Explore the vast range of titles available.

The release of extracellular vesicles (EVs) in cell cultures as well as their molecular cargo can be influenced by cell culture conditions such as the presence of foetal bovine serum (FBS). Although several studies have evaluated the effect of removing FBS‐derived EVs by ultracentrifugation (UC), less is known about the influence of FBS heat inactivation (HI) on the cell‐derived EVs. To assess this, three protocols based on different combinations of EV depletion by UC and HI were evaluated, including FBS ultracentrifuged but not heat inactivated (no‐HI FBS), FBS heat inactivated before EV depletion (HI‐before EV‐depl FBS), and FBS heat inactivated after EV depletion (HI‐after EV‐depl FBS). We isolated large (L‐EVs) and small EVs (S‐EVs) from FBS treated in the three different ways, and we found that the S‐EV pellet from HI‐after EV‐depl FBS was larger than the S‐EV pellet from no‐HI FBS and HI‐before EV‐depl FBS. Transmission electron microscopy, protein quantification, and particle number evaluation showed that HI‐after EV‐depl significantly increased the protein amount of S‐EVs but had no significant effect on L‐EVs. Consequently, the protein quantity of S‐EVs isolated from three cell lines cultured in media supplemented with HI‐after EV‐depl FBS was significantly increased. Quantitative mass spectrometry analysis of FBS‐derived S‐EVs showed that the EV protein content was different when FBS was HI after EV depletion compared to EVs isolated from no‐HI FBS and HI‐before EV‐depl FBS. Moreover, we show that several quantified proteins could be ascribed to human origin, thus demonstrating that FBS bovine proteins can mistakenly be attributed to human cell‐derived EVs. We conclude that HI of FBS performed after EV depletion results in changes in the proteome, with molecules that co‐isolate with EVs and can contaminate EVs when used in subsequent cell cultures. Our recommendation is, therefore, to always perform HI of FBS prior to EV depletion.

Heat inactivation of bovine sera is routinely performed in cell culture laboratories. Nevertheless, it remains debatable whether it is still necessary due to the improvement of the production process of bovine sera. Do the benefits balance the loss of many proteins, such as hormones and growth factors, that are very useful for cell culture? This is even truer in the case of tissue engineering, the processes of which is often very demanding. This balance is examined here, from nine populations of fibroblasts originating from three different organs, by comparing the capacity of adhesion and proliferation of cells, their metabolism, and the capacity to produce the stroma; their histological appearance, thickness, and mechanical properties were also evaluated. Overall, serum inactivation does not appear to provide a significant benefit.

Abstract Background Coronavirus disease-2019 (COVID-19) has spread widely throughout the world since the end of 2019. Nucleic acid testing (NAT) has played an important role in patient diagnosis and management of COVID-19. In some circumstances, thermal inactivation at 56°C has been recommended to inactivate severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) before NAT. However, this procedure could theoretically disrupt nucleic acid integrity of this single-stranded RNA virus and cause false negatives in real-time polymerase chain reaction (RT-PCR) tests. Methods We investigated whether thermal inactivation could affect the results of viral NAT. We examined the effects of thermal inactivation on the quantitative RT-PCR results of SARS-CoV-2, particularly with regard to the rates of false-negative results for specimens carrying low viral loads. We additionally investigated the effects of different specimen types, sample preservation times, and a chemical inactivation approach on NAT. Results Our study showed increased Ct values in specimens from diagnosed COVID-19 patients in RT-PCR tests after thermal incubation. Moreover, about half of the weak-positive samples (7 of 15 samples, 46.7%) were RT-PCR negative after heat inactivation in at least one parallel testing. The use of guanidinium-based lysis for preservation of these specimens had a smaller impact on RT-PCR results with fewer false negatives (2 of 15 samples, 13.3%) and significantly less increase in Ct values than heat inactivation. Conclusion Thermal inactivation adversely affected the efficiency of RT-PCR for SARS-CoV-2 detection. Given the limited applicability associated with chemical inactivators, other approaches to ensure the overall protection of laboratory personnel need consideration.

The major barrier to research and development of effective interventions for human noroviruses (HuNoVs) has been the lack of a robust and reproducible in vitro cultivation system. HuNoVs are the leading cause of gastroenteritis worldwide. We report the successful cultivation of multiple HuNoV strains in enterocytes in stem cell-derived, nontransformed human intestinal enteroid monolayer cultures. Bile, a critical factor of the intestinal milieu, is required for straindependent HuNoV replication. Lack of appropriate histoblood group antigen expression in intestinal cells restricts virus replication, and infectivity is abrogated by inactivation (e.g., irradiation, heating) and serum neutralization. This culture system recapitulates the human intestinal epithelium, permits human host-pathogen studies of previously noncultivatable pathogens, and allows the assessment of methods to prevent and treat HuNoV infections.

Influenza A(H5N1) virus has been found in cow’s milk, and H5N1 genetic material has been identified in the commercial milk supply. In this report, investigators assess the effect of heat inactivation on viability of the virus.

The effect of various abiotic stresses on photosynthetic apparatus is inevitably associated with formation of harmful reactive oxygen species (ROS). In this review, recent progress on ROS production by photosystem II (PSII) as a response to high light and high temperature is overviewed. Under high light, ROS production is unavoidably associated with energy transfer and electron transport in PSII. Singlet oxygen is produced by the energy transfer form triplet chlorophyll to molecular oxygen formed by the intersystem crossing from singlet chlorophyll in the PSII antennae complex or the recombination of the charge separated radical pair in the PSII reaction center. Apart to triplet chlorophyll, triplet carbonyl formed by lipid peroxidation transfers energy to molecular oxygen forming singlet oxygen. On the PSII electron acceptor side, electron leakage to molecular oxygen forms superoxide anion radical which dismutes to hydrogen peroxide which is reduced by the non-heme iron to hydroxyl radical. On the PSII electron donor side, incomplete water oxidation forms hydrogen peroxide which is reduced by manganese to hydroxyl radical. Under high temperature, dark production of singlet oxygen results from lipid peroxidation initiated by lipoxygenase, whereas incomplete water oxidation forms hydrogen peroxide which is reduced by manganese to hydroxyl radical. The understanding of molecular basis for ROS production by PSII provides new insight into how plants survive under adverse environmental conditions.

The lack of a licensed vaccine for respiratory syncytial virus (RSV) can be partly attributed to regulatory hurdles resulting from vaccine enhanced respiratory disease (ERD) subsequent to natural RSV infection that was observed in clinical trials of formalin-inactivated RSV (FI-RSV) in antigen-naïve infants. To develop an effective vaccine that does not enhance RSV illness, it is important to understand how formalin and heat inactivation affected the antigenicity and immunogenicity of FI-RSV compared to native virus. Informed by atomic structures of RSV fusion (F) glycoprotein in prefusion (pre-F) and postfusion (post-F) conformations, we demonstrate that FI-RSV predominately presents post-F on the virion surface, whereas infectious RSV presents both pre-F and post-F conformations. This significant antigenic distinction has not been previously appreciated. Thus, a stabilized pre-F antigen is more representative of live RSV than F in its post-F conformation, as displayed on the surface of FI-RSV. This finding has major implications for discriminating current pre-F-based immunogens from FI-RSV used in historical vaccine trials.

The gut microbiota is important in the pathogenesis of energy-metabolism related diseases. We focused on the interaction between intestinal bacteria and orally administered chemical drugs. Oral administration of berberine (BBR) effectively treats patients with metabolic disorders. However, because BBR exhibits poor solubility, its absorption mechanism remains unknown. Here, we show that the gut microbiota converts BBR into its absorbable form of dihydroberberine (dhBBR), which has an intestinal absorption rate 5-fold that of BBR in animals. The reduction of BBR to dhBBR was performed by nitroreductases of the gut microbiota. DhBBR was unstable in solution and reverted to BBR in intestine tissues via oxidization. Heat inactivation of intestinal homogenate did not inhibit dhBBR oxidization, suggesting the process a non-enzymatic reaction. The diminution of intestinal bacteria via orally treating KK-Ay mice with antibiotics decreased the BBR-to-dhBBR conversion and blood BBR; accordingly, the lipid- and glucose-lowering efficacy of BBR was reduced. Conclusively, the gut microbiota reduces BBR into its absorbable form of dhBBR, which then oxidizes back to BBR after absorption in intestine tissues and enters the blood. Thus, interaction(s) between the gut microbiota and orally administrated drugs may modify the structure and function of chemicals and be important in drug investigation.

Neutralisation assays are commonly used to assess vaccine-induced and naturally acquired immune responses; identify correlates of protection; and inform important decisions on the screening, development, and use of therapeutic antibodies. Neutralisation assays are useful tools that provide the gold standard for measuring the potency of neutralising antibodies, but they are not without limitations. Common methods such as the heat-inactivation of plasma samples prior to neutralisation assays, or the use of anticoagulants such as EDTA for blood collection, can inactivate the complement system. Even in non-heat-inactivated samples, the levels of complement activity can vary between samples. This can significantly impact the conclusions regarding neutralising antibody potency. Restoration of the complement system in these samples can be achieved using an exogenous source of plasma with preserved complement activity or with purified complement proteins. This can significantly enhance the neutralisation titres for some antibodies depending on characteristics such as antibody isotype and the epitope they bind, enable neutralisation with otherwise non-neutralising antibodies, and demonstrate a better relationship between in vitro and in vivo findings. In this review, we discuss the evidence for complement-mediated enhancement of antibody neutralisation against a range of viruses, explore the potential mechanisms which underpin this enhancement, highlight current gaps in the literature, and provide a brief summary of considerations for adopting this approach in future research applications.

Standard precautions to minimize the risk of SARS-CoV-2 transmission implies that infected cell cultures and clinical specimens may undergo some sort of inactivation to reduce or abolish infectivity. We evaluated three heat inactivation protocols (56 °C-30 min, 60 °C-60 min and 92 °C-15 min) on SARS-CoV-2 using (i) infected cell culture supernatant, (ii) virus-spiked human sera (iii) and nasopharyngeal samples according to the recommendations of the European norm NF EN 14476-A2. Regardless of the protocol and the type of samples, a 4 Log10 TCID50 reduction was observed. However, samples containing viral loads > 6 Log10 TCID50 were still infectious after 56 °C-30 min and 60 °C-60 min, although infectivity was < 10 TCID50. The protocols 56 °C-30 min and 60 °C-60 min had little influence on the RNA copies detection, whereas 92 °C-15 min drastically reduced the limit of detection, which suggests that this protocol should be avoided for inactivation ahead of molecular diagnostics. Lastly, 56 °C-30 min treatment of serum specimens had a negligible influence on the results of IgG detection using a commercial ELISA test, whereas a drastic decrease in neutralizing titers was observed.