to frontal cortex (FC) yielded the greatest reduction in variability of healthy grey and white matter. Hence, FC corrected for affected voxels was chosen as PRR, leading to time-activity curves of FC which were congruent to HC data (SUV60-90 0.37, U test P = 0.42). SUVR showed a very strong correlation with DVR (Pearson ρ > 0.9). Focal MS lesions exhibited a high SUVR (range, 1.3-3.2).
CONCLUSIONS: This comparison with parameters from dynamic data suggests that SUVR normalised to corrected frontal cortex as PRR is suitable for the quantification of [(18)F]GE-180 uptake in lesions and different brain regions of RRMS patients. This efficient diagnostic protocol based on static [(18)F]GE-180 PET scans acquired 60-90 min p.i. allows the semi-quantitative assessment of neuroinflammation in RRMS patients in clinical routine.
PMID: 29150726 [PubMed]
Impact of prolonged overfeeding on skeletal muscle mitochondria in healthy individuals.
Diabetologia. 2017 Nov 17;:
Authors: Toledo FGS, Johannsen DL, Covington JD, Bajpeyi S, Goodpaster B, Conley KE, Ravussin E
AIMS/HYPOTHESES: Reduced mitochondrial capacity in skeletal muscle has been observed in obesity and type 2 diabetes. In humans, the aetiology of this abnormality is not well understood but the possibility that it is secondary to the stress of nutrient overload has been suggested. To test this hypothesis, we examined whether sustained overfeeding decreases skeletal muscle mitochondrial content or impairs function.
METHODS: Twenty-six healthy volunteers (21 men, 5 women, age 25.3 ± 4.5 years, BMI 25.5 ± 2.4 kg/m(2)) underwent a supervised protocol consisting of 8 weeks of high-fat overfeeding (40% over baseline energy requirements). Before and after overfeeding, we measured systemic fuel oxidation by indirect calorimetry and performed skeletal muscle biopsies to measure mitochondrial gene expression, content and function in vitro. Mitochondrial function in vivo was measured by (31)P NMR spectroscopy.
RESULTS: With overfeeding, volunteers gained 7.7 ± 1.8 kg (% change 9.8 ± 2.3). Overfeeding increased fasting NEFA, LDL-cholesterol and insulin concentrations. Indirect calorimetry showed a shift towards greater reliance on lipid oxidation. In skeletal muscle tissue, overfeeding increased ceramide content, lipid droplet content and perilipin-2 mRNA expression. Phosphorylation of AMP-activated protein kinase was decreased. Overfeeding increased mRNA expression of certain genes coding for mitochondrial proteins (CS, OGDH, CPT1B, UCP3, ANT1). Despite the stress of nutrient overload, mitochondrial content and mitochondrial respiration in muscle did not change after overfeeding. Similarly, overfeeding had no effect on either the emission of reactive oxygen species or on mitochondrial function in vivo.
CONCLUSIONS/INTERPRETATION: Skeletal muscle mitochondria are significantly resilient to nutrient overload. The lower skeletal muscle mitochondrial oxidative capacity in human obesity is likely to be caused by reasons other than nutrient overload per se.
TRIAL REGISTRATION: ClinicalTrials.gov NCT01672632.
PMID: 29150696 [PubMed - as supplied by publisher]
Urothelial cancer proteomics provides both prognostic and functional information.
Sci Rep. 2017 Nov 17;7(1):15819
Authors: de Velasco G, Trilla-Fuertes L, Gamez-Pozo A, Urbanowicz M, Ruiz-Ares G, Sepúlveda JM, Prado-Vazquez G, Arevalillo JM, Zapater-Moros A, Navarro H, Lopez-Vacas R, Manneh R, Otero I, Villacampa F, Paramio JM, Vara JAF, Castellano D
Traditionally, bladder cancer has been classified based on histology features. Recently, some works have proposed a molecular classification of invasive bladder tumors. To determine whether proteomics can define molecular subtypes of muscle invasive urothelial cancer (MIUC) and allow evaluating the status of biological processes and its clinical value. 58 MIUC patients who underwent curative surgical resection at our institution between 2006 and 2012 were included. Proteome was evaluated by high-throughput proteomics in routinely archive FFPE tumor tissue. New molecular subgroups were defined. Functional structure and individual proteins prognostic value were evaluated and correlated with clinicopathologic parameters. 1,453 proteins were quantified, leading to two MIUC molecular subgroups. A protein-based functional structure was defined, including several nodes with specific biological activity. The functional structure showed differences between subtypes in metabolism, focal adhesion, RNA and splicing nodes. Focal adhesion node has prognostic value in the whole population. A 6-protein prognostic signature, associated with higher risk of relapse (5 year DFS 70% versus 20%) was defined. Additionally, we identified two MIUC subtypes groups. Prognostic information provided by pathologic characteristics is not enough to understand MIUC behavior. Proteomics analysis may enhance our understanding of prognostic and classification. These findings can lead to improving diagnosis and treatment selection in these patients.
PMID: 29150671 [PubMed - in process]
A streamlined cloning workflow minimising the time-to-strain pipeline for Pichia pastoris.
Sci Rep. 2017 Nov 17;7(1):15817
Authors: Royle KE, Polizzi K
Although recent advances in E. coli self-assembly have greatly simplified cloning, these have not yet been harnessed for the high-throughput generation of expression strains in the early research and discovery phases of biopharmaceutical production. Here, we have refined the technique and incorporated it into a streamlined workflow for the generation of Pichia pastoris expression strains, reducing the timeline by a third and removing the reliance on DNA editing enzymes, which often require troubleshooting and increase costs. We have validated the workflow by cloning 24 human proteins of biopharmaceutical value, either as direct therapeutics or as research targets, which span a continuous range in size and GC content. This includes demonstrating the applicability of the workflow to three-part assemblies for a monoclonal antibody and its single-chain antibody fragments derivatives. This workflow should enable future research into recombinant protein production by P. pastoris and a synthetic biology approach to this industrial host.
PMID: 29150665 [PubMed - in process]
Axonemal Lumen Dominates Cytosolic Protein Diffusion inside the Primary Cilium.
Sci Rep. 2017 Nov 17;7(1):15793
Authors: Luo W, Ruba A, Takao D, Zweifel LP, Lim RYH, Verhey KJ, Yang W
Transport of membrane and cytosolic proteins in primary cilia is thought to depend on intraflagellar transport (IFT) and diffusion. However, the relative contribution and spatial routes of each transport mechanism are largely unknown. Although challenging to decipher, the details of these routes are essential for our understanding of protein transport in primary cilia, a critically affected process in many genetic diseases. By using a high-speed virtual 3D super-resolution microscopy, we have mapped the 3D spatial locations of transport routes for various cytosolic proteins in the 250-nm-wide shaft of live primary cilia with a spatiotemporal resolution of 2 ms and <16 nm. Our data reveal two spatially distinguishable transport routes for cytosolic proteins: an IFT-dependent path along the axoneme, and a passive-diffusion route in the axonemal lumen that escaped previous studies. While all cytosolic proteins tested primarily utilize the IFT path in the anterograde direction, differences are observed in the retrograde direction where IFT20 only utilizes IFT, and approximately half of KIF17 and one third of α-tubulin utilizes diffusion besides IFT.
PMID: 29150645 [PubMed - in process]
Ml proteins from Mesorhizobium loti and MucR from Brucella abortus: an AT-rich core DNA-target site and oligomerization ability.
Sci Rep. 2017 Nov 17;7(1):15805
Authors: Baglivo I, Pirone L, Pedone EM, Pitzer JE, Muscariello L, Marino MM, Malgieri G, Freschi A, Chambery A, Roop Ii RM, Pedone PV
Mesorhizobium loti contains ten genes coding for proteins sharing high amino acid sequence identity with members of the Ros/MucR transcription factor family. Five of these Ros/MucR family members from Mesorhizobium loti (Ml proteins) have been recently structurally and functionally characterized demonstrating that Ml proteins are DNA-binding proteins. However, the DNA-binding studies were performed using the Ros DNA-binding site with the Ml proteins. Currently, there is no evidence as to when the Ml proteins are expressed during the Mesorhizobium lo ti life cycle as well as no information concerning their natural DNA-binding site. In this study, we examine the ml genes expression profile in Mesorhizobium loti and show that ml1, ml2, ml3 and ml5 are expressed during planktonic growth and in biofilms. DNA-binding experiments show that the Ml proteins studied bind a conserved AT-rich site in the promoter region of the exoY gene from Mesorhizobium loti and that the proteins make important contacts with the minor groove of DNA. Moreover, we demonstrate that the Ml proteins studied form higher-order oligomers through their N-terminal region and that the same AT-rich site is recognized by MucR from Brucella abortus using a similar mechanism involving contacts with the minor groove of DNA and oligomerization.
PMID: 29150637 [PubMed - in process]
A program for iron economy during deficiency targets specific Fe proteins.
Plant Physiol. 2017 Nov 17;:
Authors: Hantzis LJ, Kroh GE, Jahn CE, Cantrell M, Peers G, Pilon M, Ravet K
Iron (Fe) is an essential element for plants, utilized in nearly every cellular process. Because the adjustment of uptake under Fe limitation cannot satisfy all demands, plants need to acclimate their physiology and biochemistry, especially in their chloroplasts, which have a high demand for Fe. To investigate if a program exists for the utilization of Fe under deficiency, we analyzed how hydroponically grown Arabidopsis thaliana adjusts its physiology and Fe protein composition in vegetative photosynthetic tissue, during Fe deficiency. Fe deficiency first affected photosynthetic electron transport with concomitant reductions in carbon assimilation and biomass production when effects on respiration were not yet significant. Photosynthetic electron transport function and protein levels of Fe-dependent enzymes were fully recovered upon iron resupply, indicating that the Fe depletion stress did not cause irreversible secondary damage. At the protein level, ferredoxin, the cytochrome-b6f complex, and Fe-containing enzymes of the plastid sulfur assimilation pathway were major targets of Fe deficiency, whereas other Fe dependent functions were relatively less affected. In coordination, SufA and SufB, two proteins of the plastid iron-sulfur cofactor assembly pathway were also diminished early by Fe depletion. Iron depletion reduced mRNA levels for the majority of the affected proteins indicating that loss of enzyme was not just due to lack of Fe cofactors. SufB and ferredoxin were early targets of transcript down-regulation. The data reveal a hierarchy for Fe utilization in photosynthetic tissue and indicate that a program is in place to acclimate to impending Fe deficiency.
PMID: 29150559 [PubMed - as supplied by publisher]
Molecular characterization of an Endozoicomonas-like organism causing infection in king scallop Pecten maximus L.
Appl Environ Microbiol. 2017 Nov 17;:
Authors: Cano I, van Aerle R, Ross S, Verner-Jeffreys DW, Paley RK, Rimmer G, Ryder D, Hooper P, Stone D, Feist SW
One of the fastest growing fisheries in the UK is the king scallop Pecten maximus L., also currently rated as the second most valuable. Mass mortality events in scallops have been reported world-wide, often with the causative agent(s) remaining uncharacterized. In May 2013 and 2014, two mass mortality events affecting king scallops were recorded in the Lyme Bay Marine Protected Area (MPA), in South West England. Histopathological examination showed gill epithelial tissues infected with intracellular microcolonies of bacteria (IMC), resembling rickettsia-like organisms (RLOs), often with bacteria released in vascular spaces. Large colonies were associated with cellular and tissue disruption of the gills. Ultrastructural examination confirmed the intracellular location of these organisms in affected epithelial cells.16S rRNA gene sequences of the putative IMC obtained from infected king scallop gill samples, collected from both mortality events, were identical, and had a 99.4% identity to 16S rRNA gene sequences obtained from Candidatus endonucleobacter bathymodioli and 95% with Endozoicomonas spp.In situ hybridization assays using 16S rRNA gene probes confirmed the presence of the sequenced IMC gene in the gill tissues. Additional DNA sequences of the bacterium were obtained using high-throughput (Illumina) sequencing and bioinformatic analysis identified over 1000 genes with high similarity to protein sequences from Endozoicomonas spp. (ranging from 77-87% identity).Specific PCR assays were developed and applied to screen for the presence of IMC 16S rRNA gene sequences in king scallop gill tissues collected in the Lyme Bay MPA during 2015 and 2016. There was 100% prevalence of the IMC in these gill tissues and the 16S rRNA gene sequences identified were identical to the sequence found during the previous mortality event.ImportanceMolluscan mass mortalities associated with IMC have been reported worldwide for many years, however, apart from histological and ultrastructural characterization, characterization of the aetiological agents is limited. In the present work, we provide detailed molecular characterization of an Endozoicomonas-like organism (ELO) associated with an important commercial scallop species.
PMID: 29150518 [PubMed - as supplied by publisher]
Multi-omics reveal the lifestyle of the acidophilic, mineral-oxidizing model species Leptospirillum ferriphilum(T).
Appl Environ Microbiol. 2017 Nov 17;:
Authors: Christel S, Herold M, Bellenberg S, El Hajjami M, Buetti-Dinh A, Pivkin IV, Sand W, Wilmes P, Poetsch A, Dopson M
Leptospirillum ferriphilum plays a major role in acidic, metal rich environments where it represents one of the most prevalent iron oxidizers. These milieus include acid rock and mine drainage as well as biomining operations. Despite its perceived importance, no complete genome sequence of this model species' type strain is available, limiting the possibilities to investigate the strategies and adaptations Leptospirillum ferriphilum(T) applies to survive and compete in its niche. This study presents a complete, circular genome of Leptospirillum ferriphilum(T) DSM 14647 obtained by PacBio SMRT long read sequencing for use as a high quality reference. Analysis of the functionally annotated genome, mRNA transcripts, and protein concentrations revealed a previously undiscovered nitrogenase cluster for atmospheric nitrogen fixation and elucidated metabolic systems taking part in energy conservation, carbon fixation, pH homeostasis, heavy metal tolerance, oxidative stress response, chemotaxis and motility, quorum sensing, and biofilm formation. Additionally, mRNA transcript counts and protein concentrations were compared between cells grown in continuous culture using ferrous iron as substrate and bioleaching cultures containing chalcopyrite (CuFeS2). Leptospirillum ferriphilum(T) adaptations to growth on chalcopyrite included a possibly enhanced production of reducing power, reduced carbon dioxide fixation, as well as elevated RNA transcripts and proteins involved in heavy metal resistance, with special emphasis on copper efflux systems. Finally, expression and translation of genes responsible for chemotaxis and motility were enhanced.IMPORTANCELeptospirillum ferriphilum is one of the most important iron-oxidizers in the context of acidic and metal rich environments during moderately thermophilic biomining. A high-quality circular genome of Leptospirillum ferriphilum(T) coupled with functional omics data provides new insights into its metabolic properties, such as the novel identification of genes for atmospheric nitrogen fixation, and represents an essential step for further accurate proteomic and transcriptomic investigation of this acidophile model species in the future. Additionally, light is shed on Leptospirillum ferriphilum(T) adaptation strategies to growth on the copper mineral chalcopyrite. This data can be applied to deepen our understanding and optimization of bioleaching and biooxidation, techniques that present sustainable and environmentally friendly alternatives to many traditional methods for metal extraction.
PMID: 29150517 [PubMed - as supplied by publisher]
Loop of Streptomyces feruloyl esterase plays an important role in its activity of releasing ferulic acid from biomass.
Appl Environ Microbiol. 2017 Nov 17;:
Authors: Uraji M, Tamura H, Mizohata E, Arima J, Wan K, Ogawa K, Inoue T, Hatanaka T
Feruloyl esterases (FAEs) are key enzymes required for the production of ferulic acid from agricultural biomass. Previously, we identified and characterized R18, an FAE from Streptomyces cinnamoneus NBRC 12852, which showed no sequence similarity with the known FAEs. To determine the region involved in its catalytic activity, we constructed chimeric enzymes using R18 and its homolog (TH2-18) from S. cinnamoneus strain TH-2. Although R18 and TH2-18 showed 74% identity in their primary sequence, the recombinant proteins of these two FAEs (rR18 and rTH2-18) showed very different specific activities toward ethyl ferulate. By comparing the catalytic activities of the chimeras, a domain comprised of residues 140 to 154 was found to be crucial for the catalytic activity of R18. Further, we analyzed the crystal structure of rR18 at a resolution of 1.5 Å to elucidate the relationship between its activity and structure. rR18 possessed a typical catalytic triad, consisting of Ser-191, Asp-214, and His-268, which was characteristic of the serine esterase family. By structural analysis, the above domain was found to be present in a loop-like structure (the R18 loop), which possessed a disulfide bond conserved in genus Streptomyces Moreover, compared to its parental strain rTH2-18, the TH2-18 mutant, in which Pro and Gly residues were inserted into the domain responsible for forming the R18 loop, showed markedly higher kcat values using artificial substrates. We also showed that the FAE activity of TH2-18 toward corn bran, a natural substrate, was improved by the insertion of the Gly and Pro residues.IMPORTANCEStreptomyces species are widely distributed bacteria that are predominantly present in soil and function as decomposers in natural environments. They produce various enzymes, such as carbohydrate hydrolases, esterases, and peptidases, which decompose agricultural biomass. In this study, based on the genetic information of two Streptomyces cinnamoneus strains, we identified novel feruloyl esterases (FAEs) capable of producing ferulic acid from biomass. These two FAEs shared high similarity in their amino acid sequences but did not resemblance any known FAEs. By comparing chimeric proteins and performing crystal structure analysis, we confirmed that a flexible loop was important for the catalytic activity of Streptomyces FAEs. Furthermore, we determined that the catalytic activity of one FAE improved drastically by inserting only two amino acids into its loop-forming domain. Thus, differences in the amino acid sequence of the loop resulted in different catalytic activities. In conclusion, our findings provide a foundation for the development of novel enzymes for industrial use.
PMID: 29150515 [PubMed - as supplied by publisher]
High prevalence and genetic diversity of large phiCD211/phiCDIF1296T-like prophages in Clostridioides difficile.
Appl Environ Microbiol. 2017 Nov 17;:
Authors: Garneau JR, Sekulovic O, Dupuy B, Soutourina O, Monot M, Fortier LC
Clostridioides difficile (formerly Clostridium difficile) is a pathogenic bacterium displaying great genetic diversity. A significant proportion of this diversity is due to the presence of integrated prophages. Here, we provide an in-depth analysis of phiCD211, also known as phiCDIF1296T, the largest phage identified in C. difficile so far, with a genome of 131-kbp. It shares morphological and genomic similarity with other large siphophages like phage 949 infecting Lactococcus lactis and phage c-st infecting Clostridium botulinum. A PhageTerm analysis indicated the presence of 378-bp direct terminal repeats at the phiCD211 genome termini. Among striking features of phiCD211, the presence of several transposase and integrase genes suggests past recombination events with other mobile genetic elements. Several gene products potentially influence the bacterial lifestyle and fitness, including a putative AcrB/AcrD/AcrF multidrug resistance protein, an EzrA septation ring formation regulator, and a spore protease. We also identified a CRISPR locus and a cas3 gene. We screened 2,584 C. difficile genomes available and detected 149 prophages sharing ≥80% nucleotide identity with phiCD211 (5% prevalence). Overall, phiCD211-like phages were detected in C. difficile strains corresponding to 21 different MLST groups, showing their high prevalence. Comparative genomic analyses revealed the existence of several clusters of highly similar phiCD211-like phages. Of note, large chromosome inversions were observed in some members, as well as multiple gene insertions and module exchanges. This highlights the great plasticity and gene coding potential of the phiCD211/phiCDIF1296T genome. Our analyses also suggest active evolution involving recombination with other mobile genetic elements.IMPORTANCEClostridioides difficile is a clinically important pathogen representing a serious threat to human health. Our hypothesis is that genetic differences between strains caused by the presence of integrated prophages could possibly explain the apparent differences observed in the virulence of different C. difficile strains. In this study, we provide a full characterization of phiCD211, also known as phiCDIF1296T, the largest phage known to infect C. difficile so far. Screening 2,584 C. difficile genomes revealed the presence of highly similar phiCD211-like phages in 5% of the strains analyzed, showing their high prevalence. Multiple genome comparisons suggest that evolution of the phiCD211-like phage community is dynamic, and some members have acquired genes that could possibly influence bacterial biology and fitness. Our study further supports the relevance of studying phages in C. difficile to better understand the epidemiology of this clinically important human pathogen.
PMID: 29150513 [PubMed - as supplied by publisher]
Sulfolobus acidocaldarius uptakes pentoses via a cut2-type ABC transporter and metabolizes them through the aldolase-independent Weimberg pathway.
Appl Environ Microbiol. 2017 Nov 17;:
Authors: Wagner M, Shen L, Albersmeier A, van der Kolk N, Kim S, Cha J, Bräsen C, Kalinowski J, Siebers B, Albers SV
Sulfolobus spp. possess a great metabolic versatility and grow heterotrophically on various carbon sources such as different sugars and peptides. Known sugar transporters in Archaea predominantly belong to ABC transport systems. Although several ABC transporters for sugar uptake have been characterized in the crenarchaeon Sulfolobus solfataricus, only one homologue of these transporters, the maltose/maltooligomer transporter, could be identified in the closely related Sulfolobus acidocaldarius Comparison of the transcriptome of S. acidocaldarius grown on peptides alone and peptides in presence of D-xylose allowed for the identification of the ABC transporter for D-xylose and L-arabinose transport and to gain deeper insights into pentose catabolism under the respective growth conditions. The D-xylose/L-arabinose substrate binding protein (SBP) (Saci_2122) of the ABC transporter is unique in Archaea and shares more similarity to bacterial SBPs of the Carbohydrate Uptake Transporter-2 (CUT2) family than to any characterized archaeal one. The identified pentose transporter is the first CUT2 family ABC transporter analyzed in the domain of Archaea. Single gene deletion mutants of the ABC transporter subunits exemplified the importance of the transport system for D-xylose and L-arabinose uptake. Next to the transporter operon, enzymes of the aldolase-independent pentose catabolism branch were found to be upregulated in N-Z-Amine and D-xylose medium. The α-ketoglutarate semialdehyde dehydrogenase (KGSADH; Saci_1938) seemed not to be essential for growth on pentoses. However, the deletion mutant of the 2-keto-3-deoxyarabinoate/xylonate dehydratase (KDXD/KDAD; Saci_1939) was no longer able to catabolize D-xylose or L-arabinose suggesting the absence of the aldolase-dependent branch in S. acidocaldariusImportance Thermoacidophilic microorganisms are emerging model organisms for biotechnological applications as their optimal growth conditions resemble conditions used in certain biotechnologies such as plant waste industrial degradation. Because of its high genome stability Sulfolobus acidocaldarius is especially suited as a platform organism for such applications. For the use in (ligno)cellulose degradation, it was important to understand the pentose uptake and metabolism in S. acidocaldarius This study revealed that only the aldolase-independent Weimberg pathway is required for growth of S. acidocaldarius on D-xylose and L-arabinose. Moreover, S. acidocaldarius employs a CUT2 ABC transporter for pentose uptake, which is more similar to bacterial than to archaeal ABC transporters. The identification of pentose inducible promoters will expedite the metabolic engineering of S. acidocaldarius for its development into a platform organism for (ligno)cellulose degradation.
PMID: 29150511 [PubMed - as supplied by publisher]
The tape measure protein is involved in the heat stability of Lactococcus lactis phages.
Appl Environ Microbiol. 2017 Nov 17;:
Authors: Geagea H, Labrie SJ, Subirade M, Moineau S
Virulent lactococcal phages are still a major risk for milk fermentation processes as they may lead to slowdowns and low quality fermented dairy products, particularly cheeses. Some of the phage control strategies used by the industry rely on heat treatments. Recently, a few Lactococcus lactis phages were found to be highly thermo-resistant. To identify the genetic determinant(s) responsible for the thermal resistance of lactococcal phages, we used the virulent phage CB14 (sk1virus/936 group) to select for phage mutants with increased heat stability. By treating phage CB14 to successive low and high temperatures, we were able to select two CB14 derivatives with increased heat stability. Sequencing of their genome revealed the same nucleotide sequences as the wild-type phage CB14, except for a same-sized deletion (120 bp) in the gene coding for the tape measure protein (TMP) of each phage mutant, but at a different position. The TMP protein sequences of these mutant phages were compared with their homologues in other wild-type L. lactis phages with a wide diversity in heat stability. Comparative analysis showed that the same nucleotide deletion appears to have also occurred in the gene coding for the TMP of highly thermo-resistant lactococcal phages P1532 and P680. We propose that the TMP is, in part, responsible for the heat stability of the highly predominant lactococcal phages of the sk1virus group.Importance Virulent lactococcal phages still represent a major risk for milk fermentation as they may lead to slowdowns and low quality fermented dairy products. Heat treatment is one of the most commonly used methods to control these virulent phages in cheese byproducts. Recently, a few Lactococcus lactis phages, members of the sk1virus group, have emerged with high thermal stability. To our knowledge, the genetic determinant(s) responsible for this thermal resistance in lactococcal phages is unknown. A better understanding of the thermal stability of these emerging virulent lactococcal phages is needed to improve industrial control strategies. In this work, we report the identification of a phage structural protein that is involved in the heat stability of a virulent sk1virus phage. Identifying such genetic determinant for heat stability is a first step to understand the emergence of this group of thermostable phages.
PMID: 29150509 [PubMed - as supplied by publisher]
Cry64Ba and Cry64Ca, two ETX/MTX2 Bacillus thuringiensis insecticidal proteins against hemipteran pests.
Appl Environ Microbiol. 2017 Nov 17;:
Authors: Liu Y, Wang Y, Shu C, Lin K, Song F, Bravo A, Soberón M, Zhang J
Genetically modified crops that express insecticidal Bacillus thuringiensis (Bt) proteins have become a primary approach for control of lepidopteran (moth) and coleopteran (beetle) pests that feed by chewing the plants. However, the sap-sucking insects (Hemiptera) are not particularly susceptible to Bt toxins. In this study, we describe two Cry toxins (Cry64Ba and Cry64Ca) from a Bt 1012 strain showed toxicity against two important rice hemipteran pests, Laodelphax striatellus and Sogatella furcifera Both of these two proteins contain an ETX/MTX2 domain, and share common sequence features with the β-pore forming toxins. Co-expression of cry64Ba and cry64Ca genes in the acrystalliferous Bt strain HD73(-) resulted in high insecticidal activity against both hemipteran pests. No toxicity was observed on other pests such as Ostrinia furnacalis, Plutella xylostella or Colaphellus bowringi Also, no hemolytic activity or toxicity against cancer cells was detected. Binding assays showed specific binding of Cry64Ba/Cry64Ca toxin complex to brush border membrane vesicles (BBMV) isolated from L. striatellus Altogether, the Cry64Ba and Cry64Ca are Bt Cry toxins high effective against hemipteran pests and could provide a novel strategy for the environment-friendly biological control of rice planthoppers in transgenic plants.Importance: Rice is an important staple food in Asia whose production is threatened by rice planthoppers. To date, no effective Bacillus thuringiensis (Bt) protein has been identified against the rice planthoppers. We cloned two Bt toxin genes from Bt 1012 strain that showed toxicity against small brown planthopper (Laodelphax striatellus) and white-backed planthopper (Sogatella furcifera). The proteins encoded by cry64Ba and cry64Ca genes are the most efficient insecticidal Bt Cry proteins against hemipteran insects reported so far. Cry64Ba and Cry64Ca showed no toxicity against some Lepidopteran or Coleopteran pests. These two proteins should be able to be used for the integrated hemipteran pest management.
PMID: 29150505 [PubMed - as supplied by publisher]
Low molecular weight thiols and thioredoxins are important players in Hg(II) resistance in Thermus thermophilus HB27.
Appl Environ Microbiol. 2017 Nov 17;:
Authors: Norambuena J, Wang Y, Hanson T, Boyd JM, Barkay T
Mercury (Hg), one of the most toxic and widely distributed heavy metals, has a high affinity for thiol groups. Thiol groups reduce and sequester Hg. Therefore, low molecular weight and protein thiols may be important cell components used in Hg resistance. To date, the role of low molecular weight thiols in Hg-detoxification remains understudied. The mercury resistance (mer) operon of Thermus thermophilus suggests an evolutionary link between Hg(II) resistance and low molecular weight thiol metabolism. This mer operon encodes for an enzyme involved in methionine biosynthesis, Oah. Challenge with Hg(II) resulted in increased expression of genes involved in the biosynthesis of multiple low molecular weight thiols (cysteine, homocysteine, and bacillithiol), as well as the thioredoxin system. Phenotypic analysis of gene replacement mutants indicated that Oah contributes to Hg resistance under sulfur limiting conditions, and strains lacking bacillithiol and/or thioredoxins are more sensitive to Hg(II) than the wild type. Growth in presence of either a thiol oxidizing agent or a thiol alkylating agent increased sensitivity to Hg(II). Furthermore, exposure to 3 μM Hg(II) consumed all intracellular reduced bacillithiol and cysteine. Database searches indicate that oah2 is present in all Thermus spp. mer operons. The presence of a thiol related gene was also detected in some alphaprotobacterial mer operons, in which a glutathione reductase gene was present, supporting the role of thiols in Hg(II) detoxification. These results have led to a working model in which LMW thiols act as Hg(II) buffering agents while Hg is reduced by MerA.ImportanceThe survival of microorganisms in presence of toxic metals is central to life's sustainability. The affinity of thiol groups to toxic heavy metals drives microbe-metal interactions and modulate metal toxicity. Mercury detoxification (mer) genes likely originated early in microbial evolution among geothermal environments. Little is known about how mer systems interact with cellular thiol systems. Thermus spp. possess a simple mer operon in which a low molecular weight thiol biosynthesis gene is present, along with merR and merA In this study, we present experimental evidence for the role of thiol systems in mercury resistance. Our data suggest that in T. thermophilus thiolated compounds may function side-by-side with mer genes to detoxify mercury. Thus, thiol systems function in consort with mer-mediated resistance to mercury, suggesting exciting new questions for future research.
PMID: 29150497 [PubMed - as supplied by publisher]
Small, dense high-density lipoproteins display potent vasorelaxing activity, reflecting their elevated content of sphingosine-1-phosphate.
J Lipid Res. 2017 Nov 17;:
Authors: Persegol L, Darabi M, Dauteuille C, Lhomme M, Chantepie S, Rye KA, Therond P, Chapman MJ, Salvayre R, Negre-Salvayre A, Lesnik P, Monier S, Kontush A
AIMS: Functional heterogeneity of HDL is attributed to its diverse bioactive components. We evaluated whether vasodilatory effects of HDL differed across HDL subpopulations, reflecting their distinct molecular composition.
METHODS: Capacity of five major HDL subfractions to counteract inhibitory effects of oxidized low-density lipoprotein on acetylcholine-induced vasodilation was tested in a rabbit aortic rings model. Nitric oxide (NO) production, an essential pathway in endothelium-dependent vasorelaxation, was studied in SV40-transformed murine endothelial cells (SVEC).
RESULTS: Small, dense HDL3 subfractions displayed potent vasorelaxing activity (up to +31% vs. baseline, p<0.05); in contrast, large, light HDL2 did not induce aortic-ring relaxation when compared on a total protein basis. HDL3 particles were enriched with sphingosine-1-phosphate (S1P; up to 3-fold vs. HDL2), with the highest content in HDL3b and 3c that concomitantly revealed the strongest vasorelaxing properties. NO generation was enhanced by HDL3c in SVEC (1.5-fold, p<0.01), a phenomenon which was blocked by the S1P receptor antagonist VPC 23019. S1P-enriched reconstituted HDL was 1.8-fold (p<0.01) more potent vasorelaxant than control rHDL in aortic rings.
CONCLUSIONS: Small, dense HDL3 particles displayed potent protective effects against oxidative stress-associated endothelium dysfunction, potentially reflecting their elevated content of S1P that might facilitate interaction with S1P receptors and ensuing NO generation.
PMID: 29150495 [PubMed - as supplied by publisher]
Biogenesis of the bacterial cbb3 cytochrome c oxidase: active subcomplexes support a sequential assembly model.
J Biol Chem. 2017 Nov 17;:
Authors: Durand A, Bourbon ML, Steunou AS, Khalfaoui-Hassani B, Legrand C, Guitton A, Astier C, Ouchane S
The cbb3 oxidase has a high affinity for oxygen and is required for growth of bacteria, including pathogens, in oxygen-limited environments. However, the assembly of this oxidase is poorly understood. Most cbb3 are composed of four subunits: the catalytic CcoN subunit, the two cytochromes c subunits (CcoO and CcoP) involved in electron transfer and the small CcoQ subunit with an unclear function. Here, we address the role of these four subunits in cbb3 biogenesis in the purple bacterium Rubrivivax gelatinosus Analyses of membrane proteins from different mutants revealed the presence of active CcoNQO and CcoNO subcomplexes and also showed that the CcoP subunit is not essential for their assembly. However, CcoP was required for the oxygen reduction activity in the absence of CcoQ. We also found that CcoQ is dispensable for forming an active CcoNOP subcomplex in membranes. CcoNOP exhibited oxygen reductase activity, indicating that the cofactors (hemes b and copper for CcoN, cytochromes c for CcoO and CcoP) were present within the subunits. Finally, we discovered the presence of a CcoNQ subcomplex and showed that CcoN is the required anchor for the assembly of the full CcoNQOP complex. On the basis of these findings, we propose a sequential assembly model in which the CcoQ subunit is required for the early maturation step: CcoQ first associates with CcoN before the CcoNQ-CcoO interaction. CcoP associates to CcoNQO subcomplex in the late maturation step and once the CcoNQOP complex is fully formed, CcoQ is released for degradation by the FtsH protease. This model could be conserved in other bacteria including the pathogenic bacteria lacking the assembly factor CcoH as in R. gelatinosus.
PMID: 29150446 [PubMed - as supplied by publisher]
Suppression of FIP200 and autophagy by tumor-derived lactate promotes naïve T cell apoptosis and affects tumor immunity.
Sci Immunol. 2017 Nov 17;2(17):
Authors: Xia H, Wang W, Crespo J, Kryczek I, Li W, Wei S, Bian Z, Maj T, He M, Liu RJ, He Y, Rattan R, Munkarah A, Guan JL, Zou W
Naïve T cells are poorly studied in cancer patients. We report that naïve T cells are prone to undergo apoptosis due to a selective loss of FAK family-interacting protein of 200 kDa (FIP200) in ovarian cancer patients and tumor-bearing mice. This results in poor antitumor immunity via autophagy deficiency, mitochondria overactivation, and high reactive oxygen species production in T cells. Mechanistically, loss of FIP200 disables the balance between proapoptotic and antiapoptotic Bcl-2 family members via enhanced argonaute 2 (Ago2) degradation, reduced Ago2 and microRNA1198-5p complex formation, less microRNA1198-5p maturation, and consequently abolished microRNA1198-5p-mediated repression on apoptotic gene Bak1 Bcl-2 overexpression and mitochondria complex I inhibition rescue T cell apoptosis and promoted tumor immunity. Tumor-derived lactate translationally inhibits FIP200 expression by down-regulating the nicotinamide adenine dinucleotide level while potentially up-regulating the inhibitory effect of adenylate-uridylate-rich elements within the 3' untranslated region of Fip200 mRNA. Thus, tumors metabolically target naïve T cells to evade immunity.
PMID: 29150439 [PubMed - in process]
Ketogenic diet and anorexia nervosa.
Med Hypotheses. 2017 Nov;109:150-152
Authors: Scolnick B
This hypothesis suggest that starvation-induced ketosis, which leads to a fundamental biochemical change in the metabolic fuel supply of the brain, is uniquely anxiolytic and rewarding to patients prone to AN. Ketosis can easily be replicated by a unique diet marked by high fat, moderate protein, and very low carbohydrate. This diet, known as a ketogenic diet (KGD) mimics starvation, thus allowing the patient to experience the anxiolytic state of ketosis, and yet avoid the morbidity of starvation.
PMID: 29150275 [PubMed - in process]
Neuroprotective potential of high-dose biotin.
Med Hypotheses. 2017 Nov;109:145-149
Authors: McCarty MF, DiNicolantonio JJ
A recent controlled trial has established that high-dose biotin supplementation - 100 mg, three times daily - has a stabilizing effect on progression of multiple sclerosis (MS). Although this effect has been attributed to an optimization of biotin's essential cofactor role in the brain, a case can be made that direct stimulation of soluble guanylate cyclase (sGC) by pharmacological concentrations of biotin plays a key role in this regard. The utility of high-dose biotin in MS might reflect an anti-inflammatory effect of cGMP on the cerebral microvasculature, as well on oligodendrocyte differentiation and on Schwann cell production of neurotrophic factors thought to have potential for managing MS. But biotin's ability to boost cGMP synthesis in the brain may have broader neuroprotective potential. In many types of neurons and neural cells, cGMP exerts neurotrophic-mimetic effects - entailing activation of the PI3K-Akt and Ras-ERK pathways - that promote neuron survival and plasticity. Hippocampal long term potentiation requires nitric oxide synthesis, which in turn promotes an activating phosphorylation of CREB via a pathway involving cGMP and protein kinase G (PKG). In Alzheimer's disease (AD), amyloid beta suppresses this mechanism by inhibiting sGC activity; agents which exert a countervailing effect by boosting cGMP levels tend to restore effective long-term potentiation in rodent models of AD. Moreover, NO/cGMP suppresses amyloid beta production within the brain by inhibiting expression of amyloid precursor protein and BACE1. In conjunction with cGMP's ability to oppose neuron apoptosis, these effects suggest that high-dose biotin might have potential for the prevention and management of AD. cGMP also promotes neurogenesis, and may lessen stroke risk by impeding atherogenesis and hypertrophic remodeling in the cerebral vasculature. The neuroprotective potential of high-dose biotin likely could be boosted by concurrent administration of brain-permeable phosphodiesterase-5 inhibitors.
PMID: 29150274 [PubMed - in process]
Reduced gene expression of sirtuins and active AMPK levels in children and adolescents with obesity and insulin resistance.
Obes Res Clin Pract. 2017 Nov 14;:
Authors: Arab Sadeghabadi Z, Nourbakhsh M, Pasalar P, Emamgholipour S, Golestani A, Larijani B, Razzaghy-Azar M
BACKGROUND: Sirtuins, including SIRT1 and SIRT2, are longevity-associated deacetylase enzymes that modulate metabolic homeostasis in response to the cellular energy state. Adenosine monophosphate activated protein kinase (AMPK) and SIRT1 are interrelated and share several common target pathways. This study aimed to evaluate the SIRT1 and SIRT2 gene expression in peripheral blood mononuclear cells (PBMCs) as well as plasma levels of AMPK, in obese children and adolescents.
MATERIALS AND METHODS: Participants included 60 children and adolescents (30 obese and 30 age- and gender-matched control subjects). Real-time polymerase chain reaction (PCR) was used to assess the SIRT1 and SIRT2 gene expression in PBMCs. Serum phospho-AMPK and insulin were measured using enzyme-linked immunosorbent assay (ELISA), and insulin resistance (IR) was calculated by the Homeostasis Model of Assessment of Insulin Resistance (HOMA-IR). Glucose and lipid profile were also measured.
RESULTS: SIRT1 gene expression and phospho-AMPK plasma levels were significantly diminished in obese subjects compared to the control group, and both SIRT1 and SIRT2 were significantly lower in obese children with IR compared to those without IR. SIRT1 expression revealed significant negative correlations with body mass index and waist circumference as well as insulin and HOMA-IR and a positive correlation with AMPK. SIRT2 negatively correlated with SIRT1 and positively correlated with high density lipoprotein-cholesterol (HDL-C).
CONCLUSION: SIRT1 and SIRT2 expression and AMPK levels decrease in children with obesity and IR. Targeting SIRT1 can be valuable in preventing obesity-associated IR in childhood and adolescence.
PMID: 29150224 [PubMed - as supplied by publisher]
For survival, the emergence of oligoclonal bands after multiple myeloma treatment is less important than achieving complete remission.
Rev Bras Hematol Hemoter. 2017 Oct - Dec;39(4):331-336
Authors: Silva LSVD, Crusoe EQ, Souza LRG, Chiattone CS, Hungria VTM
BACKGROUND: The emergence of oligoclonal bands, proteins differing from those originally identified at diagnosis, has been reported in multiple myeloma patients after high-dose chemotherapy followed by autologous stem cell transplantation and after successful conventional chemotherapy. The clinical relevance of oligoclonal bands remains unclear, but their emergence has been associated with better prognosis. The aim of the present study was to determine the prevalence, clinical characteristics and prognostic impact of the presence of oligoclonal bands in multiple myeloma patients.
METHODS: A retrospective cohort study was conducted. The study included newly diagnosed multiple myeloma patients with at least very good partial response after conventional dose or high-dose chemotherapy followed by autologous stem cell transplantation. The emergence of oligoclonal bands was identified using serum protein electrophoresis as well as serum and urine immunofixation techniques.
RESULTS: A total of 101 patients were included with a median follow-up of 42 months. In total, 55% were male, and the median age was 58 years (29-87 years). Fifty-one (50.5%) patients developed oligoclonal bands. They comprised 60% (45/75) of patients treated with autologous stem cell transplantation and 23% (6/26) of those who were not transplanted. Patients with oligoclonal bands showed better progression-free survival than those without the emergence of oligoclonal bands (p-value=0.0075).
CONCLUSION: The prevalence of oligoclonal bands in this study population was 50.5% with its frequency being greater in cases treated with autologous stem cell transplantation and in those attaining complete remission. Complete remission was more important than the emergence of oligoclonal bands on progression-free survival.
PMID: 29150105 [PubMed]
Unique arginine array improves cytosolic localization of hydrocarbon-stapled peptides.
Bioorg Med Chem. 2017 Nov 07;:
Authors: Quach K, LaRochelle J, Li XH, Rhoades E, Schepartz A
We have previously reported that miniature proteins containing a distinct array of 5 arginine residues on a folded α-helix - a penta-arg motif - traffic with high efficiency from endosomes into the cytosol and nucleus of mammalian cells. Here we evaluate whether a penta-arg motif can improve the intracellular trafficking of an otherwise impermeant hydrocarbon-stapled peptide, SAH-p53-4(Rho). We prepared a panel of SAH-p53-4(Rho) variants containing penta-arg sequences with different spacings and axial arrangement and evaluated their overall uptake (as judged by flow cytometry) and their intracellular access (as determined by fluorescence correlation spectroscopy, FCS). One member of this panel reached the cytosol extremely well, matching the level achieved by SAH-p53-8(Rho), a previously reported and highly permeant hydrocarbon-stapled peptide. Notably, we found no relationship between cellular uptake as judged by flow cytometry and cytosolic access as determined by FCS. This result reiterates that overall uptake and endosomal release represent fundamentally different biological processes. To determine cytosolic and/or nuclear access, one must measure concentration directly using a quantitative and non-amplified tool such as FCS. As has been observed for highly cell permeant miniature proteins such as ZF5.3, optimal penetration of hydrocarbon-stapled peptides into the cell cytosol results when the penta-arg motif is located within more (as opposed to less) structured regions.
PMID: 29150077 [PubMed - as supplied by publisher]
Effects of Cyclosporine, Tacrolimus, and Rapamycin on Osteoblasts.
Transplant Proc. 2017 Nov;49(9):2219-2224
Authors: Martín-Fernández M, Rubert M, Montero M, de la Piedra C
PURPOSE: One factor that can contribute to severe bone loss after transplantation is the direct action of immunosuppressants on bone cells. The aim of this work was to study the effects of cyclosporine (CsA), tacrolimus (FK-506), and rapamycin (RAPA) on the release of three local factors directly implicated in bone-remodeling regulation and apoptosis of human osteoblasts: interleukin (IL)-6, osteoprotegerin, and receptor activator of nuclear factor κβ (RANKL).
BASIC PROCEDURES: Human osteoblasts were obtained from five different patients who underwent orthopedic surgery. These cells were treated with what are considered to be a clinically high dose and an acceptable dose of each immunosuppressant-RAPA 50 ng/mL and 12 ng/mL, FK-506 20 ng/mL and 5 ng/mL, CsA 1000 ng/mL and 250 ng/mL-or vehicle. Apoptotic cell death was quantified using flow cytometry of DNA content in permeabilized, propidium iodide-stained cells. IL-6 was measured using enzyme-linked immunosorbent assay (ELISA; Quantikine Human IL6, R&D Systems, Minneapolis, Minn, United States). Messenger RNA (mRNA) expression of osteoprotegerin, RANKL, and IL-6 was measured using quantitative RT-PCR.
MAIN FINDINGS: A significant increase in IL-6 (mRNA and released protein) was observed in the presence of FK-506 and RAPA. Addition of RAPA to the cultures of osteoblasts produced a significant increase in the OPG/RANKL ratio. A significant increase in osteoblast apoptosis was observed in the cells treated with FK-506 and RAPA 24 hours after the addition of immunosuppressants. CsA did not produce any significant changes in osteoblasts.
PRINCIPAL CONCLUSIONS: These results suggest that an increase in osteoblast apoptosis by osteoblasts may be one of the mechanisms by which bone loss occurs after RAPA and FK-506 treatments.
PMID: 29149986 [PubMed - in process]
Novel insights into chromosomal conformations in cancer.
Mol Cancer. 2017 Nov 17;16(1):173
Authors: Jia R, Chai P, Zhang H, Fan X
Exploring gene function is critical for understanding the complexity of life. DNA sequences and the three-dimensional organization of chromatin (chromosomal interactions) are considered enigmatic factors underlying gene function, and interactions between two distant fragments can regulate transactivation activity via mediator proteins. Thus, a series of chromosome conformation capture techniques have been developed, including chromosome conformation capture (3C), circular chromosome conformation capture (4C), chromosome conformation capture carbon copy (5C), and high-resolution chromosome conformation capture (Hi-C). The application of these techniques has expanded to various fields, but cancer remains one of the major topics. Interactions mediated by proteins or long noncoding RNAs (lncRNAs) are typically found using 4C-sequencing and chromatin interaction analysis by paired-end tag sequencing (ChIA-PET). Currently, Hi-C is used to identify chromatin loops between cancer risk-associated single-nucleotide polymorphisms (SNPs) found by genome-wide association studies (GWAS) and their target genes. Chromosomal conformations are responsible for altered gene regulation through several typical mechanisms and contribute to the biological behavior and malignancy of different tumors, particularly prostate cancer, breast cancer and hematologic neoplasms. Moreover, different subtypes may exhibit different 3D-chromosomal conformations. Thus, C-tech can be used to help diagnose cancer subtypes and alleviate cancer progression by destroying specific chromosomal conformations. Here, we review the fundamentals and improvements in chromosome conformation capture techniques and their clinical applications in cancer to provide insight for future research.
PMID: 29149895 [PubMed - in process]
Serum concentrations of Krebs von den Lungen-6, surfactant protein D, and matrix metalloproteinase-2 as diagnostic biomarkers in patients with asbestosis and silicosis: a case-control study.
BMC Pulm Med. 2017 Nov 17;17(1):144
Authors: Xue C, Wu N, Li X, Qiu M, Du X, Ye Q
BACKGROUND: Asbestosis and silicosis are progressive pneumoconioses characterized by interstitial fibrosis following exposure to asbestos or silica dust. We evaluated the potential diagnostic biomarkers for these diseases.
METHODS: The serum concentrations of Krebs von den Lungen-6 (KL-6), surfactant protein D (SP-D), and matrix metalloproteinase-2 (MMP-2), MMP-7, and MMP-9 were measured in 43 patients with asbestosis, 45 patients with silicosis, 40 dust-exposed workers (DEWs) without pneumoconiosis, and 45 healthy controls (HCs). Chest high-resolution computed tomography (HRCT) images were reviewed by experts blinded to the clinical data. According to the receiver operating characteristic (ROC) curve, the ideal level of each biomarker and its diagnostic sensitivity were obtained.
RESULTS: The serum KL-6 and MMP-2 concentrations were highest in patients with asbestosis, particularly in comparison with those in DEWs and HCs (P<0.05). The serum SP-D concentration was significantly higher in patients with asbestosis than in patients with silicosis, DEWs, and HCs (P<0.01), whereas no significant difference was noted among patients with silicosis, DEWs, and HCs. No significant difference in the serum MMP-7 or -9 concentration was found among patients with asbestosis, patients with silicosis, DEWs, or HCs. Among patients with asbestosis, the serum KL-6 concentration was significantly correlated with the lung fibrosis scores on HRCT and negatively correlated with the forced vital capacity (FVC) % predicted and diffusing capacity of the lung for carbon monoxide (DLCO) % predicted. The serum SP-D and MMP-2 concentrations were negatively correlated with the DLCO % predicted (all P<0.05). The order of diagnostic accuracy according to the ROC curve was KL-6, SP-D, and MMP-2 in patients with asbestosis alone and in the combination of both patients with asbestosis and those with silicosis. The combination of all three biomarkers may increase the possibility of diagnosing asbestosis (sensitivity, 93%; specificity, 57%) and both asbestosis and silicosis (sensitivity, 83%; specificity, 62%).
CONCLUSIONS: KL-6, SP-D, and MMP-2 are available biomarkers for the adjuvant diagnosis of asbestosis and silicosis. The combination of all three biomarkers may improve the diagnostic sensitivity for asbestosis and silicosis.
PMID: 29149883 [PubMed - in process]
High-density lipoprotein (HDL) Dysfunction and Future of HDL.
Curr Vasc Pharmacol. 2017 Nov 16;:
Authors: Ertek S
Although high density lipoprotein cholesterol (HDL-C) levels are inversely proportional to cardiovascular risk in many studies, recent pharmacological interventional studies with HDL-C raising strategies did not show a benefit in terms of vascular events. The HDL particle is heterogenous with anti-atherogenic functions and non-vascular effects. Many factors affect HDL components and may either cause compositional changes, post-translational modifications of proteins, or alter lipids and other cargo molecules; generally these factors cause more than one of these changes, resulting in functional differences. Therefore, the role of lipoproteins change in different physical and disease conditions. Mainly, in proteome, apolipoprotein A1 (Apo-A1), myeloperoxidase (MPO), paroxonase (PON) are affected by inflammation or glycation-related factors; and especially esterification or unesterification of lipids, changes in phospholipid or unsaturated lipid content change the HDL function. Measuring the HDL-C level is probably not a good predictor of its cardiovascular benefits, and methods to evaluate HDL functions are required. In current medical practice, it is not simple and feasible to measure different functions of this lipoprotein, but near-future strategies may be developed. Meanwhile, as we learn more about HDL structure and the role of each component, we can develop therapeutic approaches to improve HDL function. Apo-A1-mimetics, reconstituted HDL, nanoparticles and microRNA therapies could be promising as anti-atherosclerotic therapies. They may even provide useful therapies for the treatment of some non-cardiovascular diseases.
PMID: 29149817 [PubMed - as supplied by publisher]