A pathway map of signaling events triggered upon SARS-CoV infection

Severe acute respiratory syndrome coronaviruses (SARS-CoVs) caused worldwide epidemics over the past few decades. Extensive studies on various strains of coronaviruses provided a basic understanding of the pathogenesis of the disease. Presently, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is leading a global pandemic with unprecedented challenges. This is the third coronavirus outbreak of this century. A signaling pathway map of signaling events induced by SARS-CoV infection is not yet available. In this study, we present a literature-annotated signaling pathway map of reactions induced by SARS-CoV infected cells. Multiple signaling modules were found to be orchestrated including PI3K-AKT, Ras-MAPK, JAK-STAT, Type 1 IFN and NFκB. The signaling pathway map of SARS-CoV consists of 110 molecules and 101 reactions mediated by SARS-CoV proteins. The pathway reaction data are available in various community standard data exchange formats including Systems Biology Graphical Notation (SBGN). The pathway map is publicly available through the GitHub repository and data in various formats can be freely downloadable.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12079-021-00642-2.     

Engineering tolerance and hyperaccumulation of arsenic in plants by combining arsenate reductase and gamma-glutamylcysteine synthetase expression

We have developed a genetics-based phytoremediation strategy for arsenic in which the oxyanion arsenate is transported aboveground, reduced to arsenite, and sequestered in thiol-peptide complexes. The Escherichia coli arsC gene encodes arsenate reductase (ArsC), which catalyzes the glutathione (GSH)-coupled electrochemical reduction of arsenate to the more toxic arsenite. Arabidopsis thaliana plants transformed with the arsC gene expressed from a light-induced soybean rubisco promoter (SRS1p) strongly express ArsC protein in leaves, but not roots, and were consequently hypersensitive to arsenate. Arabidopsis plants expressing the E. coli gene encoding gamma-glutamylcysteine synthetase (gamma-ECS) from a strong constitutive actin promoter (ACT2p) were moderately tolerant to arsenic compared with wild type. However, plants expressing SRS1p/ArsC and ACT2p/gamma-ECS together showed substantially greater arsenic tolerance than gamma-ECS or wild-type plants. When grown on arsenic, these plants accumulated 4- to 17-fold greater fresh shoot weight and accumulated 2- to 3-fold more arsenic per gram of tissue than wild type or plants expressing gamma-ECS or ArsC alone. This arsenic remediation strategy should be applicable to a wide variety of plant species.     

Thermal Disinfection Validation: The Relationship Between A0 and Microbial Reduction

Validating a thermal disinfection process for the processing of medical devices using moist heat via direct temperature monitoring is a conservative approach and has been established as the A₀ method. Traditional use of disinfection challenge microorganisms and testing techniques, although widely used and applicable for chemical disinfection studies, do not provide as robust a challenge for testing the efficacy of a thermal disinfection process. Considerable research has been established in the literature to demonstrate the relationship between the thermal resistance of microorganisms to inactivation and the A₀ method formula. The A₀ method, therefore, should be used as the preferred method for validating a thermal disinfection process using moist heat.

Disinfection, which is defined as reducing the number of viable microorganisms on a product to a level previously specified as appropriate for its intended further handling or use, can be achieved thermally by the action of moist heat.1 Thermal disinfection during the processing of medical devices, typically performed in a washer-disinfector, is widely used for two purposes. The first is for reducing product bioburden (disinfection) either as a terminal step (e.g., for noncritical or semicritical devices) or prior to packaging and sterilization (e.g., for critical devices) in preparation for patient use. The second is to render the devices safe for handling for central service professionals during inspection and packaging.2,3 Thermal disinfection requirements therefore should consider the potential levels of microbial contamination on reusable devices after use, the desired level of reduction to render those devices safe for handling and for their intended purpose, and the reliability of the disinfection process to consistently achieve that endpoint.The microbial load on device types after patient use has been established in the literature and can vary depending on the typical clinical use of the device. For example, critical (surgical) devices, on average, have demonstrated relatively low levels of viable microorganisms (bioburden level <10² colony-forming units [CFU]/cm²).4 However, these same studies have shown the concentration of other testing analytes (e.g., protein, total organic carbon, hemoglobin) to be more noteworthy. Although the data indicate that residual clinical soil (e.g., human secretions, blood, tissue) can harbor microorganisms, the incoming product bioburden levels are far below the microbial populations challenged during an overkill sterilization process (e.g., moist heat or gaseous processes).Conservative sterilization processes have been demonstrated to achieve at least a 12-log₁₀ reduction of microorganisms with a known higher resistance versus typical bioburden.3,5 Cleaning, which is defined as the removal of contamination from an item to the extent necessary for its further processing and its intended subsequent use, is an important step to render the device ready for sterilization and will further reduce the levels of microorganisms prior to sterilization. Therefore, with critical devices, adequate cleaning followed by sterilization is the minimum requirement to ensure the device is safe for patient use.It is not likely that, for the intended use of the device, a disinfection process is strictly necessary as an intermediate step prior to sterilization. A benefit may exist to having an interim disinfection step to render the device safe for handling during inspection and packaging for sterilization. For example, the expectation in the Occupational Safety and Health Administration''s Bloodborne Pathogens standard 29 CFR 1910.1030 is that an employer will minimize the occupational exposure to bloodborne pathogens.Thermal disinfection has been used by sterile processing departments as a universal precaution to reduce the risk of exposure to processing personnel postcleaning. Although routine thermal disinfection at less than 100°C (212°F) may not be effective in deactivating all types of microorganisms (e.g., certain types of bacteria spores), it is a reliable and consistent disinfection process. As the temperature increases above a certain point (typically ≥70°C or 158°F), so does the activity against microorganisms, with variable intrinsic and acquired resistance mechanisms to heat.3 Thermal disinfection therefore will provide processing personnel with a minimized risk of bloodborne pathogens exposure.In other situations, the microbiological load can be much higher (e.g., with flexible endoscopes used in the gastrointestinal system6) or more variable (e.g., with noncritical devices or surfaces depending on their use7). Where practical, thermal disinfection is still viewed as the preferred and more reliable method to render these devices safe for use due to its known efficacy against microbial pathogens.5 Chemical disinfection generally is only considered if thermal disinfection cannot be applied (e.g., due to thermo-sensitivity of device or surface materials).     

Glc-6-PD and PKG contribute to hypoxia-induced decrease in smooth muscle cell contractile phenotype proteins in pulmonary artery

Persistent hypoxic pulmonary vasoconstriction (HPV) plays a significant role in the pathogenesis of pulmonary hypertension, which is an emerging clinical problem around the world. We recently showed that hypoxia-induced activation of glucose-6-phosphate dehydrogenase (Glc-6-PD) in pulmonary artery smooth muscle links metabolic changes within smooth muscle cells to HPV and that inhibition of Glc-6PD reduces acute HPV. Here, we demonstrate that exposing pulmonary arterial rings to hypoxia (20-30 Torr) for 12 h in vitro significantly (P < 0.05) reduces (by 30-50%) SM22α and smooth muscle myosin heavy chain expression and evokes HPV. Glc-6-PD activity was also elevated in hypoxic pulmonary arteries. Inhibition of Glc-6-PD activity prevented the hypoxia-induced reduction in SM22α expression and inhibited HPV by 80-90% (P < 0.05). Furthermore, Glc-6-PD and protein kinase G (PKG) formed a complex in pulmonary artery, and Glc-6-PD inhibition increased PKG-mediated phosphorylation of VASP (p-VASP). In turn, increasing PKG activity upregulated SM22α expression and attenuated HPV evoked by Glc-6-PD inhibition. Increasing passive tension (from 0.8 to 3.0 g) in hypoxic arteries for 12 h reduced Glc-6-PD, increased p-VASP and SM22α levels, and inhibited HPV. The present findings indicate that increases in Glc-6-PD activity influence PKG activity and smooth muscle cell phenotype proteins, all of which affect pulmonary artery contractility and remodeling.     

Evaluation of non-target effects of OMRI-listed insecticides for management of Drosophila suzukii Matsumura in berry crops

The spotted wing drosophila, Drosophila suzukii Matsumura, is an invasive pest of many fruit crops throughout North America, South America and Europe. The presence of this destructive pest has led to an increase in the number of insecticide applications. While conventional growers have an arsenal of different insecticides at their disposal, organic growers have a limited selection of effective options and rely heavily on applications of Entrust^®, the organic formulation of spinosad. An important part of research is to develop more tools for organic growers and evaluate the effects of insecticides intended to target D. suzukii on natural enemies in the system. The effects of six organic pesticides alone and in combination with three adjuvants and two phagostimulants were tested in laboratory bioassays on three common natural enemies in berry production systems including two predators, Chrysoperla rufilabris and Orius insidiosus, and a parasitoid wasp, Aphidius colemani. Under the IOBC toxicity rating scale, spinosad was rated consistently from slightly harmful to harmful across natural enemy species and residue age (the effects of pesticides over time). Sabadilla alkaloids caused mortality to O. insidiosus equal to that of spinosad. All tested pesticides were at least slightly harmful to A. colemani, and the adjuvant polyether-polymethylsiloxane-copolymer polyether caused mortality that was not significantly different from spinosad. In general, neither the addition of adjuvants nor phagostimulants increased the mortality of the insecticides tested. The exception was polyether-polymethylsiloxane-copolymer polyether, but it is unclear whether it increased the toxicity of the pesticides or was simply toxic itself since it caused high mortality to A. colemani when applied alone. Sublethal effects were measured for two predatory species by measuring eggs laid and % egg hatch. Minimal sublethal effects were observed in C. rufilabris. In contrast, all tested insecticides caused reduced egg hatch in O. insidiosus compared with the control.     

Inhibitory effects of Azadirachta indica on DMBA-induced skin carcinogenesis in Balb/c mice

Male Balb/c mice were divided into four groups on the basis of their respective treatments wherein mice of Group I served as controls. For induction of skin tumors, mice of Group II and IV were injected sub-cutaneously with 7,12-dimethylbenz(a)anthracene (DMBA). Mice of Group III and IV were administered aqueous Azadirachta indica leaf extract (AAILE) thrice a week throughout the experiment. After 14 weeks of the first DMBA injection, Group II and IV mice developed tumors. In the tumor-bearing mice that received AAILE (Group IV), a significant reduction in mean tumor burden and tumor volume was observed. The tumors were confirmed to be papillomas and interestingly, the extent of hyper-chromatia was observed to be much more in skin tumors of Group II mice vis a vis the mice receiving AAILE. An increase in the extent of lipid peroxidation was observed in tumorous tissue of Group IV when compared to that of Group II mice. Glutathione (GSH) content and the activities of GSH-based antioxidant enzymes viz. glutathione peroxidase (GPx) and glutathione reductase (GR) increased significantly in the skin tissues of all the groups of mice when compared to control counterparts. Catalase activity was found to decrease significantly in the skin of mice, which received AAILE treatment only (Group III). Activity of super-oxide dismutase (SOD) decreased significantly in all the tumorous tissues (Group II and IV mice). In light of the above observations, the role of AAILE in inhibition of DMBA-induced skin carcinogenesis is discussed in the present study.     

The Role of Histamine in the Retina: Studies on the Hdc Knockout Mouse

The role of histamine in the retina is not well understood, despite it regulating a number of functions within the brain, including sleep, feeding, energy balance, and anxiety. In this study we characterized the structure and function of the retina in mice that lacked expression of the rate limiting enzyme in the formation of histamine, histidine decarboxylase (Hdc^−/− mouse). Using laser capture microdissection, Hdc mRNA expression was assessed in the inner and outer nuclear layers of adult C57Bl6J wildtype (WT) and Hdc^−/−-retinae. In adult WT and Hdc^−/−-mice, retinal fundi were imaged, retinal structure was assessed using immunocytochemistry and function was probed by electroretinography. Blood flow velocity was assessed by quantifying temporal changes in the dynamic fluorescein angiography in arterioles and venules. In WT retinae, Hdc gene expression was detected in the outer nuclear layer, but not the inner nuclear layer, while the lack of Hdc expression was confirmed in the Hdc^−/− retina. Preliminary examination of the fundus and retinal structure of the widely used Hdc^−/−mouse strain revealed discrete lesions across the retina that corresponded to areas of photoreceptor abnormality reminiscent of the rd8 (Crb1) mutation. This was confirmed after genotyping and the strain designated Hdc^rd8/rd8. In order to determine the effect of the lack of Hdc-alone on the retina, Hdc^−/− mice free of the Crb1 mutation were bred. Retinal fundi appeared normal in these animals and there was no difference in retinal structure, macrogliosis, nor any change in microglial characteristics in Hdc^−/− compared to wildtype retinae. In addition, retinal function and retinal blood flow dynamics showed no alterations in the Hdc^−/− retina. Overall, these results suggest that histamine plays little role in modulating retinal structure and function.     

Cyclic GMP specifically suppresses Type-Iα cGMP-dependent protein kinase expression by ubiquitination

Type I cGMP-dependent protein kinase (PKG-I) mediates nitric oxide (NO) and hormone dependent smooth muscle relaxation and stimulates smooth muscle cell-specific gene expression. Expression of PKG-I in cultured smooth muscle cells depends on culture conditions and is inhibited by inflammatory cytokines such as interleukin-I and tumor necrosis factor-α, which are known to stimulate Type II NO synthase (iNOS) expression. We report here that the suppression of PKG-I protein levels in smooth muscle cells is triggered by the ubiquitin/26S proteasome pathway. Incubation of vascular smooth muscle cells with phosphodiesterase-resistant cyclic GMP analogs (e.g., 8-bromo-cGMP) decreases PKG-I protein level in a time- and concentration-dependent manner. To study this process, we tested the effects of 8-Br-cGMP on PKG-I protein level in Cos7 cells, which do not express endogenous type I PKG mRNA. 8-Br-cGMP induced the ubiquitination and down-regulation of PKG-Iα, but not PKG-Iβ. Treatment of cells with the 26S proteasome inhibitor, MG-132, increased ubiquitination of PKG. Blocking PKG-I catalytic activity using the cell-permeant specific PKG-I inhibitor, DT-2, inhibited cGMP-induced PKG-I ubiquitination and down-regulation, suggesting that PKG catalytic activity and autophosphorylation were required for suppression of PKG-I level. Mutation of the known autophosphorylation sites of PKG-Iα to alanine uncovered a specific role for autophosphorylation of serine-64 in cGMP-dependent ubiquitination and suppression of PKG-I level. The results suggest that chronic elevation of cGMP, as seen in inflammatory conditions, triggers ubiquitination and degradation of PKG-Iα in smooth muscle.