Hemoperfusion with CytoSorb as Adjuvant Therapy in Critically Ill Patients with SARS-CoV2 Pneumonia

Background: Several pro- and anti-inflammatory cytokines involved in COVID-19 and it is reasonable to speculate that their removal from blood might limit organ damage. Hemoperfusion with CytoSorb is a technique developed to adsorb molecules in the middle molecular weight range (up to 55 kDa). Studies in vitro and in vivo have shown that HP is highly effective in clearing blood from a number of cytokines. Method(s): We report a case series of 9 consecutive COVID-patients admitted to our COVID Intensive Care Unit (ICU). Five of them were treated with HP using CytoSorb (T), due to the heavy emergency overload it was impossible to deliver blood purification in the other 4 patients (C), who were also considered as potential candidates by the attending medical team. All patients had pneumonia and respiratory failure requiring continuous positive airway pressure. Different antibacterial prophylaxes, antiviral, and anti-inflammatory therapies including steroids were delivered. Result(s): Our results show a better clinical course of T compared to control patients (C), in fact all T except 1 survived, and only 2 of them were intubated, while all C required intubation and died. CRP decreased in both groups, but to a greater extent after HP. Lymphocytopenia worsened in control patient but not in treated patient after HP. Procalcitonin increased in 2 of the not treated patients. In all survived patients (n = 4) HP reduced pro-inflammatory cytokines, as IL-6, TNF-alpha, and IL-8. Notably, a striking effect was observed on IL-6 levels that at the end of the second session were decreased by a 40% than before the first treatment. Serum levels of IL-8 and TNF-alpha were lowered within normal range. In all patients the treatment was safe and there were no complications. Conclusion(s): Our study suggests a potential efficacy of HP in an early phase of viral infection not only for improving survival in the treated patients but also by the remodeling treatment-associated cytokine levels.

Blood purification treatment and cytokine removal using Cytosorb device in critically ill patients with COVID-19 infection

Introduction: Acute kidney injury (AKI) is a complication of SARS-CoV-2 disease, associated with worse clinical outcomes. Renal replacement therapy (RRT) in combination with sequential extracorporeal blood purification therapies (EBPT) might support renal function, attenuate systemic inflammation, and prevent or mitigate multiple organ dysfunctions. Method(s): We retrospectively analyzed 20 patients admitted in ICU for ARDS and who developed moderate-to-severe AKI requiring RRT. Cytokine hemadsorption with Cytosorb was performed in association with CRRT. The main indication for this treatment was the worsening of hemodynamic and respiratory conditions and suspicion of cytokine storm. The protocol consisted in the use of 3-4 cartridges in total;among these, the first 2 were changed after 12 hours of treatment to maximize cytokine removal, while the others after 24 hours. We examined comorbidities, clinical and laboratory characteristics and the impact of treatment in terms of mortality rate and changes in data before and after treatment. Result(s): Nineteen patients (95%) had an AKI at any time during their ICU stay. Of these, 5 patients (25%) had AKI stage II and 14 patients (70%) had AKI stage III. All patients included in this subgroup were mechanical ventilated and required vasopressor's use. Mean prescribed CRRT dose was 31.2 +/- 11.7 ml/kg/h. The median time to strating RRT after ICU admission was 7 days (IQR 3.5-15 days) and the median duration was 7 days (IQR 2.5-12.5 days). Mean SOFA score at the time of RRT start was extremely high (14.5 +/- 2.8). Mortality rate was important (18 patients, 90%) in our cohort. Comparing clinical and laboratory data before and after treatment, a significant improvement of inflammatory markers was reported, with the reduction of C-reactive protein (CRP, 143 [62.1- 328.5] vs 83.5 [66.7-153.5] mg/L);however, no significant changes in IL-6, WBC and PCT values were observed. A slight increase of PaO2/FiO2 were described, although not statistically significant (PaO2/FiO2 ratio 144 [82.7-174.2] vs 183 [132-355.5] mmHg). Conclusion(s): Our experience supports the need of an adequate timing for the use of Cytosorb in critically ill patients with Covid-19. Although a discrete efficacy in improving inflammatory cascade, the late use of EBPT, when organ dysfunction was already ongoing, didn't impact survival.

Production and characterisation of a SARSCoV-2 S-protein RBD homodimer with increased avidity for specific antibodies.

Monitoring of the proportion of immune individuals and the effectiveness of vaccination in a population involves evaluation of several important parameters, including the level of virus-neutralising antibodies. In order to combat the COVID-19 pandemic, it is essential to develop approaches to detecting SARS-CoV-2 neutralising antibodies by safe, simple and rapid methods that do not require live viruses. To develop a test system for enzyme-linked immunosorbent assay (ELISA) that detects potential neutralising antibodies, it is necessary to obtain a highly purified recombinant receptor-binding domain (RBD) of the spike (S) protein with high avidity for specific antibodies. The aim of the study was to obtain and characterise a SARSCoV-2 S-protein RBD homodimer and a recombinant RBD-expressing cell line, as well as to create an ELISA system for detecting potential neutralising antibodies. Material(s) and Method(s): the genetic construct was designed in silico. To generate a stable producer cell line, the authors transfected CHO-S cells, subjected them to antibiotic pressure, and selected the optimal clone. To isolate monomeric and homodimeric RBD forms, the authors purified the recombinant RBD by chromatographic methods. Further, they analysed the activity of the RBD forms by Western blotting, bio-layer interferometry, and indirect ELISA. The analysis involved monoclonal antibodies GamXRH19, GamP2C5, and h6g3, as well as serum samples from volunteers vaccinated with Gam-COVID-Vac (Sputnik V) and unvaccinated ones. Result(s): the authors produced the CHO-S cell line for stable expression of the recombinant SARS-CoV-2 S-protein RBD. The study demonstrated the recombinant RBD's ability to homodimerise after fed-batch cultivation of the cell line for more than 7 days due to the presence of unpaired cysteines. The purified recombinant RBD yield from culture broth was 30-50 mg/L. Monomeric and homodimeric RBD forms were separated using gel-filtration chromatography and characterised by their ability to interact with specific monoclonal antibodies, as well as with serum samples from vaccinated volunteers. The homodimeric recombinant RBD showed increased avidity for both monoclonal and immune sera antibodies. Conclusion(s): the homodimeric recombinant RBD may be more preferable for the analysis of levels of antibodies to the receptor-binding domain of the SARS-CoV-2 S protein.Copyright © 2023 Authors. All rights reserved.

Validating saliva as a biological sample for cost-effective, rapid and routine screening for SARS-CoV-2

Purpose: Compared to nasopharyngeal/oropharyngeal swabs (N/OPS-VTM), non-invasive saliva samples have enormous potential for scalability and routine population screening of SARS-CoV-2. In this study, we investigate the efficacy of saliva samples relative to N/OPS-VTM for use as a direct source for RT-PCR based SARS-CoV-2 detection. Method(s): We collected paired nasopharyngeal/oropharyngeal swabs and saliva samples from suspected positive SARS-CoV-2 patients and tested using RT-PCR. We used generalized linear models to investigate factors that explain result agreement. Further, we used simulations to evaluate the effectiveness of saliva-based screening in restricting the spread of infection in a large campus such as an educational institution. Result(s): We observed a 75.4% agreement between saliva and N/OPS-VTM, that increased drastically to 83% in samples stored for less than three days. Such samples processed within two days of collection showed 74.5% test sensitivity. Our simulations suggest that a test with 75% sensitivity, but high daily capacity can be very effective in limiting the size of infection clusters in a workspace. Guided by these results, we successfully implemented a saliva-based screening in the Bangalore Life Sciences Cluster (BLiSC) campus. Conclusion(s): These results suggest that saliva may be a viable alternate source for SARS-CoV-2 surveillance if samples are processed immediately. Although saliva shows slightly lower sensitivity levels when compared to N/OPS-VTM, saliva collection is logistically advantageous. We strongly recommend the implementation of saliva-based screening strategies for large workplaces and in schools, as well as for population-level screening and routine surveillance as we learn to live with the SARS-CoV-2 virus.Copyright © 2023 Indian Association of Medical Microbiologists

On-Chip Nucleic Acid Purification Followed by ddPCR for SARS-CoV-2 Detection.

We developed a microfluidic chip integrated with nucleic acid purification and droplet-based digital polymerase chain reaction (ddPCR) modules to realize a 'sample-in, result-out' infectious virus diagnosis. The whole process involved pulling magnetic beads through drops in an oil-enclosed environment. The purified nucleic acids were dispensed into microdroplets by a concentric-ring, oil-water-mixing, flow-focusing droplets generator driven under negative pressure conditions. Microdroplets were generated with good uniformity (CV = 5.8%), adjustable diameters (50-200 µm), and controllable flow rates (0-0.3 µL/s). Further verification was provided by quantitative detection of plasmids. We observed a linear correlation of R2 = 0.9998 in the concentration range from 10 to 105 copies/µL. Finally, this chip was applied to quantify the nucleic acid concentrations of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The measured nucleic acid recovery rate of 75 ± 8.8% and detection limit of 10 copies/µL proved its on-chip purification and accurate detection abilities. This chip can potentially be a valuable tool in point-of-care testing.

The Benefits of Nursing Home Air Purification on COVID-19 Outcomes: A Natural Experiment.

OBJECTIVES: Improving indoor air quality is one potential strategy to reduce the transmission of SARS-CoV-2 in any setting, including nursing homes, where staff and residents have been disproportionately and negatively affected by the COVID-19 pandemic. DESIGN: Single group interrupted time series. SETTING AND PARTICIPANTS: A total of 81 nursing homes in a multifacility corporation in Florida, Georgia, North Carolina, and South Carolina that installed ultraviolet air purification in their existing heating, ventilation, and air conditioning systems between July 27, 2020,k and September 10, 2020. METHODS: We linked data on the date ultraviolet air purification systems were installed with the Nursing Home COVID-19 Public Health File (weekly data reported by nursing homes on the number of residents with COVID-19 and COVID-19 deaths), public data on data on nursing home characteristics, county-level COVID-19 cases/deaths, and outside air temperature. We used an interrupted time series design and ordinary least squares regression to compare trends in weekly COVID-19 cases and deaths before and after installation of ultraviolet air purification systems. We controlled for county-level COVID-19 cases, death, and heat index. RESULTS: Compared with pre-installation, weekly COVID-19 cases per 1000 residents (-1.69; 95% CI, -4.32 to 0.95) and the weekly probability of reporting any COVID-19 case (-0.02; 95% CI, -0.04 to 0.00) declined in the post-installation period. We did not find any difference pre- and post-installation in COVID-19-related mortality (0.00; 95% CI, -0.01 to 0.02). CONCLUSIONS AND IMPLICATIONS: Our findings from this small number of nursing homes in the southern United States demonstrate the potential benefits of air purification in nursing homes on COVID-19 outcomes. Intervening on air quality may have a wide impact without placing significant burden on individuals to modify their behavior. We recommend a stronger, experimental design to estimate the causal effect of installing air purification devices on improving COVID-19 outcomes in nursing homes.

A one-enzyme rt-qpcr assay for sars-cov-2, and procedures for reagent production

Given the scale of the ongoing COVID-19 pandemic, the need for reliable, scalable testing, and the likelihood of reagent shortages, especially in resource-poor settings, we have developed an RTqPCR assay that relies on an alternative to conventional viral reverse transcriptases, a thermostable reverse transcriptase/DNA polymerase (RTX) (Ellefson et al., 2016). Here we show that RTX performs comparably to the other assays sanctioned by the CDC and validated in kit format. We demonstrate two modes of RTX use - (i) dye-based RT-qPCR assays that require only RTX polymerase, and (ii) TaqMan RT-qPCR assays that use a combination of RTX and Taq DNA polymerases (as the RTX exonuclease does not degrade a TaqMan probe). We also provide straightforward recipes for the purification of this alternative reagent RTX. We anticipate that in low resource or point-of-need settings researchers could obtain the available constructs and begin to develop their own assays, within whatever regulatory framework exists for them.Copyright © 2021 Bio-protocol LLC. All Rights Reserved.

Efficient Purification of Polyhistidine-Tagged Recombinant Proteins Using Functionalized Corundum Particles.

Immobilized metal affinity chromatography (IMAC) is a popular and valuable method for the affinity purification of polyhistidine-tagged recombinant proteins. However, it often shows practical limitations, which might require cumbersome optimizations, additional polishing, and enrichment steps. Here, we present functionalized corundum particles for the efficient, economical, and fast purification of recombinant proteins in a column-free format. The corundum surface is first derivatized with the amino silane APTES, then EDTA dianhydride, and subsequently loaded with nickel ions. The Kaiser test, well known in solid-phase peptide synthesis, was used to monitor amino silanization and the reaction with EDTA dianhydride. In addition, ICP-MS was performed to quantify the metal-binding capacity. His-tagged protein A/G (PAG), mixed with bovine serum albumin (BSA), was used as a test system. The PAG binding capacity was around 3 mg protein per gram of corundum or 2.4 mg per 1 mL of corundum suspension. Cytoplasm obtained from different E. coli strains was examined as examples of a complex matrix. The imidazole concentration was varied in the loading and washing buffers. As expected, higher imidazole concentrations during loading are usually beneficial when higher purities are desired. Even when higher sample volumes, such as one liter, were used, recombinant protein down to a concentration of 1 µg/mL could be isolated selectively. Comparing the corundum material with standard Ni-NTA agarose beads indicated higher purities of proteins isolated using corundum. His6-MBP-mSA2, a fusion protein consisting of monomeric streptavidin and maltose-binding protein in the cytoplasm of E. coli, was purified successfully. To show that this method is also suitable for mammalian cell culture supernatants, purification of the SARS-CoV-2-S-RBD-His8 expressed in human Expi293F cells was performed. The material cost of the nickel-loaded corundum material (without regeneration) is estimated to be less than 30 cents for 1 g of functionalized support or 10 cents per milligram of isolated protein. Another advantage of the novel system is the corundum particles' extremely high physical and chemical stability. The new material should be applicable in small laboratories and large-scale industrial applications. In summary, we could show that this new material is an efficient, robust, and cost-effective purification platform for the purification of His-tagged proteins, even in challenging, complex matrices and large sample volumes of low product concentration.

Circulation of respiratory viruses during the COVID-19 pandemic in The Gambia

Background: In many countries, non-pharmaceutical interventions to limit severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission resulted in significant reductions in other respiratory viruses. However, similar data from Africa are limited. We explored the extent to which viruses such as influenza and rhinovirus co-circulated with SARS-CoV-2 in The Gambia during the COVID-19 pandemic. Methods: Between April 2020 and March 2022, respiratory viruses were detected using RT-PCR in nasopharyngeal swabs from 1397 participants with influenza-like illness. An assay to detect SARS-CoV-2 and a viral multiplex RT-PCR assay was used as previously described to detect influenza A and B, respiratory syncytial virus (RSV) A and B, parainfluenza viruses 1-4, human metapneumovirus (HMPV), adenovirus, seasonal coronaviruses (229E, OC43, NL63) and human rhinovirus. Result(s): Overall virus positivity was 44.2%, with prevalence higher in children <5 years (80%) compared to children aged 5-17 years (53.1%), adults aged 18-50 (39.5%) and >50 years (39.9%), p<0.0001. After SARS-CoV-2 (18.3%), rhinoviruses (10.5%) and influenza viruses (5.5%) were the most prevalent. SARS-CoV-2 positivity was lower in children <5 (4.3%) and 5-17 years (12.7%) than in adults aged 18-50 (19.3%) and >50 years (24.3%), p<0.0001. In contrast, rhinoviruses were most prevalent in children <5 years (28.7%), followed by children aged 5-17 (15.8%), adults aged 18-50 (8.3%) and >50 years (6.3%), p<0.0001. Four SARS-CoV-2 waves occurred, with 36.1%-52.4% SARS-CoV-2 positivity during peak months. Influenza infections were observed in both 2020 and 2021 during the rainy season as expected (peak positivity 16.4%-23.5%). Peaks of rhinovirus were asynchronous to the months when SARS-CoV-2 and influenza peaked. Conclusion(s): Our data show that many respiratory viruses continued to circulate during the COVID-19 pandemic in The Gambia, including human rhinoviruses, despite the presence of NPIs during the early stages of the pandemic, and influenza peaks during expected months.Copyright: © 2023 Jarju S et al.

SARS-CoV-2 VARIANTS EVOLVED CONVERGENT STRATEGIES TO REMODEL THE HOST RESPONSE

Background: Five variants of concern (VOCs) have dominated COVID-19 disease etiology since 2020-Alpha, Beta, Gamma, Delta, and Omicron-possessing over 150 defining genomic alterations. Here, we used global proteomic and genomic approaches to study the host responses and selective forces driving VOC evolution. Method(s): We infected Calu-3 human lung epithelial cells with 5 VOCs and 2 wave 1 (W1) controls and performed mass spectrometry abundance proteomics, phosphoproteomics, and mRNA sequencing at 10 and 24 hours post infection. We additionally performed affinity purification mass spectrometry (APMS) by individually expressing all VOC mutant viral proteins (52) and corresponding W1 forms in human cells to quantify differential virus-host protein-protein interactions. Data was integrated using network modeling and bioinformatics to pinpoint VOC-specific differences. Four novel mutant viruses were developed using reverse genetics technology to validate the impact of specific genomic alterations. Result(s): We discovered VOCs evolved convergent molecular strategies to remodel the host response by modulating viral RNA and protein levels (most notably of N, Orf9b, and Orf6), altering nucleocapsid phosphorylation, and rewiring virus-host protein complexes. Integrative systems analyses revealed that Alpha, Beta, Gamma, and Delta ultimately converged in the suppression of interferon stimulated genes (ISGs) relative to W1 viruses, but Omicron BA.1 did not, and Delta induced more pro-inflammatory genes compared to other VOCs. Altered regulation of ISGs correlated with the expression of viral innate immune antagonist proteins, including Orf6, N, and Orf9b;for example, Omicron BA.1 depicted a 2-fold decrease in Orf6 expression. We identified mutations that alter expression of Orf9b (N D3L and N -3A del) and the novel VOC protein N* (N R203K/G204R), and confirmed Orf6 innate immune antagonism using recombinant virus technology. Remarkably, Omicron BA.4 and BA.5 regained strengthened innate immune antagonism compared to BA.1, which again correlated with enhanced Orf6 expression, though dampened in BA.4 by a mutation (D61L) that we discovered disrupts the Orf6-nuclear pore interaction. Conclusion(s): Collectively, our findings suggest SARS-CoV-2 convergent evolution overcomes human innate immune barriers, laying the groundwork to understand future coronavirus evolution associated with immune escape and enhanced human-to-human transmission.

Evaluation of low-cost SARS-CoV-2 RNA purification methods for viral quantification by RT-qPCR and next-generation sequencing analysis: Implications for wider wastewater-based epidemiology adoption.

Based Epidemiology (WBE) consists of quantifying biomarkers in sewerage systems to derive real-time information on the health and/or lifestyle of the contributing population. WBE usefulness was vastly demonstrated in the context of the COVID-19 pandemic. Many methods for SARS-CoV-2 RNA determination in wastewater were devised, which vary in cost, infrastructure requirements and sensitivity. For most developing countries, implementing WBE for viral outbreaks, such as that of SARS-CoV-2, proved challenging due to budget, reagent availability and infrastructure constraints. In this study, we assessed low-cost methods for SARS-CoV-2 RNA quantification by RT-qPCR, and performed variant identification by NGS in wastewater samples. Results showed that the effect of adjusting pH to 4 and/or adding MgCl2 (25 mM) was negligible when using the adsorption-elution method, as well as basal physicochemical parameters in the sample. In addition, results supported the standardized use of linear rather than plasmid DNA for a more accurate viral RT-qPCR estimation. The modified TRIzol-based purification method in this study yielded comparable RT-qPCR estimation to a column-based approach, but provided better NGS results, suggesting that column-based purification for viral analysis should be revised. Overall, this work provides evaluation of a robust, sensitive and cost-effective method for SARS-CoV-2 RNA analysis that could be implemented for other viruses, for a wider WEB adoption.

Experimental study of the purification performance of a MopFan-based photocatalytic air cleaning system.

Severe acute respiratory syndrome coronavirus (SARS-CoV)-2, the virus that causes the coronavirus disease (COVID)-19, is primarily transmitted through respiratory droplets which linger in enclosed spaces, often exacerbated by HVAC systems. Although research to improve HVAC handling of SARS-CoV-2 is progressing, currently installed HVAC systems cause problems because they recirculate air and use ineffective filters against virus. This paper details the process of developing a novel method of eliminating air pollutants and suspended pathogens in enclosed spaces using Photocatalytic Oxidation (PCO) technology. It has been previously employed to remove organic contaminants and compounds from air streams using the irradiation of titanium dioxide (TiO2) surfaces with ultraviolet (UV) lights causing the disintegration of organic compounds by reactions with oxygen (O) and hydroxyl radicals (OH). The outcome was two functional prototypes that demonstrate the operation of PCO-based air purification principle. These prototypes comprise a novel TiO2 coated fibre mop system, which provide very large surface area for UV irradiation. Four commercially accessible materials were used for the construction of the mop: Tampico, Brass, Coco, and Natural synthetic. Two types of UV lights were used: 365 nm (UVA) and 270 nm (UVC). A series of tests were conducted that proved the prototype's functionality and its efficiency in lowering volatile organic compounds (VOCs) and formaldehyde (HCHO). The results shown that a MopFan with rotary mop constructed with Coco fibres and utilising UVC light achieves the best VOC and HCHO purification performance. Within 2 h, this combination lowered HCHO by 50% and VOCs by 23% approximately.

Development of Equipment for Air Decontamination in the Ventilation and Air Conditioning Systems of Public Buildings with the Use of the Photocatalysis and Plasmochemistry Methods

Introduction. Seasonal waves of SARS outbreaks, including COVID-19, necessitate the development of measures to create health-safe conditions in crowded places.Problem Statement. The existing supply and exhaust systems of the centralized heating, ventilation and air conditioning (HVAC) do not protect against infection, moreover, they serve as a source for the accumulation and spread of pathogenic microorganisms. Finding effective ways to clean the air in places of mass gathering of people as a component of anti-epidemic measures is an urgent task. Purpose. The purpose of this research is to develop and create equipment for cleaning and disinfecting air from airborne pathogenic microflora in the HVAC systems, which can be installed in the centralized ventilation systems of buildings without their reconstruction and modifications in technological parameters. Material and Methods. A complex of physical and chemical methods, which includes analytical and experimental techniques with the use of the theory of electrogas dynamics of dispersed systems and the raster scanning microscopy methods, and the methods for comparing the same quality indicators of specimens and initial samples have been used.Results. To study the efficiency of both the individual plasma-chemical , photocatalytic modules, as well as the equipment as a whole under the operating conditions that simulate those of the centralized ventilation system, an experimental stand has been created. The optimal technological parameters of the processes for raising the efficiency of air disinfection and purification in the HVAC systems by the plasma photocatalysis methods have been determined. Technical solutions for increasing the energy efficiency of the experimental stand for the complex air purification and disinfection from a wide class of air pollutants in the supply and exhaust ventilation systems of buildings have been proposed.ent, as well as to determine the required level of innova-tion factor by maximizing the hidden innovation capacity.Conclusions. Air disinfection by the method of combined plasma-photocatalytic effect on the air flow with a system for catalytic-thermal decomposition of excess ozone ensures effectively removing pollutants and allows reducing the microbiological contamination of the air to a safe level.

Ergonomics-oriented operation, maintenance and control of indoor air environment for public buildings

In response to the construction process of Healthy China. it is rather important to create a safe, healthy and energy-efficient indoor environment for public buildings. The public building space is often densely populated, with a large flow of people and many types of air pollution, which presents non-uniform dynamic distribution characteristics. This brings great challenges to the control of indoor air safety, especially during the pandemic period of COVID-19. Excessive ventilation may not only cause large energy waste. but also lead to cross-contamination and even a cluster of infection. In this paper, an operation and maintenance (O&M) control system for indoor air safety is developed based on the core concepts and basic methods of human ergonomics. In this system, one of the important human environmental variables is focused for control, i.e.. indoor air pollution level. Especially after the outbreak of COVID-19. droplets and droplet nuclei from respiration are the most significant air pollution categories required for mitigation. Towards the efficient control of air pollution in large public buildings. it should further take into account the interaction of human, equipment and machines (i.e., ventilation_ air purification and disinfection and intelligent control system) and building environment. Firstly, on the basis of the online monitoring of indoor air pollution concentration and personnel flow, the non-uniform dynamic distribution of indoor pollutants and personnel can be obtained by using the non-uniform and low-dimensional rapid prediction models and computer vision processing. Then, the optimal setting results of ventilation parameters (e.g., ventilation modes, supply air rate. etc.) can be outputted by the environmental control decision system. Finally, based on a combination of monitoring sensors, controllers and actuator hardware equipment (at the location of fans or dampers), the intelligent regulation and control of ventilation system can be realized, aimed at minimizing energy consumption and reducing pollutant concentration and exposure level. Meanwhile, the air purification and disinfection system (especially for the disinfection of virus particles) are operated under the condition of the ventilated environment, which can serve as a powerful auxiliary to the maintenance of indoor air safety. The workflow and effect of the O&M control system are demonstrated by an engineering application case of the front hall in the International Convention and Exhibition Center. The results indicate that the non-uniform and low-dimensional rapid prediction model for pollutant concentration is effective for the ventilation control with the average prediction difference of 11.9%. The implementation of the intelligent ventilation system can reduce the risk of human infection to less than 4%. and its energy-saving ratio for the ventilation can be as high as about 45%. Through optimizing the layout strategies of disinfection devices based on the intelligent ventilation control, the space accessibility of negative oxygen ions can be well accepted, to further increase the removal efficiency of air pollution. The calculated value of space disinfection rate is more than 99%, which can further reduce the risk of infection by 1-2 orders of magnitude. This study can provide an important reference for the promotion and upgrading of O&M control system for indoor air safety.

The Role of Separation Techniques in the Analysis of mRNA Therapeutic Drug Substances and Drug Products

Messenger ribonucleic acids (mRNA) therapeutics are becoming more widespread pharmaceutical tools to treat a wide range of diseases or infections, as highlighted by regulatory approval of two vaccines for SARS-CoV-2. Alongside their use as vaccines, they also play a role in protein replacement therapy to ensure therapeutic protein is synthesized within the patient. Structural elements, such as the 5' cap, UTR regions, reading frame, and poly A tail are considered as critical quality attributes (CQAs) that are subject to a range of analytical techniques. However, chromatography and other separation methods are commonly used for characterization and quantification of the drug substance and drug product. This article reviews a range of techniques available for separative analysis of mRNA therapeutics, their associated impurities, and delivery vehicles.

Year 2022 in review - Critical care nephrology.

The intensive care medicine literature continued to resonate with the fading COVID-19 pandemic in 2022. Nevertheless, several interesting scientific publications have appeared in the field of critical care nephrology, the contents of which are worth mentioning. This summary article aims to guide the reader through the most fundamental aspects of nephrotoxicity, acute kidney injury, and extracorporeal purification methods.Copyright © 2022, Czech Medical Association J.E. Purkyne. All rights reserved.

Airborne SARS-CoV- 2 detection in outdoor air

Background: The potential aerosol spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV- 2) has been suggested. While indoor air sampling for SARS-CoV- 2 has demonstrated detectable viral RNA and has been related to virus transmission, the contribution of outdoor air to the spread of the viral infection is not yet known. We aimed at developing a methodology to detect the virus in outdoor air. Method(s): T he s ampling w as p erformed u sing a C HEMVOL v olumetric impactor (Butraco) equipped with 2 stages (PM > 10 & 2.5 > PM > 10um). Filters were collected and preserved at -80 degreeC. Total RNA extraction was performed directly from the collected filters with the Phenol-Chloroform method using TRItidy GTM reagent according to the manufacturer's instructions. For total RNA purification samples were purified with the commercial kit E.Z.N.A. Total RNA Kit I. Real-Time Reverse Transcription PCR was executed to detect the N gene from the Sarbecovirus family and RdRp gene from SARS-CoV- 2 using the ViroReal Kit SARS-CoV- 2 Multiplex. A protein-rich fraction was obtained with ammonium bicarbonate buffer extraction followed by lyophilization. SARS-CoV- 2 spike protein was assessed by specific immunological detection (SARS-CoV- 2 Antigen Test Kit). Result(s): RT-PCR for N gene results, identifying Sarbecovirus family, were positive and Cq > 33, in the samples from the last week of December 2020 and the first and second weeks of January 2021, in both PM > 10 and 2.5 > PM > 10. The RdRp gene was undetectable, probably due to low virus concentration. The protein samples from the same days tested positive for the specific antigen spike protein. All results combined confirm the detection of SARS-CoV- 2 in outdoor air. Conclusion(s): Airborne SARS-CoV- 2 was detected in ambient air. These results will contribute to an early detection of SARS-Cov- 2 in ambient air, thus eventually providing the base for early alert systems allowing the implementation of preventive measures to control outbreaks.

Production of SARS-CoV-2 nucleocapsid protein in Escherichia coli and its characterization

SARS-CoV-2 encodes a nucleocapsid protein that binds to the single-stranded RNA genome present inside the viral particles. It is one of the predominant proteins expressed during infection and tends to be highly immunogenic in infected individuals. Consequently, it has been recognized as an important diagnostic marker for the development of antigen or antibody detection-based diagnostic platforms for COVID-19. In this work, we have described cloning, autoinduction-based expression, and purification of full-length SARS-CoV-2 nucleocapsid protein using Escherichia coli as the heterologous host. We have characterized the purified protein using commercial rapid antigen test kits and indirect ELISA to demonstrate its suitability as bait for diagnostic assays. The purified recombinant protein reported in this study can be useful for numerous applications including the development of monoclonal or polyclonal antibodies, studying protein structure and function, and mapping B-and T-cell epitopes for designing suitable vaccines and therapeutics. © 2023 Verma and Abhishek.

Antioxidant and Anti-Breast Cancer Properties of Hyaluronidase from Marine Staphylococcus aureus (CASMTK1)

This work studied the antioxidant and anti-breast cancer properties of hyaluronidase, extracted from a potential marine strain, Staphylococcus aureus (CASMTK1), isolated from Parangipettai coastal waters in southeast coast of India. The Staphylococcal enzyme production was tested under different carbon and nitrogen sources;and recorded the maximum production when the microbial strain was cultured with starch as the carbon source and ammonium sulphate as the inorganic nitrogen source with the enzyme production of 92.5 U/mL and 95.0 U/mL, respectively. The hyaluronidase enzyme production was also tested in different pH and temperature;and recorded the maximum yield of 102.5 U/mL in pH 5 and that of 95.5 U/mL in 45 °C. The partially purified enzyme was subjected to FTIR and FT Raman technique and found the presence of the amide- I and II, Carboxyl, N-H bending, C-H stretching and α-helices and β-sheet proteins between wave number 1500–1700 cm−1. The partially purified enzyme also exhibited strong antioxidant and in-vitro breast cancer properties. The enzyme showed the highest hydroxyl radical scavenging activity of 79% at the 50 µg/mL concentration, and this activity increased in a dose-dependent manner. The enzyme inhibited proliferation of the breast cancer cell line of MCF-7, and it caused 100% cell death at the concentration of 80 µg/mL. The enzyme generated capacity of producing free radicles that damage the cancer cells, and this effect was very nearer to the standard drug, paclitaxel. The enzyme damaged the cancer cells and induced apoptosis in 78% of cancer cells as evident by condensed or fragmented chromatin at 40 µg/mL. Further purification of the enzyme, analysis of its molecular aspects, and elucidation of exact mechanisms of its biological activities will throw new light on the utility of staphylococcal hyaluronidase in anticancer chemotherapy.