Translational efficiency of BVDV IRES and EMCV IRES for T7 RNA polymerase driven cytoplasmic expression in mammalian cell lines

Mammalian T7 polymerase-based cytoplasmic expression systems are common tool for molecular studies. The majority of these systems include the internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV). To carry out a cap-independent translation process, this type of IRES might require the expression of an extensive array of host factors, what is a disadvantage. Other IRESes might be less dependent on the host cell factors, but their biology is characterized to a lesser degree. Here, we compare the translational efficiencies of bovine viral diarrhea virus (BVDV) IRES with that of ECMV. Both IRESes were tested in reporter vectors containing the T7 promoter, an IRES of choice and the coding sequence of the enhanced green fluorescent protein (EGFP). To provide for the expression of T7 RNA polymerase, the corresponding gene was isolated from Escherichia coli and inserted into pCDNA3.1-Hygro(+). After co-transfection of the T7 RNA polymerase encoding vector with either of the two IRES-containing reporter vectors into T7 baby hamster kidney (T7-BHK), human embryonic kidney (HEK) 293T, chinese hamster ovary (CHO) and HeLa cells, the translational efficiency of the reporter construct was studied by fluorescence microscopy and flow cytometry. In T7-BHK, HEK 293T and HeLa cells the translational efficiency of BVDV IRES was two to three times higher than that of EMCV IRES. In CHO cells, BVDV IRES and EMCV IRES were equally efficient. An analysis of the secondary structure of respective mRNAs showed that their ΔG values were–544.00 and–469.40 kcal/mol for EMCV IRES and BVDV IRES harboring molecules, respectively. As EMCV IRES-containing mRNA is more stable, it is evident that other, still unidentified factors should be held responsible for the enhanced translational efficiency of BDVD IRES. Taken together, our results indicate the potential of BVDV IRES as a replacement for EMCV IRES, which is now commonly used for T7 polymerase driven cytoplasmic expression of genes of interest or virus cDNA rescue experiments.     

In vitro evaluation of effects of metformin on morphine and methadone tolerance through mammalian target of rapamycin signaling pathway

The chronic use of opioids leads to tolerance, psychological, and physical dependence that limits their use as an effective long-term pain control. Several studies have shown that mammalian target of rapamycin (mTOR) plays a crucial role in the development of opioid tolerance. Metformin activates 5′ adenosine monophosphate-activated protein kinase (AMPK) which directly suppresses the mTOR complex 1 signaling pathway. On the other hand, metformin can also inhibit mTOR directly and in an AMPK-independent manner. Thus, in the current study, we aimed to investigate the effects of metformin on the development of morphine and/or methadone-induced tolerance in human glioblastoma (T98G) cell line. We examined the effects of chronic treatment of morphine and/or methadone in the presence or absence of metformin with or without AMPK inhibitor (dorsomorphin hydrochloride) on levels of nitric oxide and cyclic adenosine monophosphate (cAMP), phosphorylated and dephosphorylated ribosomal protein S6 kinase β-1 (S6K1) and 4E-binding protein 1 (4E-BP1) in T98G cells. Pretreatment of cells with metformin (40 µM) with or without AMPK inhibitor (dorsomorphin hydrochloride; 1 µM) before adding of morphine (2.5 µM) or methadone (1 µM) revealed a protective effects on the development of opioid tolerance. Prior administration of metformin reversed the elevation of nitric oxide levels induced by morphine (p < 0.001) and methadone (p < 0.001) and also prevented the raise of cAMP levels induced by morphine in T98G cells (p < 0.05). Contribution of mTOR signaling pathway in metformin-induced effect was shown by the inhibition of phosphorylation of S6K1 and 4E-BP1, the downstream targets of mTOR. mTOR activation suppresses opioid-induced antinociception, and its activity has also been increased during opioid tolerance.     

Load transfer along the bone–dental implant interface

In this paper the variation of normal and shear stresses along a path defined on the bone–dental implant interface is investigated. In particular, the effects of implant diameter, collar length and slope, body length, and the effects of four different types of external threads on the interfacial stress distribution are studied. The geometry of the bone is digitized from a CT scan of a mandibular incisor and the surrounding bone. The bone and the implant are assumed to be perfectly bonded. The finite element method with 2D plane strain assumption is used to compute interfacial stresses. Highest continuous interfacial stresses are encountered in the region where the implant collar engages the cortical region, and near the apex of the implant in the subcortical region. Stress concentrations in the interfacial stresses occur near the geometric discontinuities on the implant contour, and jumps in stress values occur where the elastic modulus of the bone transitions between the cortical and trabecular bone values. Among the six contour parameters, the slope and the length of the implant collar, and the implant diameter influence the interfacial stress levels the most, and the effects of changing these parameters are significantly noticed only in the cortical bone (alveolar ridge) area. External threads cause significant stress concentrations in interfacial stresses in otherwise smoothly varying regions. This work shows that the presence of external threads could cause significant variations in both normal and shear stresses along the bone–implant interface, but not reduction in shear stress as previously thought.     

Characterization and risk assessment of the invasive papaya mealybug,Paracoccus marginatus,in Kenya under changing climate

The present study was conducted to characterize the newly invasive papaya mealybug Paracoccus marginatus Williams and Granara de Willink in Kenya using molecular techniques and to establish the potential risk of spread of the pest. Although abundant literature of P. marginatus outbreaks exists in other parts of the world, studies from Africa are rare. Our results revealed significant similarity between Kenyan samples with GenBank accession number KP692333.1 of P. marginatus. Phylogenetic analyses generated a tree that was paraphyletic with two clusters showing low genetic distance values for the P. marginatus sequences, which diverged from that of Planococcus citri. Our models displayed an optimal performance with mean area under the curve value of 0.82 and 0.98 for Genetic Algorithm for Rule-Set Production (GARP) and maximum entropy modelling (MaxEnt), respectively. Isothermality was the most influential variable in determining the potential distribution of P. marginatus with a 69% contribution to the models. Other variables included temperature mean diurnal range temperature seasonality, temperature annual range and annual precipitation in decreasing order of contribution. Current prediction by both models exceeded the existing range of P. marginatus, exacerbating the potential threat of the pest. GARP was more conservative in predicting suitable areas for P. marginatus, while MaxEnt showed further expansion by the year 2050. Our findings provide important information to guide biosecurity agencies in decision-making to prevent the spread and enhance control efforts of P. marginatus.     

Investigation of the effect of homocysteinylation of substance P on its binding to the NK1 receptor using molecular dynamics simulation

Substance P is a neurotransmitter or modulator in both the central and peripheral nervous systems. In this work, modifications of the lysine in SP by homocysteine and an acetyl group as well as the conformational dynamics of the native and modified SP peptides and their complexes with the NK1 receptor were studied via MD simulation. It was found that modifying SP stabilizes the peptide structure, but the modified SP peptides are less likely to bind to the NK1 receptor, so the resulting complexes are less stable. The RMSD of native SP (~0.33 nm) is about twice as large as that of the modified SP peptides (~0.18 nm), while the RMSD for the receptor complexed with native SP is ~0.3 nm, and that for the receptor complexed with either of the modified peptides is ~0.35 nm, which demonstrates the high stability of the modified SP peptides as well as the receptor complexed with native SP. Such behavior was also observed in other structural analyses. The binding free energies of the native and modified SP peptides with the NK1 receptor were also compared. The ΔG_bind values for the binding of homocysteinylated SP to the NK1 receptor and the binding of the acetylated SP and native SP to the NK1 receptor were ?38.89, ?64.46, and???264.52 kJ mol^?1, respectively. Modification of the lysine of SP decreases the binding affinity of the peptide to the NK1 receptor. In other words, homocysteinylation or acetylation of SP leads to weaker interactions of the peptide with the NK1 receptor compared to those between native SP and NK1. We propose that this phenomenon leads to increased levels of homocysteinylated SP in plasma in many diseases such as breast cancer.

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Graphical abstract Substance P (SP) is a neuropeptide which binds to the NK1 receptor. SP is of great pharmacological interest, as agonists and antagonists of SP can potentially be used to treat many chronic diseases. Therefore, in this work, the lysine (LYS) in SP was theoretically modified with a homocysteine or acetyl group to explore the effects of such a modification on the binding affinity of this peptide with the NK1 receptor and the structural dynamics of the resulting complex

     

Use of bacterial acc deaminase to increase oil (especially poly aromatic hydrocarbons) phytoremediation efficiency for maize (zea mays) seedlings

Oil presence in soil, as a stressor, reduces phytoremediation efficiency through an increase in the plant stress ethylene. Bacterial 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase, as a plant stress ethylene reducer, was employed to increase oil phytoremediation efficiency. For this purpose, the ability of ACC deaminase-producing Pseudomonas strains to grow in oil-polluted culture media and withstand various concentrations of oil and also their ability to reduce plant stress ethylene and enhance some growth characteristics of maize and finally their effects on increasing phytoremediation efficiency of poly aromatic hydrocarbons (PAHs) in soil were investigated. Based on the results, of tested strains just P9 and P12 were able to perform oil degradation. Increasing oil concentration from 0 to 10% augmented these two strains population, 15.7% and 12.9%, respectively. The maximum increase in maize growth was observed in presence of P12 strain. Results of high-performance liquid chromatography (HPLC) revealed that PAHs phytoremediation efficiency was higher for inoculated seeds than uninoculated. The highest plant growth and PAHs removal percentage (74.9%) from oil-polluted soil was observed in maize inoculated with P12. These results indicate the significance of ACC deaminase producing bacteria in alleviation of plant stress ethylene in oil-polluted soils and increasing phytoremediation efficiency of such soils.     

A Microassay for Determination of the Cytopathogenicity of Human Immunodeficiency Virus Type-1 Isolates

Correlations between the in vitro biological properties of HIV strains isolated from patients and the prognosis of their disease have been reported. We developed a technique to study the phenotype of HIV strains isolated from patients. We used the P4 cell line, derived from HeLa cells, which has been transfected with receptor CD4 gene. HIV laboratory strain (HIV_LAI) and peripheral blood leukocytes (PBLs) from donors infected with HIV_LAI induce syncytium in P4 cell cultures in vitro. The presence of reporter gene (LacZ gene) under the control of the HIV-1 long terminal repeat (LTR) in these cells allows colorimetric visualization of syncytia in the cytoplasm using a β-galactosidase (βgal) assay in the presence of X-gal. We cocultivated 1 × 10⁶ patient PBLs with 2 × 10⁶ normal PHA-activated normal PBLs for 4 days in the presence of IL-2 in 24-well plates. Half of the medium was replaced twice a week and PHA-activated normal PBLs were added every 7 days. HIV-1 was isolated from cocultured PBLs of 18 patients with advanced-stage HIV infection as assessed by the production of HIV p24 detected with a commercially available HIV-1 p24 ELISA. Supernatant and 10⁵ cells were collected twice a week from cocultured PBLs and were added to P4 cells in 96-well microtiter plates. The cultures were observed every day for 3 days and then the βgal assay was performed. We did not observe any effect with cells and supernatant from 8 patients, harvested from cultures incubated for as long as 28 days. The phenotype of these isolates was called NC (noncytopathic). In cells from 2 patients, we obtained blue multinucleated giant cells; the phenotype of these strains was called SI (syncytium inducing). In cultures from 8 other patients, we obtained the death of P4 cells without syncytium formation, and the phenotype of these strains was called CI (cell-killing inducing). In every case, the cytopathic effect of HIV-1 isolates could be detected with cocultured PBLs collected as early as day 4 of culture. Cocultured PBLs from 13 healthy controls did not alter the P4 cells. We displayed the replication of CI strains of HIV-1, but not the one of NC strains in P4 cell line. Our micromethod allowed the detection of cytopathic effects of HIV isolates. Further investigations should define the clinical applications of this method.     

High Levels of sTNFR p75 and TNFα in Dengue-Infected Patients

Soluble forms of the two molecular species of the cell surface TNF receptors (sTNFR p55 and sTNFR p75) can reduce the activity of TNFα but they may also enhance its function by stabilizing the active TNFα oligomer. Considering the pathophysiological importance of sTNFR p75 for the regulation of the bioavailability of TNFα in the body, we determined the serum levels of sTNFR p75 and TNFα in 45 children and 28 adults with laboratory-confirmed dengue infection by using immunoassays. The serum samples were obtained from day 1 to day 15 after the onset of the disease during the 1989–1990 outbreak of dengue-3 in Tahiti, French Polynesia. The patients were clinically classified as having dengue hemorrhagic fever (DHF) and graded according to the criteria of the World Health Organization (WHO) into four grades from less severe (grade I) to severe (grade IV). The sera of both children and adult patients of all severity grades contained higher levels of sTNFR p75 than the sera of control subjects. Although high levels of TNFα were also detected in children and adults among grade I, II, III and IV patients, we found no correlation between sTNFR p75 and TNFα. We observed in adults a moderate elevation of sTNFR p75 and TNFα in sera compared with that observed in children. The raised levels of immunoreactive sTNFR p75 and TNFα in all clinical groups of dengue-infected patients strongly indicate activation of the TNFα system during dengue infection. The balance between sTNFR p75 and TNFα may be altered in dengue infection. Further investigations are needed to understand the role of sTNFR p75 and TNFα in the pathogenesis of DHF and to improve the management of dengue infection.