SARS-CoV-2 subgenomic RNA kinetics in longitudinal clinical samples

Background Given the persistence of viral RNA in clinically recovered COVID-19 patients, subgenomic RNAs (sgRNA) have been reported as potential molecular viability markers for SARS-CoV-2. However, few data are available on their longitudinal kinetics, compared with genomic RNA (gRNA), in clinical samples. Methods We analyzed 536 samples from 205 patients with COVID-19 from placebo-controlled, outpatient trials of Peginterferon Lambda-1a (Lambda; n=177) and favipiravir (n=359). Nasal swabs were collected at three time points in the Lambda (Day 1, 4 and 6) and favipiravir (Day 1, 5, and 10) trials. N-gene gRNA and sgRNA were quantified by RT-qPCR. To investigate the decay kinetics in vitro, we measured gRNA and sgRNA in A549 ACE2+ cells infected with SARS-CoV-2, following treatment with remdesivir or DMSO control. Results At six days in the Lambda trial and ten days in the favipiravir trial, sgRNA remained detectable in 51.6% (32/62) and 49.5% (51/106) of the samples, respectively. Cycle threshold (Ct) values for gRNA and sgRNA were highly linearly correlated (marginal R 2, 0.83) and the rate of increase did not differ significantly in Lambda (1.36 cycles/day vs 1.36 cycles/day; p = 0.97) or favipiravir (1.03 cycles/day vs 0.94 cycles/day; p=0.26) trials. From samples collected 15-21 days after symptom onset, sgRNA was detectable in 48.1% (40/83) of participants. In SARS-CoV-2 infected A549 ACE2+ cells treated with remdesivir, the rate of Ct increase did not differ between gRNA and sgRNA. Conclusions In clinical samples and in vitro, sgRNA was highly correlated with gRNA and did not demonstrate different decay patterns to support its application as a viability marker.

Viability markers for determination of desiccation tolerance and critical stages during dehydration in Selaginella species

Plants can tolerate some degree of dehydration but below a threshold of water content most plants die. However, some species display specific physiological, biochemical, and molecular responses that allow survival to desiccation. Some of these responses are activated at critical stages during water loss and could represent the difference between desiccation tolerance (DT) and death. Here, we report the development of a simple and reproducible system to determine DT in Selaginella species. This system is based on the use of excised tissue (explants), exposed to a dehydration agent inside small containers, rather than whole plants making it faster, better controlled, and potential use under field conditions. We also report that the triphenyltetrazolium chloride test is a simple and accurate assay to determine tissue viability. The explant system is particularly useful to identify critical points during the dehydration process and was applied to identify novel desiccation-tolerant Selaginella species. Our data suggest that desiccation-sensitive Selaginella species have a change in viability when dehydrated to 40% RWC, indicating the onset of a critical condition at this water content. Comparative studies at these critical stages could provide a better understanding of DT mechanisms and unravel insights into the key responses to survive desiccation.

Anticoagulant Management and Synthesis of Hemostatic Proteins during Machine Preservation of Livers for Transplantation

Liver transplantation remains the only curative treatment for patients with end-stage liver disease. Despite a steadily increasing demand for suitable donor livers, the current pool of donor organs fails to meet this demand. To resolve this discrepancy, livers traditionally considered to be of suboptimal quality and function are increasingly utilized. These marginal livers, however, are less tolerant to the current standard cold preservation of donor organs. Therefore, alternative preservation methods have been sought and are progressively applied into clinical practice. Ex situ machine perfusion is a promising alternative preservation modality particularly for suboptimal donor livers as it provides the ability to resuscitate, recondition, and test the viability of an organ prior to transplantation. This review addresses the modalities of machine perfusion currently being applied, and particularly focuses on the hemostatic management employed during machine perfusion. We discuss the anticoagulant agents used, the variation in dosage, and administration, as well as the implications of perfusion for extended periods of time in terms of coagulation activation associated with production of coagulation factors during perfusion. Furthermore, in regard to viability testing of an organ prior to transplantation, we discuss the possibilities and limitations of utilizing the synthesis of liver-derived coagulation factors as potential viability markers.

Updates on the applications of flow cytometry in microbiology

Flow cytometry (FCM) was first developed for medical and clinical applications such as hematology and oncology. Although these areas still account for the vast majority of publications on this technique, during the past few years it has been also introduced in other areas, such as optimization and monitoring of biotechnological and environmental processes, pharmacology, toxicology, bacteriology and virology. In the food and drinks industries, the time required for conventional microbiology tests can lead to substantial delays in product release to the market. FCM has been used in conjunction with viability markers for rapid counting of yeast, molds and bacterial cells, including foodborne pathogens and microbial contaminants, in food products as well as for monitoring and improving the final products quality. FCM is an excellent tool, still unexplored in clinical microbiology, allowing for detection of cellular and non-cellular components in different clinical specimens, the study of antimicrobial activity, allowing for rapid and direct antimicrobial susceptibility testing and for the investigation of resistance mechanisms. Recent FCM developments important for addressing questions in environmental microbiology include the study of microbial physiology under environmentally relevant conditions, the development of new methods to identify active microbial populations and to isolate previously uncultured microorganisms and of high-throughput autofluorescence bioreporter assays. Moreover, the technological advancements will make possible the miniaturisation and automation of FCM devices, allowing to revolutionize their applications in the near future. The purpose of this minireview is to update the current applications of FCM in different fields of applied microbiology, and to highlight the main advantages and pitfalls for each of them.

Potential Neuroprotective and Anti-Apoptotic Properties of a Long-Lasting Stable Analog of Ghrelin: an In Vitro Study Using SH-SY5Y Cells

Neurodegenerative disorders, such as Alzheimer’s disease (AD) and Parkinson’s disease (PD), are increasing in prevalence. Currently, there are no effective and specific treatments for these disorders. Recently, positive effects of the orexigenic hormone ghrelin on memory and learning were demonstrated in mouse models of AD and PD. In this study, we tested the potential neuroprotective properties of a stable and long-lasting ghrelin analog, Dpr3ghrelin (Dpr3ghr), in SH-SY5Y neuroblastoma cells stressed with 1.2 mM methylglyoxal (MG), a toxic endogenous by-product of glycolysis, and we examined the impact of Dpr3ghr on apoptosis. Pre-treatment with both 10-5 and 10-7 M Dpr3ghr resulted in increased viability in SH-SY5Y cells (determined by MTT staining), as well as reduced cytotoxicity of MG in these cells (determined by LDH assay). Dpr3ghr increased viability by altering pro-apoptotic and viability markers: Bax was decreased, Bcl-2 was increased, and the Bax/Bcl-2 ratio was attenuated. The ghrelin receptor GHS-R1 and Dpr3ghr-induced activation of PBK/Akt were immuno-detected in SH-SY5Y cells to demonstrate the presence of GHS-R1 and GHS-R1 activation, respectively. We demonstrated that Dpr3ghr protected SH-SY5Y cells against MG-induced neurotoxicity and apoptosis. Our data suggest that stable ghrelin analogs may be candidates for the effective treatment of neurodegenerative disorders.

Measuring embryo metabolism to predict embryo quality

Measuring the metabolism of early embryos has the potential to be used as a prospective marker for post-transfer development, either alone or in conjunction with other embryo quality assessment tools. This is necessary to maximise the opportunity of couples to have a healthy child from assisted reproduction technology (ART) and for livestock breeders to efficiently improve the genetics of their animals. Nevertheless, although many promising candidate substrates (e.g. glucose uptake) and methods (e.g. metabolomics using different spectroscopic techniques) have been promoted as viability markers, none has yet been widely used clinically or in livestock production. Herein we review the major techniques that have been reported; these are divided into indirect techniques, where measurements are made from the embryo’s immediate microenvironment, or direct techniques that measure intracellular metabolic activity. Both have strengths and weaknesses, the latter ruling out some from contention for use in human ART, but not necessarily for use in livestock embryo assessment. We also introduce a new method, namely multi- (or hyper-) spectral analysis, which measures naturally occurring autofluorescence. Several metabolically important molecules have fluorescent properties, which we are pursuing in conjunction with improved image analysis as a viable embryo quality assessment methodology.