In silico identification and characterization of putative differentially expressed genes involved in common bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L.) seed development

Two genotypes of common bean (Phaseolus vulgaris L.) were studied to determine the structural cause of seed abortion in this species. In the non-abortive control (wild-type, cultivar BAT93), the histological analysis revealed a classical pattern of seed development and showed coordinated differentiation of the embryo proper, suspensor, endosperm tissue and seed coat. In contrast, the ethyl methanesulfonate (EMS) mutant (cultivar BAT93) showed disruption in the normal seed development leading to embryo abortion. Aborted embryos from these degenerate seeds showed abnormalities in suspensor and cotyledons at the globular, heart, torpedo and cotyledon stages. Exploring the feasibility of incorporating the available online bioinformatics databases, we identified 22 genes revealing high homology with genes involved in Arabidopsis thaliana embryo development and expressed in common bean immature seeds. The expression patterns of these genes were confirmed by RT–PCR. All genes were highly expressed in seed tissues. To study the expression profiles of isolated genes during Phaseolus embryogenesis, six selected genes were examined by quantitative RT–PCR analysis on the developing embryos of wild-type and EMS mutant plants. All selected genes were expressed differentially at different stages of embryo development. These results could help to improve understanding of the mechanism of common bean embryogenesis.     

Morphology of three new marine Frontonia species (Ciliophora; Peniculida) with note on the phylogeny of this genus

Members of the ciliate genus Frontonia are common colonizers of periphytic communities in aquatic biotopes. Recent studies indicate that their species diversity is higher than previously supposed. In this study the morphology and infraciliature of three new species, Frontonia sinica spec. nov., F. pusilla spec. nov., and F. elegans spec. nov., isolated from coastal waters of China, were investigated using live observation and silver impregnation methods. Frontonia sinica differs from its congeners by the following combination of characters: ellipsoidal body, about 116 somatic and five or six vestibular kineties, peniculi 1 and 2 four-rowed, peniculus 3 two-rowed, and a single contractile vacuole. Frontonia pusilla has about 72 somatic kineties, four-rowed peniculi 1 and 2, a two-rowed peniculus 3, and two contractile vacuoles. Frontonia elegans has 73 somatic kineties, four-rowed peniculi 1 and 2, a three-rowed peniculus 3, and two contractile vacuoles. In the present work, six new small-subunit rRNA gene sequences of six Frontonia species are used to construct the phylogenetic trees. Our phylogenetic analysis supports that the genus Frontonia may be paraphyletic. Meanwhile, no pattern of correlation could be found between the structures of peniculi and the phylogenetic relationships of Frontonia species in the present study.     

Designing an immunoinformatic vaccine for peri-implantitis using a structural biology approach

ObjectivesPeri-implantitis is a destructive inflammatory process that affects the soft and hard tissues around dental implants. porphyromonas gingivalis, an anaerobic gram-negative bacterium, appears to be the main culprit. Since there is no efficient and specific vaccine to treat peri-implantitis, the goal of our research has been to develop a multi-epitope vaccination utilizing an immunoinformatics approach that targeted P. gingivalis type I fim A.Materials and methodsP. gingivalis peptides 6JKZ and 6KMF are suitable for vaccine development. B- and T-cell epitopes from 6KMF and 6JKZ were detected and evaluated based on critical factors to produce a multi-epitope vaccine construct. It was assessed based on allergenicity, antigenicity, stability. The vaccine's dual major histocompatibility complex (MHC-I and MHC-II) binding epitopes allowed it to reach a larger population. P. gingivalis fimbriae induce immune subversion through TLR -CXCR4 receptor complex pathway. The ClusPro 2.0 server was used to do the molecular docking using TLR2 - CXCR4 and vaccine epitopes as receptor and ligand respectively.ResultsThe designed vaccine was non-allergenic and had a high antigenicity, solubility, and stability. The 3D structure of the vaccine revealed strong interaction with CXCR4(TLR2) using molecular docking. The vaccine-CXCR4 interface was more consistent, possibly because the vaccination has a higher affinity for the CXCR4-TLR2 complex.ConclusionThis study details the vaccine's distinct and sustained interaction with the CXCR4(TLR2) immunological receptor and its consistent and effective utterance in the bacterial system. As a result, our vaccine formulation will evoke a significant memory response and induce an adaptive immune response against P. gingivalis.     

Morphology and Phylogenetic Placement of Three New Zoothamnium species (Ciliophora: Peritrichia) from Coastal Waters of Southern China

The morphology, infraciliature, and silverline system of three peritrichous ciliates, Zoothamnium bucciniiformum sp. n., Zoothamnium florens sp. n., and Zoothamnium zhanjiangense sp. n., were investigated based on both living and silver‐stained specimens. Zoothamnium bucciniiformum sp. n., collected from coastal waters (salinity 30‰) off Zhanjiang, southern China, can be distinguished by the following characters: dichotomously branched stalk, peristomial lip with medial circumferential infolding, contractile vacuole apically positioned, 32–49 silverlines between the anterior end and the aboral trochal band, 15–26 between the aboral trochal band and the scopula; two kineties in peniculus 3, not parallel to each other. Zoothamnium florens sp. n., collected from a mangrove wetland (salinity 13‰) off Zhanjiang, is characterized by its large conical zooid, tuberculate peristomial lip, asymmetrical dichotomously branched colony, 59–81 silverlines between the anterior end and the aboral trochal band and 29–36 between the aboral trochal band and the scopula. Zoothamnium zhanjiangense, collected from a mangrove wetland (salinity about 9.5‰) off Zhanjiang, differs from its congeners by the alternately branched stalk, peristomial lip with medial circumferential infolding, 40–63 silverlines from the peristomial area to the aboral trochal band and 13–24 from the aboral trochal band to the scopula. The comparison and analysis of SSU rDNA sequences also support present identifications.     

Laboratory Test Surveillance following Acute Kidney Injury

Background

Patients with hospitalized acute kidney injury (AKI) are at increased risk for accelerated loss of kidney function, morbidity, and mortality. We sought to inform efforts at improving post-AKI outcomes by describing the receipt of renal-specific laboratory test surveillance among a large high-risk cohort.

Methods

We acquired clinical data from the Electronic health record (EHR) of 5 Veterans Affairs (VA) hospitals to identify patients hospitalized with AKI from January 1^st, 2002 to December 31^st, 2009, and followed these patients for 1 year or until death, enrollment in palliative care, or improvement in renal function to estimated GFR (eGFR) ≥60 L/min/1.73 m². Using demographic data, administrative codes, and laboratory test data, we evaluated the receipt and timing of outpatient testing for serum concentrations of creatinine and any as well as quantitative proteinuria recommended for CKD risk stratification. Additionally, we reported the rate of phosphorus and parathyroid hormone (PTH) monitoring recommended for chronic kidney disease (CKD) patients.

Results

A total of 10,955 patients admitted with AKI were discharged with an eGFR<60 mL/min/1.73 m². During outpatient follow-up at 90 and 365 days, respectively, creatinine was measured on 69% and 85% of patients, quantitative proteinuria was measured on 6% and 12% of patients, PTH or phosphorus was measured on 10% and 15% of patients.

Conclusions

Measurement of creatinine was common among all patients following AKI. However, patients with AKI were infrequently monitored with assessments of quantitative proteinuria or mineral metabolism disorder, even for patients with baseline kidney disease.     

C6ORF66 is an assembly factor of mitochondrial complex I

Homozygosity mapping was performed in five patients from a consanguineous family who presented with infantile mitochondrial encephalomyopathy attributed to isolated NADH:ubiquinone oxidoreductase (complex I) deficiency. This resulted in the identification of a missense mutation in a conserved residue of the C6ORF66 gene, which encodes a 20.2 kDa mitochondrial protein. The mutation was also detected in a patient who presented with antenatal cardiomyopathy. In muscle of two patients, the levels of the C6ORF66 protein and of the fully assembled complex I were markedly reduced. Transfection of the patients' fibroblasts with wild-type C6ORF66 cDNA restored complex I activity. These data suggest that C6ORF66 is an assembly factor of complex I. Interestingly, the C6ORF66 gene product was previously shown to promote breast cancer cell invasiveness.     

Identification of GBF1 as a Cellular Factor Required for Hepatitis C Virus RNA Replication

In infected cells, hepatitis C virus (HCV) induces the formation of membrane alterations referred to as membranous webs, which are sites of RNA replication. In addition, HCV RNA replication also occurs in smaller membrane structures that are associated with the endoplasmic reticulum. However, cellular mechanisms involved in the formation of HCV replication complexes remain largely unknown. Here, we used brefeldin A (BFA) to investigate cellular mechanisms involved in HCV infection. BFA acts on cell membranes by interfering with the activation of several members of the family of ADP-ribosylation factors (ARF), which can lead to a wide range of inhibitory actions on membrane-associated mechanisms of the secretory and endocytic pathways. Our data show that HCV RNA replication is highly sensitive to BFA. Individual knockdown of the cellular targets of BFA using RNA interference and the use of a specific pharmacological inhibitor identified GBF1, a guanine nucleotide exchange factor for small GTPases of the ARF family, as a host factor critically involved in HCV replication. Furthermore, overexpression of a BFA-resistant GBF1 mutant rescued HCV replication in BFA-treated cells, indicating that GBF1 is the BFA-sensitive factor required for HCV replication. Finally, immunofluorescence and electron microscopy analyses indicated that BFA does not block the formation of membranous web-like structures induced by expression of HCV proteins in a nonreplicative context, suggesting that GBF1 is probably involved not in the formation of HCV replication complexes but, rather, in their activity. Altogether, our results highlight a functional connection between the early secretory pathway and HCV RNA replication.Hepatitis C virus (HCV) is an important human pathogen. It mainly infects human hepatocytes, and this often leads to chronic hepatitis, cirrhosis, or hepatocarcinoma. HCV studies have been hampered for many years by the difficulty in propagating this virus in vitro. Things have recently changed with the development of a cell culture model referred to as HCVcc (34, 60, 65), which allows the study of the HCV life cycle in cell culture and facilitates studies of the interactions between HCV and the host cell.HCV is an enveloped positive-strand RNA virus belonging to the family Flaviviridae (35). The viral genome contains a single open reading frame, which is flanked by two noncoding regions that are required for translation and replication. All viral proteins that are produced after proteolytic processing of the initially synthesized polyprotein are membrane associated (15, 43). This reflects the fact that virtually all steps of the viral life cycle occur in close association with cellular membranes.Interactions of HCV with cell membranes begin during entry. Several receptors, coreceptors, and other entry factors have been discovered over the years, which link HCV entry to specialized domains of the plasma membrane, such as tetraspanin-enriched microdomains and tight junctions (8, 16, 59). The internalization of the viral particle occurs by clathrin-mediated endocytosis (5, 40). The fusion of the viral envelope with the membrane of an acidic endosome likely mediates the transfer of the viral genome to the cytosol of the cell (5, 40, 57). However, little is known regarding the pre- and postfusion intracellular transport steps of entering viruses in the endocytic pathway.HCV RNA replication is also associated with cellular membranes. Replication begins with the translation of the genomic RNA of an incoming virus. This leads to the production of viral proteins, which in turn initiate the actual replication of the viral RNA. Mechanisms regulating the transition from the translation of the genomic RNA to its replication are not yet known. All viral proteins are not involved in RNA replication. Studies performed with subgenomic replicons demonstrated that proteins NS3-4A, NS4B, NS5A, and NS5B are necessary and sufficient for replication (6, 27, 37). RNA replication proceeds through the synthesis of a cRNA strand (negative strand), catalyzed by the RNA-dependent RNA polymerase activity of NS5B, which is then used as a template for the synthesis of new positive strands.Electron microscopy studies using a subgenomic replicon model suggested that replication takes place in membrane structures made of small vesicles, referred to as “membranous webs,” which are induced by the virus (26). Membranous webs are detectable not only in cells carrying subgenomic replicons but also in infected cells (50). They appear to be associated with the endoplasmic reticulum (ER) (26). In addition to the membranous webs, a second type of ER-associated replicase that is smaller and more mobile has recently been described (63). Cellular mechanisms leading to these membrane alterations are still poorly understood. In cells replicating and secreting infectious viruses effectively, the situation appears to be even more complex, since replicase components appear to be, at least in part, associated with cytoplasmic lipid droplets (41, 50, 56). This association depends on the capsid protein (41) and may reflect a coupling between replication and assembly. Indeed, HCV assembly and secretion show some similarities with very-low-density lipoprotein (VLDL) maturation and secretion (24, 64).Our knowledge of the cellular membrane mechanisms involved in the HCV life cycle is still limited. The expression of NS4B alone induces membrane alterations that are reminiscent of membranous webs (19). However, cellular factors that participate in this process are still unknown. On the other hand, several cellular proteins potentially involved in the HCV life cycle have been identified through their interactions with viral proteins. For some of these proteins, a functional role in infection was recently confirmed using RNA interference (48). It is very likely that other cellular factors critical to HCV infection have yet to be identified.To gain more insight into cellular mechanisms underlying HCV infection, we made use of brefeldin A (BFA), a macrocyclic lactone of fungal origin that exhibits a wide range of inhibitory actions on membrane-associated mechanisms of the secretory and endocytic pathways (30). BFA acts on cell membranes by interfering with the activation of several members of the family of ADP-ribosylation factors (ARFs). ARFs are small GTP-binding proteins of the Ras superfamily. They function as regulators of vesicular traffic, actin remodeling, and phospholipid metabolism by recruiting effectors to membranes. BFA does not actually interfere directly with ARF GTPases but rather interferes with their activation by regulators known as guanine nucleotide exchange factors (GEFs) (14, 25). We now report the identification of an ARF GEF as a cellular BFA-sensitive factor that is required for HCV replication.     

Glutathione Provides a Source of Cysteine Essential for Intracellular Multiplication of Francisella tularensis

Francisella tularensis is a highly infectious bacterium causing the zoonotic disease tularemia. Its ability to multiply and survive in macrophages is critical for its virulence. By screening a bank of HimarFT transposon mutants of the F. tularensis live vaccine strain (LVS) to isolate intracellular growth-deficient mutants, we selected one mutant in a gene encoding a putative γ-glutamyl transpeptidase (GGT). This gene (FTL_0766) was hence designated ggt. The mutant strain showed impaired intracellular multiplication and was strongly attenuated for virulence in mice. Here we present evidence that the GGT activity of F. tularensis allows utilization of glutathione (GSH, γ-glutamyl-cysteinyl-glycine) and γ-glutamyl-cysteine dipeptide as cysteine sources to ensure intracellular growth. This is the first demonstration of the essential role of a nutrient acquisition system in the intracellular multiplication of F. tularensis. GSH is the most abundant source of cysteine in the host cytosol. Thus, the capacity this intracellular bacterial pathogen has evolved to utilize the available GSH, as a source of cysteine in the host cytosol, constitutes a paradigm of bacteria–host adaptation.