A recombinant antigen with potential for serodiagnosis of Echinococcus granulosus infection in dogs

Antibodies specific for Echinococcus granulosus were affinity purified from dog serum on transfer blots containing putative serodiagnostic antigens. These antibodies and serum pools derived from dogs with E. granulosus infections were used to screen a lambda gt11 cDNA library constructed using E. granulosus protoscolex mRNA. Nine definitive antigenic clones were isolated and characterized, of which one (c10P1) gave strong specific reactions in plaque immunoassay with sera from E. granulosus infected dogs. These clones were all subcloned into the plasmid vector pGEX-1. Antigenicity of clones was confirmed in colony immunoassay and/or immunoblot. Glutathione S-transferase (GST) fusion proteins of individual subclones were produced in Escherichia coli, purified by affinity chromatography and evaluated in ELISA using sera from dogs with infections of E. granulosus, Taenia spp. or nematodes, and helminth-free dog sera. The GST fusion protein 10P1 showed a specificity of 100% in ELISA for diagnosis of E. granulosus infection in dogs despite its relatively low sensitivity. Further investigations aim to identify additional recombinant antigens and test 10P1 expressed in alternative expression systems to increase diagnostic sensitivity of the ELISA.     

Import of proteins into mitochondria. Energy-dependent, two-step processing of the intermembrane space enzyme cytochrome b2 by isolated yeast mitochondria

Import of in vitro-synthesized cytochrome b2 (a soluble intermembrane space enzyme) was studied wih isolated yeast mitochondria. Import requires an electrochemical gradient across the inner membrane and is accompanied by cleavage of the precursor to the corresponding mature form. This conversion proceeds via an intermediate form of cytochrome b2, which can be detected as a transient species when mitochondria are incubated with the cytochrome b2 precursor for short times or at low temperatures. Conversion of the precursor to the intermediate form is energy-dependent and catalyzed by an o-phenanthroline-sensitive protease located in the soluble matrix. The intermediate is subsequently converted to mature cytochrome b2 in a reaction which is o-phenanthroline-insensitive and requires neither an energized inner membrane nor a soluble component of the intermembrane space. Whereas mature cytochrome b2 is soluble, the intermediate formed by isolated mitochondria is membrane-bound and exposed to the intermembrane space. The same intermediate is detected as a transient species during cytochrome b2 maturation in intact yeast cells (Reid, G. A., Yonetani, T., and Schatz, G (1982) J. Biol. Chem. 257, 13068-13074). The in vitro studies reported here suggest that a part of the cytochrome b2 precursor polypeptide chain is transported to the matrix where it is cleaved to a membrane-bound intermediate form by the same protease that processes polypeptides destined for the matrix space or for the inner membrane. In a second reaction, the cytochrome b2 intermediate is converted to mature cytochrome b2 which is released into the intermembrane space. The binding of heme is not necessary for converting the intermediate to the mature polypeptide.     

The Arabidopsis HUELLENLOS gene, which is essential for normal ovule development, encodes a mitochondrial ribosomal protein.

The HUELLENLOS (HLL) gene participates in patterning and growth of the Arabidopsis ovule. We have isolated the HLL gene and shown that it encodes a protein homologous to the L14 proteins of eubacterial ribosomes. The Arabidopsis genome also includes a highly similar gene, HUELLENLOS PARALOG (HLP), and genes for both cytosolic (L23) and chloroplast ribosome L14 proteins. Phylogenetic analysis shows that HLL and HLP differ significantly from these other two classes of such proteins. HLL and HLP fusions to green fluorescent protein were localized to mitochondria. Ectopic expression of HLP complemented the hll mutant, indicating that HLP and HLL share redundant functions. We conclude that HLL and HLP encode L14 subunits of mitochondrial ribosomes. HLL mRNA was at significantly higher levels than HLP mRNA in pistils, with the opposite pattern in leaves. This differential expression can explain the confinement of effects of hll mutations to gynoecia and ovules. Our elucidation of the nature of HLL shows that metabolic defects can have specific effects on developmental patterning.     

The Degree and Length of O-Glycosylation of Recombinant Proteins Produced in Pichia pastoris Depends on the Nature of the Protein and the Process Type

The methylotrophic yeast Pichia pastoris is known as an efficient host for the production of heterologous proteins. While N-linked protein glycosylation is well characterized in P. pastoris there is less knowledge of the patterns of O-glycosylation. O-glycans produced by P. pastoris consist of short linear mannose chains, which in the case of recombinant biopharmaceuticals can trigger an immune response in humans. This study aims to reveal the influence of different cultivation strategies on O-mannosylation profiles in P. pastoris. Sixteen different model proteins, produced by different P. pastoris strains, are analyzed for their O-glycosylation profile. Based on the obtained data, human serum albumin (HSA) is chosen to be produced in fast and slow growth fed batch fermentations by using common promoters, P_GAP and P_AOX1. After purification and protein digestion, glycopeptides are analyzed by LC/ESI-MS. In the samples expressed with P_GAP it is found that the degree of glycosylation is slightly higher when a slow growth rate is used, regardless of the efficiency of the producing strain. The highest glycosylation intensity is observed in HSA produced with P_AOX1. The results indicate that the O-glycosylation level is markedly higher when the protein is produced in a methanol-based expression system.     

Mitochondrial Genome of the Eyeworm,Thelazia callipaeda (Nematoda: Spirurida), as the First Representative from the Family Thelaziidae

Human thelaziosis is an underestimated parasitic disease caused by Thelazia species (Spirurida: Thelaziidae). The oriental eyeworm, Thelazia callipaeda, infects a range of mammalian definitive hosts, including canids, felids and humans. Although this zoonotic parasite is of socio-economic significance in Asian countries, its genetics, epidemiology and biology are poorly understood. Mitochondrial (mt) DNA is known to provide useful genetic markers to underpin fundamental investigations, but no mt genome had been characterized for any members of the family Thelaziidae. In the present study, we sequenced and characterized the mt genome of T. callipaeda. This AT-rich (74.6%) mt genome (13,668 bp) is circular and contains 12 protein-coding genes, 22 transfer RNA genes and two ribosomal RNA genes, but lacks an atp8 gene. All protein-coding genes are transcribed in the same direction; the gene order is the same as those of Dirofilaria immitis and Setaria digitata (Onchocercidae), but distinct from Dracunculus medinensis (Dracunculidae) and Heliconema longissimum (Physalopteridae). Phylogenetic analyses of the concatenated amino acid sequence data for all 12 protein-coding genes by Bayesian inference (BI) showed that T. callipaeda (Thelaziidae) is related to the family Onchocercidae. This is the first mt genome of any member of the family Thelaziidae and should represent a new source of genetic markers for studying the epidemiology, ecology, population genetics and systematics of this parasite of humans and other mammals.     

Getting to the guts of the matter: The status and potential of ‘omics’ research of parasitic protists of the human gastrointestinal system

Parasitic protists are a major cause of diarrhoeal illnesses in humans globally. Collectively, enteric pathogens exceed all other forms of infectious disease, in terms of their estimated global prevalence and socioeconomic impact. They have a disproportionately high impact on children in impoverished communities, leading to acute (diarrhoea, vomiting, dehydration and death) and chronic disease (malabsorption, malnutrition, physical and cognitive stunting and predisposition to chronic, non-communicable disease) consequences. However, historically, investment in research and disease control measures has been disproportionately poor, leading to their current classification as neglected pathogens. A sound understanding of their biology is essential in underpinning detection, treatment and control efforts. One major tool in rapidly improving our knowledge of these parasites is the use of biological systems, including ‘omic’ technologies. In recent years, these tools have shown significant success when applied to enteric protists. This review summarises much of this knowledge and highlights the significant remaining knowledge gaps. A major focus of the present review was to provide a perspective on a way forward to address these gaps using advanced biotechnologies.     

Molecular optimization of rabies virus glycoprotein expression in Pichia pastoris

In this work, different approaches were investigated to enhance the expression rabies virus glycoprotein (RABV‐G) in the yeast Pichia pastoris; this membrane protein is responsible for the synthesis of rabies neutralizing antibodies. First, the impact of synonymous codon usage bias was examined and an optimized RABV‐G gene was synthesized. Nevertheless, data showed that the secretion of the optimized RABV‐G gene was not tremendously increased as compared with the non‐optimized one. In addition, similar levels of RABV‐G were obtained when α‐factor mating factor from Saccharomyces cerevisiae or the acid phosphatase PHO1 was used as a secretion signal. Therefore, sequence optimization and secretion signal were not the major bottlenecks for high‐level expression of RABV‐G in P. pastoris. Unfolded protein response (UPR) was induced in clones containing high copy number of RABV‐G expression cassette indicating that folding was the limiting step for RABV‐G secretion. To circumvent this limitation, co‐overexpression of five factors involved in oxidative protein folding was investigated. Among these factors only PDI1, ERO1 and GPX1 proved their benefit to enhance the expression. The highest expression level of RABV‐G reached 1230 ng ml^?1. Competitive neutralizing assay confirmed that the recombinant protein was produced in the correct conformational form in this host.     

Risk factors of peripheral arterial disease: a case control study in Sri Lanka

Background

Peripheral artery disease (PAD) is an important global health problem and contributes to notable proportion of morbidity and mortality. This particular manifestation of systemic atherosclerosis is largely under diagnosed and undertreated. For sustainable preventive strategies in a country, it is mandatory to identify country-specific risk factors. We intended to assess the risk factors of PAD among adults aged 40–74 years.

Methods

This case control study was conducted in 2012–2013 in Sri Lanka. Seventy-nine cases and 158 controls in the age group of 40–74 years were selected for the study in order to have case to control ratio 1:2. The criterion for selecting cases and control was based on Ankle brachial pressure index (ABPI). Cases were selected from those who had ABPI 0.85 or less (ABPI ≤0.85) in either lower limb. Controls were selected from those ABPI score between 1.18 and 1.28 in both lower limbs. Only newly identified individuals with PAD were selected as cases. Controls were selected from the same geographical location and within the 5 year age group as cases.

Results

The history of diabetes mellitus more than 10 years (OR 5.8, 95% CI 2.2–14.2), history of dyslipidemia for more than 10 years (OR 4.9, 95% CI 2.1–16.2), history of hypertension for more than 10 years (OR 3.8, 95% CI 1.8–12.7) and smoking (OR 2.9, 95% CI 1.2–6.9), elevated HsCRP (OR 3.7, 95% CI 1.2–12.0) and hyperhomocysteinemia (OR 3.0, 95% CI 1.1–8.1) were revealed as country specific significant risk factor of PAD.

Conclusions

Diabetes mellitus, hypertension, dyslipidemia, smoking as well as elevated homocysteine and HsCRP found as risk factors of PAD. Longer the duration or higher level exposure to these risk factors has increased the risk of PAD. These findings emphasis the need for routine screening of PAD among patients with the identified risk factors.