Controlled Heat and Humidity-Based Treatment for the Reuse of Personal Protective Equipment: A Pragmatic Proof-of-Concept to Address the Mass Shortage of Surgical Masks and N95/FFP2 Respirators and to Prevent the SARS-CoV2 Transmission.

Background: The coronavirus infectious disease-2019 (COVID-19) pandemic has led to an unprecedented shortage of healthcare resources, primarily personal protective equipment like surgical masks, and N95/filtering face piece type 2 (FFP2) respirators. Objective: Reuse of surgical masks and N95/FFP2 respirators may circumvent the supply chain constraints and thus overcome mass shortage. Methods, design, setting, and measurement: Herein, we tested the effects of dry- and moist-air controlled heating treatment on structure and chemical integrity, decontamination yield, and filtration performance of surgical masks and FFP2 respirators. Results: We found that treatment in a climate chamber at 70°C during 1 h with 75% humidity rate was adequate for enabling substantial decontamination of both respiratory viruses, oropharyngeal bacteria, and model animal coronaviuses, while maintaining a satisfying filtering capacity. Limitations: Further studies are now required to confirm the feasibility of the whole process during routine practice. Conclusion: Our findings provide compelling evidence for the recycling of pre-used surgical masks and N95/FFP2 respirators in case of imminent mass shortfall.

BK Polyomavirus Hijacks Extracellular Vesicles for En Bloc Transmission.

Most people are asymptomatic carriers of the BK polyomavirus (BKPyV), but the mechanisms of persistence and immune evasion remain poorly understood. Furthermore, BKPyV is responsible for nephropathies in kidney transplant recipients. Unfortunately, the sole therapeutic option is to modulate immunosuppression, which increases the risk of transplant rejection. Using iodixanol density gradients, we observed that Vero and renal proximal tubular epithelial infected cells release two populations of infectious particles, one of which cosediments with extracellular vesicles (EVs). Electron microscopy confirmed that a single vesicle could traffic tens of viral particles. In contrast to naked virions, the EV-associated particles (eBKPyVs) were not able to agglutinate red blood cells and did not use cell surface sialylated glycans as an attachment factor, demonstrating that different entry pathways were involved for each type of infectious particle. However, we also observed that naked BKPyV and eBKPyV were equally sensitive to neutralization by the serum of a seropositive patient or commercially available polyvalent immunoglobulin preparations, which occurred at a postattachment step, after endocytosis. In conclusion, our work shows a new mechanism that likely plays a critical role during the primary infection and in the persistence, but also the reactivation, of BKPyV.IMPORTANCE Reactivation of BKPyV is responsible for nephropathies in kidney transplant recipients, which frequently lead to graft loss. The mechanisms of persistence and immune evasion used by this virus remain poorly understood, and a therapeutic option for transplant patients is still lacking. Here, we show that BKPyV can be released into EVs, enabling viral particles to infect cells using an alternative entry pathway. This provides a new view of BKPyV pathogenesis. Even though we did not find any decreased sensitivity to neutralizing antibodies when comparing EV-associated particles and naked virions, our study also raises important questions about developing prevention strategies based on the induction or administration of neutralizing antibodies. Deciphering this new release pathway could enable the identification of therapeutic targets to prevent BKPyV nephropathies. It could also lead to a better understanding of the pathophysiology of other polyomaviruses that are associated with human diseases.

Virus detection by transmission electron microscopy: Still useful for diagnosis and a plus for biosafety.

Transmission electron microscopy (TEM) is the only imaging technique allowing the direct visualization of viruses, due to its nanometer-scale resolution. Between the 1960s and 1990s, TEM contributed to the discovery of many types of viruses and served as a diagnostic tool for identifying viruses directly in biological samples, either in suspension or in sections of tissues or mammalian cells grown in vitro in contact with clinical samples. The diagnosis of viral infections improved considerably during the 1990s, with the advent of highly sensitive techniques, such as enzyme-linked immunosorbent assay (ELISA) and PCR, rendering TEM obsolete for this purpose. However, the last 20 years have demonstrated the utility of this technique in particular situations, due to its "catch-all" nature, making diagnosis possible through visualization of the virus, without the need of prior assumptions about the infectious agent sought. Thus, in several major outbreaks in which molecular techniques failed to identify the infectious agent, TEM provided the answer. TEM is also still occasionally used in routine diagnosis to characterize infections not diagnosed by molecular assays. It is also used to check the microbiological safety of biological products. Many biopharmaceuticals are produced in animal cells that might contain little-known, difficult-to-detect viruses. In this context, the "catch-all" properties of TEM make it possible to document the presence of viruses or virus-like particles in these products.

How Klingler's dissection permits exploration of brain structural connectivity? An electron microscopy study of human white matter.

The objective of this study is to explore histological and ultrastructural changes induced by Klingler's method. Five human brains were prepared. First, the effects of freezing-defrosting on white matter were explored with optical microscopy on corpus callosum samples of two brains; one prepared in accordance with the description of Klingler (1956) and the other without freezing-defrosting. Then, the combined effect of formalin fixation and freezing-defrosting was explored with transmission electron microscopy (EM) on samples of cingulum from one brain: samples from one hemisphere were fixed in paraformaldehyde-glutaraldehyde (para/gluta), other samples from the other hemisphere were fixed in formalin; once fixed, half of the samples were frozen-defrosted. Finally, the effect of dissection was explored from three formalin-fixed brains: one hemisphere of each brain was frozen-defrosted; samples of the corpus callosum were dissected before preparation for scanning EM. Optical microscopy showed enlarged extracellular space on frozen samples. Transmission EM showed no significant alteration of white matter ultrastructure after formalin or para/gluta fixation. Freezing-defrosting created extra-axonal lacunas, larger on formalin-fixed than on para/gluta-fixed samples. In all cases, myelin sheaths were preserved, allowing maintenance of axonal integrity. Scanning EM showed the destruction of most of the extra-axonal structures after freezing-defrosting and the preservation of most of the axons after dissection. Our results are the first to highlight the effects of Klingler's preparation and dissection on white matter ultrastructure. Preservation of myelinated axons is a strong argument to support the reliability of Klingler's dissection to explore the structure of human white matter.

Voltage-gated sodium channels potentiate the invasive capacities of human non-small-cell lung cancer cell lines.

Ionic channel activity is involved in fundamental cellular behaviour and participates in cancerous features such as proliferation, migration and invasion which in turn contribute to the metastatic process. In this study, we investigated the expression and role of voltage-gated sodium channels in non-small-cell lung cancer cell lines. Functional voltage-gated sodium channels expression was investigated in normal and non-small-cell lung cancer cell lines. The measurement, in patch-clamp conditions, of tetrodotoxin-inhibitable sodium currents indicated that the strongly metastatic cancerous cell lines H23, H460 and Calu-1 possess functional sodium channels while normal and weakly metastatic cell lines do not. While all the cell lines expressed mRNA for numerous sodium channel isoforms, only H23, H460 and Calu-1 cells had a 250 kDa protein corresponding to the functional channel. The other cell lines also had another protein of 230 kDa which is not addressed to the membrane and might act as a dominant negative isoform to prevent channel activation. At the membrane potential of these cells, channels are partially open. This leads to a continuous entry of sodium, disrupting sodium homeostasis and down-stream signaling pathways. Inhibition of the channels by tetrodotoxin was responsible for a 40-50% reduction of in vitro invasion. These experiments suggest that the functional expression of voltage-gated sodium channels might be an integral component of the metastatic process in non-small-cell lung cancer cells probably through its involvement in the regulation of intracellular sodium homeostasis. These channels could serve both as novel markers of the metastatic phenotype and as potential new therapeutic targets.