Genuine Selective Caspase-2 Inhibition with new Irreversible Small Peptidomimetics

Caspase-2 (Casp2) is a promising therapeutic target in several human diseases including nonalcoholic steatohepatitis (NASH) and Alzheimer's disease (AD). However, the design of active-site-directed inhibitor selective to individual caspase family members is challenging because caspases have extremely similar active sites. Here we present new peptidomimetics derived from the VDVAD pentapeptide structure, harboring non-natural modifications at the P2 position and an irreversible warhead. Enzyme kinetics shows that these new compounds, such as LJ2 or its specific isomer LJ2a, and LJ3a, strongly and irreversibly inhibit Casp2 with genuine selectivity. According to Casp2 role in cellular stress responses, LJ2 inhibits cell death induced by microtubule destabilization or hydroxamic acid-based deacetylase inhibition. The most potent peptidomimetic, LJ2a, inhibits human Casp2 with a remarkably high inactivation rate (k3/Ki ~ 5 500 000 M-1s-1) and the most selective inhibitor, LJ3a, has a near to 1000 times higher inactivation rate on Casp2 as compared to Casp3. Structural analysis of LJ3a shows that spatial configuration of C[alpha]; at the P2 position determines inhibitor efficacy. In transfected human cell lines overexpressing site-1 protease (S1P), sterol regulatory element-binding protein 2 (SREBP2) and Casp2, LJ2a and LJ3a fully inhibit SREBP2 activation, suggesting a potential to prevent NASH development. Furthermore, in primary hippocampal neurons treated with [beta]-amyloid oligomers, submicromolar concentrations of LJ2a and of LJ3a prevent synapse loss, indicating a potential for further investigations in AD treatment.

UV Inactivation of SARS-CoV-2 across the UVC spectrum: KrCl* excimer, mercury-vapor, and LED sources

Effective disinfection technology to combat severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can help reduce viral transmissions during the on-going COVID-19 global pandemic and in the future. Ultraviolet (UV) devices emitting UVC irradiation (200-280 nm) have proven to be effective for virus disinfection, but limited information is available for SARS-CoV-2 due to the safety requirements of testing, which is limited to biosafety level (BSL) 3 laboratories. In this study, inactivation of SARS-CoV-2 in thin-film buffered aqueous solution (pH 7.4) was determined across UVC irradiation wavelengths (222 nm to 282 nm) from krypton chloride (KrCl*) excimers, a low-pressure mercury-vapor lamp, and two UVC light emitting diodes. Our results show that all tested UVC devices can effectively inactivate SARS-CoV-2, among which the KrCl* excimer had the best disinfection performance (i.e., highest inactivation rate). The inactivation rate constants of SARS-CoV-2 across wavelengths are similar to those for murine hepatitis virus (MHV) from our previous investigation, suggesting that MHV can serve as a reliable surrogate of SARS-CoV-2 with a lower BSL requirement (BSL-2) during UV disinfection tests. This study provides fundamental information for UVC action on SARS-CoV-2 and guidance for achieving reliable disinfection performance of UVC devices. IMPORTANCE UV light is an effective tool to help stem the spread of respiratory viruses and protect public health in commercial, transportation and healthcare settings. For effective use of UV, there is a need to determine the efficiency of different UV wavelengths in killing pathogens, specifically SARS-CoV-2, to support efforts to control the on-going COVID-19 global pandemic and future coronavirus-caused respiratory virus pandemics. We found that SARS-CoV-2 can be inactivated effectively using a broad range of UVC wavelengths, and 222nm provided the best disinfection performance. Interestingly, 222 nm irradiation has been found to be safe for human exposure up to thresholds that are beyond effective for inactivating viruses. Therefore, applying UV light from KrCl* excimers in public spaces can effectively help reduce viral aerosol or surface transmissions.

Heat-treated virus inactivation rate depends strongly on treatment procedure: illustration with SARS-CoV-2

Decontamination helps limit environmental transmission of infectious agents. It is required for the safe re-use of contaminated medical, laboratory and personal protective equipment, and for the safe handling of biological samples. Heat treatment is a common decontamination method, notably used for viruses. We show that for liquid specimens (here, solution of SARS-CoV-2 in cell culture medium), virus inactivation rate under heat treatment at 70°C can vary by almost two orders of magnitude depending on the treatment procedure, from a half-life of 0.86 min (95% credible interval: [0.09, 1.77]) in closed vials in a heat block to 37.00 min ([12.65, 869.82]) in uncovered plates in a dry oven. These findings suggest a critical role of evaporation in virus inactivation via dry heat. Placing samples in open or uncovered containers may dramatically reduce the speed and efficacy of heat treatment for virus inactivation. Given these findings, we reviewed the literature temperature-dependent coronavirus stability and found that specimen containers, and whether they are closed, covered, or uncovered, are rarely reported in the scientific literature. Heat-treatment procedures must be fully specified when reporting experimental studies to facilitate result interpretation and reproducibility, and must be carefully considered when developing decontamination guidelines. Importance Heat is a powerful weapon against most infectious agents. It is widely used for decontamination of medical, laboratory and personal protective equipment, and for biological samples. There are many methods of heat treatment, and methodological details can affect speed and efficacy of decontamination. We applied four different heat-treatment procedures to liquid specimens containing SARS-CoV-2. Our results show that the container used to store specimens during decontamination can substantially affect inactivation rate: for a given initial level of contamination, decontamination time can vary from a few minutes in closed vials to several hours in uncovered plates. Reviewing the literature, we found that container choices and heat treatment methods are only rarely reported explicitly in methods sections. Our study shows that careful consideration of heat-treatment procedure — in particular the choice of specimen container, and whether it is covered — can make results more consistent across studies, improve decontamination practice, and provide insight into the mechanisms of virus inactivation.

Decontamination Of Endospores By Plasma Sources On Dried Surfaces: A Review Of Key Parameters And Inactivation Results

The efficiency of plasmas sources for the decontamination of heat-sensitive devices has been proven for more than 20 years, but commercial plasma-based sterilizers still have a narrow range of applications. This can be partially explained by difficulties to determine reliable bio-indicators and standardized microbiological test procedures required by industrial uses. In this paper, we examine the influence of environmental factors on the inactivation rate of microorganisms deposited on surfaces and treated by plasma sources. In addition, we present a literature review showing that several in-discharge and afterglow plasma sterilizers offer shorter treatment times than conventional low-temperature sterilizers to reduce the concentration of endospores on contaminated surfaces by 6-log. Finally we make a few recommendations for future plasma decontamination standards.

Neuronal junctophilins recruit specific CaV and RyR isoforms to ER-PM junctions and functionally alter CaV2.1 and CaV2.2

Junctions between the endoplasmic reticulum and plasma membrane that are induced by the neuronal junctophilins are of demonstrated importance, but their molecular architecture is still poorly understood and challenging to address in neurons. This is due to the small size of the junctions and the multiple isoforms of candidate junctional proteins in different brain areas. Using colocalization of tagged proteins expressed in tsA201 cells, and electrophysiology, we compared the interactions of JPH3 and JPH4 with different calcium channels. We found that JPH3 and JPH4 caused junctional accumulation of all the tested high-voltage-activated CaV isoforms, but not a low-voltage-activated CaV. Also, JPH3 and JPH4 noticeably modify CaV2.1 and CaV2.2 inactivation rate. RyR3 moderately colocalized at junctions with JPH4, whereas RyR1 and RyR2 did not. By contrast, RyR1 and RyR3 strongly colocalized with JPH3, and RyR2 moderately. Likely contributing to this difference, JPH3 binds to cytoplasmic domain constructs of RyR1 and RyR3, but not of RyR2.

Inactivation of E. coli Using Atmospheric Pressure Plasma Jet with Dry and Wet Argon Discharges

The acceleration of inactivating viable cells of Escherichia coli (E. coli), by using new direct and indirect innovative methods, is the targeted method of using an atmospheric pressure plasma jet (APPJ) operated by an AC high-voltage power source with variable frequency up to 60 kHz and voltage ranging from 2.5 to 25 kV. Discharges using dry argon (0% O2) discharges and different wet argon discharges using admixtures with O2/Ar ratios ranging from 0.25% to 1.5% were studied. The combined effects of dry and wet argon discharges, direct and indirect exposure using a mesh controller, and hollow magnets were studied to reach a complete bacterial inactivation in short application times. Survival curves showed that the inactivation rate increased as the wettability increased. The application of magnetized non-thermal plasma discharge with a 1.5% wetness ratio causes a fast inactivation rate of microbes on surfaces, and a dramatic decrease of the residual survival of the bacterial ratio due to an increase in the jet width and the enhanced ability of fast transport of the charges to viable cells, especially at the edge of the Petri dish. The membrane damage of E. coli mechanism factors in the activation process by APPJ is discussed.

Predictive modeling of virus inactivation by UV

Disinfection strategies are commonly applied to inactivate pathogenic viruses in water, food, air, and on surfaces to prevent the spread of infectious diseases. Determining how quickly viruses are inactivated to mitigate health risks is not always feasible due to biosafety restrictions or difficulties with virus culturability. Therefore, methods that would rapidly predict kinetics of virus inactivation by UV254 would be valuable, particularly for emerging and difficult-to-culture viruses. We conducted a rapid systematic literature review to collect high-quality inactivation rate constants for a wide range of viruses. Using these data and basic virus information (e.g., genome sequence attributes), we developed and evaluated four different model classes, including linear and non-linear approaches, to find the top performing prediction model. For both the (+) ssRNA and dsDNA virus types, multiple linear regressions were the top performing model classes. In both cases, the cross-validated root mean squared relative prediction errors were similar to those associated with experimental rate constants. We tested the models by predicting and measuring inactivation rate constants for two viruses that were not identified in our systematic review, including a (+) ssRNA mouse coronavirus and a dsDNA marine bacteriophage; the predicted rate constants were within 7% and 71% of the experimental rate constants, respectively. Finally, we applied our models to predict the UV254 rate constants of several viruses for which high-quality UV254 inactivation data are not available. Our models will be valuable for predicting inactivation kinetics of emerging or difficult-to-culture viruses.

Mechanistic theory predicts the effects of temperature and humidity on inactivation of SARS-CoV-2 and other enveloped viruses

Environmental conditions affect virus inactivation rate and transmission potential. Understanding those effects is critical for anticipating and mitigating epidemic spread. Ambient temperature and humidity strongly affect the inactivation rate of enveloped viruses, but a mechanistic, quantitative theory of those effects has been elusive. We measure the stability of the enveloped respiratory virus SARS-CoV-2 on an inert surface at nine temperature and humidity conditions and develop a mechanistic model to explain and predict how temperature and humidity alter virus inactivation. We find SARS-CoV-2 survives longest at low temperatures and extreme relative humidities; median estimated virus half-life is over 24 hours at 10 °C and 40 % RH, but approximately 1.5 hours at 27 °C and 65 % RH. Our mechanistic model uses simple chemistry to explain the increase in virus inactivation rate with increased temperature and the U-shaped dependence of inactivation rate on relative humidity. The model accurately predicts quantitative measurements from existing studies of five different human coronaviruses (including SARS-CoV-2), suggesting that shared mechanisms may determine environmental stability for many enveloped viruses. Our results indicate scenarios of particular transmission risk, point to pandemic mitigation strategies, and open new frontiers in the mechanistic study of virus transmission.

Heat-treated virus inactivation rate depends strongly on treatment procedure

Decontamination of objects and surfaces can limit transmission of infectious agents via fomites or biological samples. It is required for the safe re-use of potentially contaminated personal protective equipment and medical and laboratory equipment. Heat treatment is widely used for the inactivation of various infectious agents, notably viruses. We show that for liquid specimens (here suspension of SARS-CoV-2 in cell culture medium), virus inactivation rate under heat treatment at 70°C can vary by almost two orders of magnitude depending on the treatment procedure, from a half-life of 0.86 min (95% credible interval: [0.09, 1.77]) in closed vials in a heat block to 37.0 min ([12.65, 869.82]) in uncovered plates in a dry oven. These findings suggest a critical role of evaporation in virus inactivation using dry heat. Placing samples in open or uncovered containers may dramatically reduce the speed and efficacy of heat treatment for virus inactivation. Heating procedures must be carefully specified when reporting experimental studies to facilitate result interpretation and reproducibility, and carefully considered when designing decontamination guidelines.