Expression in animal cells and characterization of the hepatitis E virus structural proteins

Hepatitis E virus (HEV) is a major human pathogen in much of the developing world. It is a positive-strand RNA virus with a 7.5-kb polyadenylated genome consisting of three open reading frames (ORFs). In the absence of an in vitro culture system, the replication and expression strategy of HEV and the nature of its encoded polypeptides are not well understood. We have expressed the two ORFs constituting the structural portion of the HEV genome in COS-1 cells by using simian virus 40-based expression vectors and in vitro by using a coupled transcription-translation system. We show here that the major capsid protein, encoded by ORF2, is an 88-kDa glycoprotein which is expressed intracellularly as well as on the cell surface and has the potential to form noncovalent homodimers. It is synthesized as a precursor (ppORF2) which is processed through signal sequence cleavage into the mature protein (pORF2), which is then glycosylated (gpORF2). The minor protein, pORF3, encoded by ORF3 is a 13.5-kDa nonglycosylated protein expressed intracellularly and does not show any major processing. pORF3 interacts with a cellular protein of about 18 kDa which we call 3IP, the pORF3-interacting protein. The significance of these findings are discussed in light of an existing model of HEV genome replication and expression

Hendra and Nipah virus infection in cultured human olfactory epithelial cells

Henipaviruses are emerging zoonotic viruses and causative agents of encephalitis in humans. However, the mechanisms of entry into the central nervous system (CNS) in humans are not known. Here, we evaluated the possible role of olfactory epithelium in virus entry into the CNS. We characterized Hendra virus (HeV) and Nipah virus (NiV) infection of primary human olfactory epithelial cultures. We show that henipaviruses can infect mature olfactory sensory neurons. Henipaviruses replicated efficiently, resulting in cytopathic effect and limited induction of host responses. These results show that human olfactory epithelium is susceptible to infection with henipaviruses, suggesting that this could be a pathway for neuroinvasion in humans

The effects of cadmium on the biochemical and physiological parameters of Eruca sativa

In this study, Eruca sativa (Rocket) seedlings were treated with different cadmium (Cd) concentrations (0, 150, 300 and 450 μg ∙ g−1). The effects of Cd on lipid peroxidation, enzymatic (APx, CAT, GPX, SOD) and non-enzymatic antioxidants (total ascorbate, dehydroascorbate, ascorbate, non-protein thiol), fresh and dry masses, water content were determined. Also, Cd content of the leaves and the roots were analysed. The highest cadmium accumulation of leaves was at 450 μg ∙ g−1 Cd treatment and the accumulation was 2.62 times greater than those in the roots. The translocation factor was 3.89 at 300 μg ∙ g−1 Cd treatment. Cd treatments caused decreases of fresh, dry mass and water content of leaves and roots. Malondialdehyde content, which is an index of lipid peroxidation, was increased in proportion with the increase in Cd. While there was not change in the activity of GPX according to control, a decrease in activities of SOD, CAT and APX were observed with the increase of cadmium concentration. Although a significant increase in the amounts of non-protein thiol groups and proline were observed in 450 μg ∙ g−1 Cd treated plants, Cd did not lead to a significant change in AsA, DHA and total AsA contents. According to the results of the research, E. sativa may be a Cd hyperaccumulator plant and we suggest that the plant may be a candidate plant for remediation of Cd-contaminated soil

Developing a Comprehensive Power Simulation Model for the MEMESat-1 CubeSat Using Orbital Dynamics

The University of Georgia’s Small Satellite Research Lab’s Mission for Education and Multimedia Engagement Satellite (MEMESat-1) requires the use of variables such as power generation, power draw, orbital path, packet size, and data processing times. As power generation and charge varies, MEMESat-1 will automatically transition through three operational modes to prevent battery depletion and halt system processes in case of anomalies. Taking these variables and operational modes into account, the MEMESat-1 Mission Operations (MOPS) team will use FreeFlyer software to analyze power generation and draw during MEMESat-1’s orbital cycle. The power limitations of MEMESat-1 are budgeted based on battery and solar cell specifications implying the necessity of power simulations by MOPS

Difficult birth is the main contributor to birth‐related fracture and accidents to other neonatal fractures

Aim Specific birth-related fractures have been studied; underestimates might be a problem. We aimed to assess all fractures diagnosed as birth-related as well as other neonatal fractures. Methods A population-based study on all infants born in Sweden 1997-2014; data were retrieved from the Swedish Health Registers (10th version of International Classification of Diseases. Outcome measures were birth-related fractures (ICD-10 P-codes) and other neonatal fractures (ICD-10 S-codes). Results The overall fracture incidence was 2.9 per 1000 live birth (N = 5336); 92.6% had P-codes and 7.4% (S-codes). Some birth-related fractures were diagnosed beyond the neonatal period. Other neonatal fractures could have been birth-related. Clavicle fracture (88.8%) was associated with adverse maternal and infant anthropometrics and birth complications. The few neonates with rib fractures all had concomitant clavicle fracture. For skull fractures, a minor part was birth-related and most were associated with accidents. Half of the long bone fractures were associated with accidents. Birth-related femur fractures were associated with bone fragility risk factors. Five infants with abuse diagnoses had fractures: skull (4), long bone (2) and rib (1). Conclusion Birth-related and other neonatal fractures are rarely diagnosed. Difficult birth is the main contributor to birth-related fracture and accidents to other neonatal fractures.Peer reviewe

Functional role of the spatial proximity of Asp114(2.50) in TMH 2 and Asn332(7.49) in TMH 7 of the μ opioid receptor

AbstractWe examined whether a proposed spatial proximity between Asp114(2.50) and Asn332(7.49) affected the functional properties of the μ opioid receptor. The D114(2.50)N mutant had reduced binding affinities for morphine, DAMGO and CTAP, but not for naloxone and [3H]diprenorphine; this mutation also abolished agonist-induced increase in [35S]GTPγS binding. The N332(7.49)D mutation eliminated detectable binding of either [3H]diprenorphine or [3H]DAMGO. The combined D114(2.50)N-N332(7.49)D mutation restored high affinity binding for [3H]diprenorphine, CTAP and naloxone, and restored partially the binding affinities, potencies and efficacies of morphine and DAMGO. Thus, reciprocal mutations of Asp114(2.50) and Asn332(7.49) compensate for the detrimental effects of the single mutations, indicating that the residues are adjacent in space and that their chemical functionalities are important for ligand binding and receptor activation

Assessment of smoking status based on cotinine levels in nasal lavage fluid

Cotinine is a principal metabolite of nicotine with a substantially longer half-life, and cotinine levels in saliva, urine or serum are widely used to validate self-reported smoking status. The nasal cavity and olfactory system are directly exposed to tobacco smoke in smokers and in non-smokers who live with or work around smokers. However, despite the potential for a direct impact of tobacco smoke on the nasal epithelium and olfactory neurons, no prior studies have assessed cotinine levels in nasal mucus. We sought to determine whether cotinine levels in nasal lavage fluid (NLF) would provide a reasonable estimate of smoke exposure. We assayed cotinine using a competitive immunoassay in NLF from 23 smokers, 10 non-smokers exposed to tobacco smoke (ETS) and 60 non-smokers who did not report smoke exposure. NLF cotinine levels were significantly higher in smokers than in non-smokers, regardless of their exposure to ambient tobacco smoke. Cotinine levels in this small group of exposed non-smokers were not significantly different than those of non-exposed non-smokers. A cutoff of 1 ng/ml provided a sensitivity of 91% and a specificity of 99% for smoking status in this sample. Data were consistent with self-reported smoking status, and a cutoff of 1.0 ng/ml NLF cotinine may be used to classify smoking status. While saliva is the most easily obtained body fluid, NLF can be used to provide an objective and precise indication of smoking status and more directly reflects smoke exposure in the nasal and olfactory mucosa

Genetically Engineered Microvesicles Carrying Suicide mRNA/Protein Inhibit Schwannoma Tumor Growth

Microvesicles (MVs) play an important role in intercellular communication by carrying mRNAs, microRNAs (miRNAs), non-coding RNAs, proteins, and DNA from cell to cell. To our knowledge, this is the first report of delivery of a therapeutic mRNA/protein via MVs for treatment of cancer. We first generated genetically engineered MVs by expressing high levels of the suicide gene mRNA and protein–cytosine deaminase (CD) fused to uracil phosphoribosyltransferase (UPRT) in MV donor cells. MVs were isolated from these cells and used to treat pre-established nerve sheath tumors (schwannomas) in an orthotopic mouse model. We demonstrated that MV-mediated delivery of CD-UPRT mRNA/protein by direct injection into schwannomas led to regression of these tumors upon systemic treatment with the prodrug (5-fluorocytosine (5-FC)), which is converted within tumor cells to 5-fluorouracil (5-FU)–an anticancer agent. Taken together, these studies suggest that MVs can serve as novel cell-derived “liposomes” to effectively deliver therapeutic mRNA/proteins to treatment of diseases

Novel regulation of PLCζ activity via its XY-linker

The XY-linker region of somatic cell PLC (phospholipase)-β, -γ, -δ and -ϵ isoforms confers potent catalytic inhibition, suggesting a common auto-regulatory role. Surprisingly, the sperm PLCζ XY-linker does not mediate auto-inhibition. Unlike for somatic PLCs, the absence of the PLCζ XY-linker significantly diminishes both in vitro PIP2 (phosphatidylinositol 4,5-bisphosphate) hydrolysis and in vivo Ca2+-oscillation-inducing activity, revealing evidence for a novel PLCζ enzymatic mechanism

Subcutaneous CERA for the treatment of chronic renal anemia in predialysis patients

Background: We investigated the efficacy, safety and tolerability of once-monthly administration of C.E.R.A. in erythropoiesis stimulating agents (ESAs) naive predialysis patients with CKD for anemia treatmentStudy Design: Single arm, open label study.Methods: A total of 75 patients (mean (SD) age was 52.8 (16.4) years, 76.0% were female) were included in this study conducted between 12 August 2008 and 30 October 2009 in 9 centers across Turkey. The mean change in Hb concentration (g/dL) between baseline (week 0) and the efficacy evaluation period (EEP) was the primary efficacy parameter evaluated in three consecutive periods including a dose titration period (DTP; with initial 1.2 mu g/kg dose of C.E.R.A., subcutaneously, 28 weeks), EEP (8 weeks) and a long-term safety period (16 weeks).Results: Our analysis revealed an improvement in Hb levels from baseline value of 9.4 (0.4) g/dL to time adjusted average level of 11.4 (0.7) g/dL in EEP in the per protocol (PP) population and from 9.3 (0.5) g/dL to 11.1 (1.0) g/dL in intent-to-treat (ITT) population. Mean (SD) change in Hb levels from baseline to EEP was 2.0 (0.7) g/dl in the PP population (primary endpoint) and 1.7 (1.1) g/dL in the ITT population. The percentage of patients whose Hb concentrations remained within the target range of 10.0-12.0 g/dL throughout the EEP was 43.9% (95% CI: 28.5-60.3%) in the PP population and 38.7% (95% CI: 27.6% to 50.6%) in the ITP population. A total of 206 adverse events (AE) were reported in 77.0% of patients with hypertension (20%) as the most frequent AE.Conclusion: Once-monthly subcutaneous C.E.R.A. administration is effective and safe in the treatment of anemia in pre-dialysis patients with CKD, who are not currently treated with ESAs