Mesenchymal Stem Cell-Derived Extracellular Vesicles to the Rescue of Renal Injury.

Acute kidney injury (AKI) and chronic kidney disease (CKD) are rising in global prevalence and cause significant morbidity for patients. Current treatments are limited to slowing instead of stabilising or reversing disease progression. In this review, we describe mesenchymal stem cells (MSCs) and their constituents, extracellular vesicles (EVs) as being a novel therapeutic for CKD. MSC-derived EVs (MSC-EVs) are membrane-enclosed particles, including exosomes, which carry genetic information that mimics the phenotype of their cell of origin. MSC-EVs deliver their cargo of mRNA, miRNA, cytokines, and growth factors to target cells as a form of paracrine communication. This genetically reprograms pathophysiological pathways, which are upregulated in renal failure. Since the method of exosome preparation significantly affects the quality and function of MSC-exosomes, this review compares the methodologies for isolating exosomes from MSCs and their role in tissue regeneration. More specifically, it summarises the therapeutic efficacy of MSC-EVs in 60 preclinical animal models of AKI and CKD and the cargo of biomolecules they deliver. MSC-EVs promote tubular proliferation and angiogenesis, and inhibit apoptosis, oxidative stress, inflammation, the epithelial-to-mesenchymal transition, and fibrosis, to alleviate AKI and CKD. By reprogramming these pathophysiological pathways, MSC-EVs can slow or even reverse the progression of AKI to CKD, and therefore offer potential to transform clinical practice.

Methods Evaluation for Rapid Concentration and Quantification of SARS-CoV-2 in Raw Wastewater Using Droplet Digital and Quantitative RT-PCR.

Wastewater surveillance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an emerging public health tool to understand the spread of Coronavirus Disease 2019 (COVID-19) in communities. The performance of different virus concentration methods and PCR methods needs to be evaluated to ascertain their suitability for use in the detection of SARS-CoV-2 in wastewater. We evaluated ultrafiltration and polyethylene glycol (PEG) precipitation methods to concentrate SARS-CoV-2 from sewage in wastewater treatment plants and upstream in the wastewater network (e.g., manholes, lift stations). Recovery of viruses by different concentration methods was determined using Phi6 bacteriophage as a surrogate for enveloped viruses. Additionally, the presence of SARS-CoV-2 in all wastewater samples was determined using reverse transcription quantitative PCR (RT-qPCR) and reverse transcription droplet digital PCR (RT-ddPCR), targeting three genetic markers (N1, N2 and E). Using spiked samples, the Phi6 recoveries were estimated at 2.6-11.6% using ultrafiltration-based methods and 22.2-51.5% using PEG precipitation. There was no significant difference in recovery efficiencies (p < 0.05) between the PEG procedure with and without a 16 h overnight incubation, demonstrating the feasibility of obtaining same day results. The SARS-CoV-2 genetic markers were more often detected by RT-ddPCR than RT-qPCR with higher sensitivity and precision. While all three SARS-CoV-2 genetic markers were detected using RT-ddPCR, the levels of E gene were almost below the limit of detection using RT-qPCR. Collectively, our study suggested PEG precipitation is an effective low-cost procedure which allows a large number of samples to be processed simultaneously in a routine wastewater monitoring for SARS-CoV-2. RT-ddPCR can be implemented for the absolute quantification of SARS-CoV-2 genetic markers in different wastewater matrices.

A multi-omics investigation of the composition and function of extracellular vesicles along the temporal trajectory of COVID-19.

Exosomes represent a subtype of extracellular vesicle that is released through retrograde transport and fusion of multivesicular bodies with the plasma membrane1. Although no perfect methodologies currently exist for the high-throughput, unbiased isolation of pure plasma exosomes2,3, investigation of exosome-enriched plasma fractions of extracellular vesicles can confer a glimpse into the endocytic pathway on a systems level. Here we conduct high-coverage lipidomics with an emphasis on sterols and oxysterols, and proteomic analyses of exosome-enriched extracellular vesicles (EVs hereafter) from patients at different temporal stages of COVID-19, including the presymptomatic, hyperinflammatory, resolution and convalescent phases. Our study highlights dysregulated raft lipid metabolism that underlies changes in EV lipid membrane anisotropy that alter the exosomal localization of presenilin-1 (PS-1) in the hyperinflammatory phase. We also show in vitro that EVs from different temporal phases trigger distinct metabolic and transcriptional responses in recipient cells, including in alveolar epithelial cells, which denote the primary site of infection, and liver hepatocytes, which represent a distal secondary site. In comparison to the hyperinflammatory phase, EVs from the resolution phase induce opposing effects on eukaryotic translation and Notch signalling. Our results provide insights into cellular lipid metabolism and inter-tissue crosstalk at different stages of COVID-19 and are a resource to increase our understanding of metabolic dysregulation in COVID-19.

Extraction of Carotenoids from Tomato Pomace via Water-Induced Hydrocolloidal Complexation.

Agro-industrial waste is a largely untapped natural resource of bioactive compounds including carotenoids and pectin. However, conventional solvent extraction involves the excessive use of organic solvents, costly equipment, and tedious operation. These limitations of conventional extraction methods could be prospectively overcome by the carotenoid-pectin hydrocolloidal complexation. The complexation of lycopene and pectin was efficiently promoted in an aqueous environment, resulting in the colloidal complexes that can be subsequently recovered by sedimentation or centrifugation. In this study, the potential of carotenoid-pectin complexation on tomato pomace containing carotenoids and pectin was evaluated. Tomato pomace is a rich source of lycopene, ß-carotene as well as pectin, making it suitable as the raw material for the carotenoid extraction. The extraction of carotenoid and pectin from tomato pomace was optimized using response surface methodology. The maximum recovery was 9.43 mg carotenoid fractions/100 g tomato pomace, while the purity of carotenoid-rich fractions was 92%. The antioxidant capacity of carotenoids extracted from the complexation method was found to be higher than that from the solvent extraction method. Moreover, extraction yield and antioxidant capacity of carotenoid obtained from the carotenoid-pectin complexation were comparable to that from solvent extraction. The carotenoid-pectin complexation is a promising green approach to valorize agro by-products for the extraction of valuable carotenoids.

Production of hyperimmune anti-SARS-CoV-2 intravenous immunoglobulin from pooled COVID-19 convalescent plasma.

Background: This study assesses the feasibility of producing hyperimmune anti-COVID-19 intravenously administrable immunoglobulin (C-IVIG) from pooled convalescent plasma (PCP) to provide a safe and effective passive immunization treatment option for COVID-19. Materials & methods: PCP was fractionated by modified caprylic acid precipitation followed by ultrafiltration/diafiltration to produce hyperimmune C-IVIG. Results: In C-IVIG, the mean SARS-CoV-2 antibody level was found to be threefold (104 ± 30 cut-off index) that of the PCP (36 ± 8.5 cut-off index) and mean protein concentration was found to be 46 ± 3.7 g/l, comprised of 89.5% immunoglobulins. Conclusion: The current method of producing C-IVIG is feasible as it uses locally available PCP and simpler technology and yields a high titer of SARS-CoV-2 antibody. The safety and efficacy of C-IVIG will be evaluated in a registered clinical trial (NCT04521309).

A comparison of DNA/RNA extraction protocols for high-throughput sequencing of microbial communities.

One goal of microbial ecology researchers is to capture the maximum amount of information from all organisms in a sample. The recent COVID-19 pandemic, caused by the RNA virus SARS-CoV-2, has highlighted a gap in traditional DNA-based protocols, including the high-throughput methods the authors previously established as field standards. To enable simultaneous SARS-CoV-2 and microbial community profiling, the authors compared the relative performance of two total nucleic acid extraction protocols with the authors' previously benchmarked protocol. The authors included a diverse panel of environmental and host-associated sample types, including body sites commonly swabbed for COVID-19 testing. Here the authors present results comparing the cost, processing time, DNA and RNA yield, microbial community composition, limit of detection and well-to-well contamination between these protocols.

Detection and quantification of Covid-19 antiviral drugs in biological fluids and tissues.

Since coronavirus disease 2019 (COVID-19) started as a fast-spreading pandemic, causing a huge number of deaths worldwide, several therapeutic options have been tested to counteract or reduce the clinical symptoms of patients infected with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Currently, no specific drugs for COVID-19 are available, but many antiviral agents have been authorised by several national agencies. Most of them are under investigation in both preclinical and clinical trials; however, pharmacokinetic and metabolism studies are needed to identify the most suitable dose to achieve the desired effect on SARS-CoV-2. Therefore, the efforts of the scientific community have focused on the screening of therapies able to counteract the most severe effects of the infection, as well as on the search of sensitive and selective analytical methods for drug detection in biological matrices, both fluids and tissues. In the last decade, many analytical methods have been proposed for the detection and quantification of antiviral compounds currently being tested for COVID-19 treatment. In this review, a critical discussion on the overall analytical procedure is provided, i.e (a) sample pre-treatment and extraction methods such as protein precipitation (PP), solid-phase extraction (SPE), liquid-liquid extraction (LLE), ultrasound-assisted extraction (UAE) and QuEChERS (quick, easy, cheap, effective, rugged and safe), (b) detection and quantification methods such as potentiometry, spectrofluorimetry and mass spectrometry (MS) as well as (c) methods including a preliminary separation step, such as high performance liquid chromatography (HPLC) and capillary electrophoresis (CE) coupled to UV-Vis or MS detection. Further current trends, advantages and disadvantages and prospects of these methods have been discussed, to help the analytical advances in reducing the harm caused by the SARS-CoV-2 virus.

Advances in point-of-care nucleic acid extraction technologies for rapid diagnosis of human and plant diseases.

Global health and food security constantly face the challenge of emerging human and plant diseases caused by bacteria, viruses, fungi, and other pathogens. Disease outbreaks such as SARS, MERS, Swine Flu, Ebola, and COVID-19 (on-going) have caused suffering, death, and economic losses worldwide. To prevent the spread of disease and protect human populations, rapid point-of-care (POC) molecular diagnosis of human and plant diseases play an increasingly crucial role. Nucleic acid-based molecular diagnosis reveals valuable information at the genomic level about the identity of the disease-causing pathogens and their pathogenesis, which help researchers, healthcare professionals, and patients to detect the presence of pathogens, track the spread of disease, and guide treatment more efficiently. A typical nucleic acid-based diagnostic test consists of three major steps: nucleic acid extraction, amplification, and amplicon detection. Among these steps, nucleic acid extraction is the first step of sample preparation, which remains one of the main challenges when converting laboratory molecular assays into POC tests. Sample preparation from human and plant specimens is a time-consuming and multi-step process, which requires well-equipped laboratories and skilled lab personnel. To perform rapid molecular diagnosis in resource-limited settings, simpler and instrument-free nucleic acid extraction techniques are required to improve the speed of field detection with minimal human intervention. This review summarizes the recent advances in POC nucleic acid extraction technologies. In particular, this review focuses on novel devices or methods that have demonstrated applicability and robustness for the isolation of high-quality nucleic acid from complex raw samples, such as human blood, saliva, sputum, nasal swabs, urine, and plant tissues. The integration of these rapid nucleic acid preparation methods with miniaturized assay and sensor technologies would pave the road for the "sample-in-result-out" diagnosis of human and plant diseases, especially in remote or resource-limited settings.