Multiplexed orthogonal genome editing and transcriptional activation by Cas12a.

CRISPR-Cas9-based combinatorial perturbation approaches for orthogonal knockout and gene activation have been impeded by complex vector designs and co-delivery of multiple constructs. Here, we demonstrate that catalytically active CRISPR-Cas12a fused to a transcriptional-activator domain enables flexible switching between genome editing and transcriptional activation by altering guide length. By leveraging Cas12a-mediated CRISPR-RNA array processing, we illustrate that Cas12a-VPR enables simplified multiplexed knockout and transcriptional activation in vitro and in vivo.

Fate and behavior of ZnO- and Ag-engineered nanoparticles and a bacterial viability assessment in a simulated wastewater treatment plant.

The fate and behaviour assessment of ZnO- and Ag-engineered nanoparticles (ENPs) and bacterial viability in a simulated wastewater treatment plant (WWTP) fed with municipal wastewater was investigated through determination of ENPs stability at varying pH and continuous exposure of ENPs to wastewater, respectively. The ENPs were introduced to a 3-L bioreactor (simulated WWTP) with a hydraulic residence time (HRT) of 6 h at a dose rate of 0.83 mg/min for 240 h. The stability of the ENPs was found to be dependent on their dissolution and aggregation at different pH, where ZnO ENPs exhibited the highest dissolution at low pH compared to Ag ENPs. The results also showed that both ENPs had high affinity for the sewage sludge as they undergo aggregation under typical wastewater conditions. Results of effluent monitored daily showed mean COD removal efficiencies of 71 ± 7% and 74 ± 8% for ZnO and Ag ENPs in test units, respectively. The treated effluent had low mean concentrations of Zn (1.39 ± 0.54 mg/L) and Ag (0.12 ± 0.06 mg/L); however, elevated mean concentrations of Zn (54 ± 39 mg/g dry sludge) and Ag (57 ± 42 mg/g dry sludge) were found in the sludge - suggesting removal of the ENPs from the wastewater by biosorption and biosolid settling mechanisms. Using X-ray diffraction (XRD) and transmission electron microscopy (TEM), the mineral identities of ZnO and Ag ENPs in the sludge from the test units were found comparable to those of commercial ENPs, but larger due to agglomeration. The bacterial viability assessment after exposure to ENPs using the Live/Dead BacLight kit, although not quantitatively assessed, suggested high resilience of the bacteria useful for biodegradation of organic material in the simulated wastewater treatment system.

The role of wastewater-based epidemiology for SARS-CoV-2 in developing countries: Cumulative evidence from South Africa supports sentinel site surveillance to guide public health decision-making.

The uptake of wastewater-based epidemiology (WBE) for SARS-CoV-2 as a complementary tool for monitoring population-level epidemiological features of the COVID-19 pandemic in low-and-middle-income countries (LMICs) is low. We report on the findings from the South African SARS-CoV-2 WBE surveillance network and make recommendations regarding the implementation of WBE in LMICs. Eight laboratories quantified influent wastewater collected from 87 wastewater treatment plants in all nine South African provinces from 01 June 2021 to 31 May 2022 inclusive, during the 3rd and 4th waves of COVID-19. Correlation and regression analyses between wastewater levels of SARS-CoV-2 and district laboratory-confirmed caseloads were conducted. The sensitivity and specificity of novel 'rules' based on WBE data to predict an epidemic wave were determined. Amongst 2158 wastewater samples, 543/648 (85 %) samples taken during a wave tested positive for SARS-CoV-2 compared with 842 positive tests from 1512 (55 %) samples taken during the interwave period. Overall, the regression-co-efficient was 0,66 (95 % confidence interval = 0,6-0,72, R2 = 0.59), ranging from 0.14 to 0.87 by testing laboratory. Early warning of the 4th wave of SARS-CoV-2 in Gauteng Province in November-December 2021 was demonstrated. A 50 % increase in log copies of SARS-CoV-2 compared with a rolling mean over the previous five weeks was the most sensitive predictive rule (58 %) to predict a new wave. Our findings support investment in WBE for SARS-CoV-2 surveillance in LMICs as an early warning tool. Standardising test methodology is necessary due to varying correlation strengths across laboratories and redundancy across testing plants. A sentinel site model can be used for surveillance networks without affecting WBE finding for decision-making. Further research is needed to identify optimal test frequency and the need for normalisation to population size to identify predictive and interpretive rules to support early warning and public health action.

Impact of SARS-CoV-2 on the mobility behaviour in Germany.

Background: The COVID-19 pandemic and the measures taken to combat it led to severe constraints for various areas of life, including mobility. To study the effects of this disruptive situation on the mobility behaviour of entire subgroups, and how they shape their mobility in reaction to the special circumstances, can help to better understand, how people react to external changes. Methodology: Aim of the study presented in this article was to investigate to what extent, how and in what areas mobility behaviour has changed during the outbreak of SARS-CoV-2 in Germany. In addition, a focus was put on the comparison of federal states with and without lockdown in order to investigate a possible contribution of this measure to changes in mobility. We asked respondents via an online survey about their trip purposes and trip frequency, their choice of transport mode and the reasons for choosing it in the context of the COVID-19 crisis. For the analyses presented in this paper, we used the data of 4157survey participants (2512 without lockdown, 1645 with lockdown). Results: The data confirmed a profound impact on the mobility behaviour with a shift away from public transport and increases in car usage, walking and cycling. Comparisons of federal states with and without lockdown revealed only isolated differences. It seems that, even if the lockdown had some minor effects, its role in the observed behavioural changes was minimal.

Are you an ambitious cyclist? Results of the cyclist profile questionnaire in Germany.

Objective: A number of studies have already grouped cyclists according to different aspects of their mobility behavior. This could be used e.g., to improve the bicycle infrastructure planning, to detect critical spots and, to reduce obstacles for cycling. This wide, preexisting, range of cyclist typologies usually concentrates on one or two influence factors and differs, content-wise, in both factors used, as well as, methodically. Based on existing cyclist typologies we extracted all possible influence factors to integrate them in one single questionnaire. The objective of this study, using an empirical, based approach, is to compare this typology of cyclists with existing ones, integrating all known influence factors of recent studies.Methods: To address these issues, we conducted a Germany-wide online survey on cycling behavior, covering all relevant aspects we derived from both literature and especially, former cyclist typology studies including: social factors; the impact of environmental, individual; and route factors; as well as motives. The main goal was to identify distinct types of cyclists, and describe them as detailed as possible. The heterogeneous sample included a total of 10,294 responses.Results: Using factor and cluster analyses, a multidimensional typology with four groups of cyclists was derived which were interpreted as: ambitious, functional, pragmatic, and passionate cyclists. In addition, socio-economic factors, cyclist's motivation, and crash history were analyzed.Conclusion: The results produced by grouping different characteristics of cyclists can lead to policy recommendations or communal bicycle traffic planning. Policy planners can estimate reactions of the different types on interventions and adjust their decisions which can serve to support already passionate cyclists or, encourage normally under-represented infrequent cyclists to cycle more. The extent of perceived safety plays here an important role in the classification, e.g., the handling of high-risk areas for crashes.

Change in religious beliefs, parental pressure, and attitudes of college students toward higher education as related to religious fundamentalism.

Men (n = 55) and women (n = 99) college students (M age = 22.3 yr., SD = 6.1, range 18 to 58 years), from a moderate-sized midwestern university reported attitudes toward the goals and purposes of higher education, perceptions of parental pressure and support, and change in religious beliefs. The Religious Fundamentalist Scale, the Quest Scale, Faith-keeping, and Obedience to Parents Scales were also administered. Students classified as religious fundamentalists had more negative attitudes toward the goals and purposes of higher education goals and toward faculty. An interaction of Sex x Fundamentalist Classification indicated that nonfundamentalist college men reported greater change in their religious beliefs, relative to other groups. Perceptions of parental pressure or support were unrelated to scores on fundamentalism. The implications of students' religious backgrounds in relation to academic success were discussed.

Abundance of Pathogenic Escherichia coli Virulence-Associated Genes in Well and Borehole Water Used for Domestic Purposes in a Peri-Urban Community of South Africa.

In the absence of pipe-borne water, many people in Africa, especially in rural communities, depend on alternative water sources such as wells, boreholes and rivers for household and personal hygiene. Poor maintenance and nearby pit latrines, however, lead to microbial pollution of these sources. We evaluated the abundance of Escherichia coli and the prevalence of pathogenic E. coli virulence genes in water from wells, boreholes and a river in a South African peri-urban community. Monthly samples were collected between August 2015 and November 2016. In all, 144 water samples were analysed for E. coli using the Colilert 18 system. Virulence genes (eagg, eaeA, stx1, stx2, flichH7, ST, ipaH, ibeA) were investigated using real-time polymerase chain reaction. Mean E. coli counts ranged between 0 and 443.1 Most Probable Number (MPN)/100 mL of water sample. Overall, 99.3% of samples were positive for at least one virulence gene studied, with flicH7 being the most detected gene (81/140; 57.6%) and the stx2 gene the least detected gene (8/140; 5.7%). Both intestinal and extraintestinal pathogenic E. coli genes were detected. The detection of virulence genes in these water sources suggests the presence of potentially pathogenic E. coli strains and is a public health concern.

Extracellular superoxide dismutase in biology and medicine.

Accumulated evidence has shown that reactive oxygen species (ROS) are important mediators of cell signaling events such as inflammatory reactions (superoxide) and the maintenance of vascular tone (nitric oxide). However, overproduction of ROS such as superoxide has been associated with the pathogenesis of a variety of diseases including cardiovascular diseases, neurological disorders, and pulmonary diseases. Antioxidant enzymes are, in part, responsible for maintaining low levels of these oxygen metabolites in tissues and may play key roles in controlling or preventing these conditions. One key antioxidant enzyme implicated in the regulation of ROS-mediated tissue damage is extracellular superoxide dismutase (EC-SOD). EC-SOD is found in the extracellular matrix of tissues and is ideally situated to prevent cell and tissue damage initiated by extracellularly produced ROS. In addition, EC-SOD is likely to play an important role in mediating nitric oxide-induced signaling events, since the reaction of superoxide and nitric oxide can interfere with nitric oxide signaling. This review will discuss the regulation of EC-SOD and its role in a variety of oxidant-mediated diseases.

Resveratrol and its metabolites inhibit pro-inflammatory effects of lipopolysaccharides in U-937 macrophages in plasma-representative concentrations.

Resveratrol has been shown to exploit various biological activities, including an anti-inflammatory activity. However, resveratrol is metabolized by phase II enzymes post-absorption to predominantly form glucuronides and sulfates. To investigate the anti-inflammatory effects of resveratrol and its dominating sulfated and glucuronated metabolites formed in vivo, U-937 macrophages were chosen as an immune-competent model system, known to release cytokines upon lipopolysaccharide stimulation. U-937 cells were stimulated with lipopolysaccharides from Escherichia coli (E. coli-LPS) to evoke an inflammatory reaction, and pre- or co-incubated with 1 or 10 µM of resveratrol (RES), resveratrol-3-sulfate (R3S), resveratrol-disulfates (RDS), resveratrol-3-glucuronide or resveratrol-4'-glucuronide. Time dependent gene expression of IL-6, IL-1α/ß and IL-1R by qPCR was studied at 1 h, 3 h, 6 h, 9 h, and 24 h of incubation, and the release of IL-6 and TNF-α, after 6 h was analysed by means of non-magnetic or magnetic bead analysis. As a result, 10 µM resveratrol completely inhibited the E. coli-LPS-induced release of IL-6, while resveratrol-3-sulfate and resveratrol-disulfates decreased it by respective 84.2 ± 29.4% and 52.3 ± 39.5%. Whereas TNF-α release was reduced by 48.1 ± 15.4%, 33.0 ± 10.0% and 46.7 ± 8.7% by RES, R3S and RDS, respectively. These results show that not only resveratrol but also resveratrol-3-sulfate and resveratrol-disulfates exhibit an anti-inflammatory potential by counteracting an inflammatory challenge in U-937 macrophages at plasma representative concentrations.

Depletion of pulmonary EC-SOD after exposure to hyperoxia.

Extracellular superoxide dismutase (EC-SOD) is highly expressed in lung tissue. EC-SOD contains a heparin-binding domain that is sensitive to proteolysis. This heparin-binding domain is important in allowing EC-SOD to exist in relatively high concentrations in specific regions of the extracellular matrix and on cell surfaces. EC-SOD has been shown to protect the lung against hyperoxia in transgenic and knockout studies. This study tests the hypothesis that proteolytic clearance of EC-SOD from the lung during hyperoxia contributes to the oxidant-antioxidant imbalance that is associated with this injury. Exposure to 100% oxygen for 72 h resulted in a significant decrease in EC-SOD levels in the lungs and bronchoalveolar lavage fluid of mice. This correlated with a significant depletion of EC-SOD from the alveolar parenchyma as determined by immunofluorescence and immunohistochemistry. EC-SOD mRNA was unaffected by hyperoxia; however, there was an increase in the ratio of proteolyzed to uncut EC-SOD after hyperoxia, which suggests that hyperoxia depletes EC-SOD from the alveolar parenchyma by cutting the heparin-binding domain. This may enhance hyperoxic pulmonary injury by altering the oxidant-antioxidant balance in alveolar spaces.

Purification and characterization of mouse soluble receptor for advanced glycation end products (sRAGE).

The receptor for advanced glycation end products (RAGE) is a member of the immunoglobulin superfamily of cell surface proteins that has been implicated as a progression factor in a number of pathologic conditions from chronic inflammation to cancer to Alzheimer's disease. In such conditions, RAGE acts to facilitate pathogenic processes. Its secreted isoform, soluble RAGE or sRAGE, has the ability to prevent RAGE signaling by acting as a decoy. sRAGE has been used successfully in animal models of a range of diseases to antagonize RAGE-mediated pathologic processes. In humans, sRAGE results from alternative splicing of RAGE mRNA. This study was aimed to determine whether the same holds true for mouse sRAGE and, in addition, to biochemically characterize mouse sRAGE. The biochemical characteristics examined include glycosylation and disulfide patterns. In addition, sRAGE was found to bind heparin, which may mediate its distribution in the extracellular matrix and cell surfaces of tissues. Finally, our data indicated that sRAGE in the mouse is likely produced by carboxyl-terminal truncation, in contrast to the alternative splicing mechanism reported in humans.