Shaping Nanobodies and Intrabodies against Proteoforms.

Proteoforms expand genomic diversity and direct developmental processes. While high-resolution mass spectrometry has accelerated characterization of proteoforms, molecular techniques working to bind and disrupt the function of specific proteoforms have lagged behind. In this study, we worked to develop intrabodies capable of binding specific proteoforms. We employed a synthetic camelid nanobody library expressed in yeast to identify nanobody binders of different SARS-CoV-2 receptor binding domain (RBD) proteoforms. Importantly, employment of the positive and negative selection mechanisms inherent to the synthetic system allowed for amplification of nanobody-expressing yeast that bind to the original (Wuhan strain RBD) but not the E484 K (Beta variant) mutation. Nanobodies raised against specific RBD proteoforms were validated by yeast-2-hybrid analysis and sequence comparisons. These results provide a framework for development of nanobodies and intrabodies that target proteoforms.

Upper-Room Germicidal Ultraviolet (UR-GUV) Application in High-Ceiling Indoor Settings for Disinfection of Airborne Viruses

This study investigated upper-room germicidal ultraviolet (UR-GUV) light application in a music rehearsal room with a high ceiling (7.5 m). The focus was on the influences of the elevation and height of UV zone on disinfection of airborne viruses. This study assumed a uniform UV fluence rate of 0.2 W/m2 in the UV irradiation zone. According to the Computational Fluid Dynamics (CFD) results, average viral concentrations (Ca), fraction remaining (FR), and equivalent air exchange rate (λe) attributed to GUV, have power relationships with UV zone height. Ca and FR decreased with UV zone height, while λe did the opposite. UV zone elevation showed little influence on UR-GUV performance, indicating well-mixed air in the rehearsal room. High ceiling makes it possible to achieve adequate UV dose by increasing both UV zone height and UV light intensity. Using open fixtures improved energy efficiency and reduced operational costs of the UR-GUV system. © 2022 17th International Conference on Indoor Air Quality and Climate, INDOOR AIR 2022. All rights reserved.

Multi-omics of NET formation and correlations with CNDP1, PSPB, and L-cystine levels in severe and mild COVID-19 infections

The detailed mechanisms of COVID-19 infection pathology remain poorly understood. To improve our understanding of SARS-CoV-2 pathology, we performed a multi-omics and correlative analysis of an immunologically naïve SARS-CoV-2 clinical cohort from blood plasma of uninfected controls, mild, and severe infections. Consistent with previous observations, severe patient populations showed an elevation of pulmonary surfactant levels. Intriguingly, mild patients showed a statistically significant elevation in the carnosine dipeptidase modifying enzyme (CNDP1). Mild and severe patient populations showed a strong elevation in the metabolite L-cystine (oxidized form of the amino acid cysteine) and enzymes with roles in glutathione metabolism. Neutrophil extracellular traps (NETs) were observed in both mild and severe populations, and NET formation was higher in severe vs. mild samples. Our correlative analysis suggests a potential protective role for CNDP1 in suppressing PSPB release from the pulmonary space whereas NET formation correlates with increased PSPB levels and disease severity. In our discussion we put forward a possible model where NET formation drives pulmonary occlusions and CNDP1 promotes antioxidation, pleiotropic immune responses, and vasodilation by accelerating histamine synthesis.

Semi-virtual patch testing: a review of the clinical impact, practicalities, patient experience and environmental impact in a tertiary dermatology centre

The COVID-19 pandemic has influenced the way patch testing is carried out at our tertiary centre. From July 2020 we introduced 'semi-virtual patch testing', where suitable patients remove patches at home on day 2 (D2) and take photos, which were reviewed during the D4 face-to-face appointment. Our aim was to assess the continued efficacy and practicality of this service, as well as the photo quality, patient satisfaction and environmental impact of the changes. Data were collected from patients attending patch testing at the Bristol Royal Infirmary from August to December 2021. Patients/carers were asked at their D4 clinic appointment to complete a pre-prepared questionnaire, with their consent, regarding their patch test experience. A clinician (consultant or clinical fellow) concurrently filled out a questionnaire on image quality. These data were collated with data obtained via telephone questionnaires (July-November 2020). Data were available for 181 patients (age range 0-89 years, 74.8% female and 25.2% male). Ninety-seven per cent (n = 135/139) were happy to have patch testing during the pandemic. Ninety-three per cent (n = 130/140) removed their patches at home on D2. Only 3.9% (n = 5/129) would have preferred to attend hospital. Information was provided by multiple sources: verbally by a nurse (95.2%), a doctor (60.7%), a written information sheet (88.1%) and a video (29.8%). Almost all patients (n = 127/ 136) felt confident/fairly confident removing the patches at home, and 85.0% (n = 108/127) reported it saved > 1 h. Seventy-four per cent (n = 95/129) would have had to take time off work/caring commitments to attend. Almost all patients (n = 131/135) reported that their overall experience was good or very good. Seventy per cent (n = 59/84) of patients travelled independently to their appointment, and 79.8% (n = 67/84) came by car/taxi. The mean mileage (one way) to attend the appointment was 13.3 (range 0.2-46.5;n = 98). Ninety-seven percent (n = 89/92) used a smartphone device for D2 photos. The overall presentation of the D2 reading was clear in 91.2% (n = 83/91), adequate in 6.6% (n = 6/91) and inadequate in 2.2% (n = 2/91). Image focus and colour exposure was good/adequate in 97.8% (n = 90/92) and 97.2% (n = 70/72), respectively. Virtual reading negatively affected the final interpretation of the patch test in 2.2% (n = 2/89) of patients and 9.4% (n = 16/170) would have had additional tests added on D2 if they had attended, the results of which were followed-up virtually. Our review of semi-virtual patch testing in our centre demonstrates this method as being an excellent option for most patients, with very limited adverse clinical impact. There were high levels of patient satisfaction, a reduced need for time away from work/home and a reduction in travel required. The vast majority of patients can competently remove their patch tests and produce high-quality images with good focus and colour exposure, mostly using smartphone technology.

Multi-omics of NET formation and correlations with CNDP1, PSPB, and L-cystine levels in severe and mild COVID-19 infections.

The detailed mechanisms of COVID-19 infection pathology remain poorly understood. To improve our understanding of SARS-CoV-2 pathology, we performed a multi-omics and correlative analysis of an immunologically naïve SARS-CoV-2 clinical cohort from blood plasma of uninfected controls, mild, and severe infections. Consistent with previous observations, severe patient populations showed an elevation of pulmonary surfactant levels. Intriguingly, mild patients showed a statistically significant elevation in the carnosine dipeptidase modifying enzyme (CNDP1). Mild and severe patient populations showed a strong elevation in the metabolite L-cystine (oxidized form of the amino acid cysteine) and enzymes with roles in glutathione metabolism. Neutrophil extracellular traps (NETs) were observed in both mild and severe populations, and NET formation was higher in severe vs. mild samples. Our correlative analysis suggests a potential protective role for CNDP1 in suppressing PSPB release from the pulmonary space whereas NET formation correlates with increased PSPB levels and disease severity. In our discussion we put forward a possible model where NET formation drives pulmonary occlusions and CNDP1 promotes antioxidation, pleiotropic immune responses, and vasodilation by accelerating histamine synthesis.

Students' Learning Interests During the Covid-19 Pandemic Period of Google Meet Application Implementation

This research explores the impact of the Google Meet application on learners' motivation to participate in the institution using two parallel classrooms: one for experimental treatments and the other as a control class. Both courses are identical in every way except for the use of Google Meet. The experimental class studied via the Google Meet program and differed dramatically from the control class, which was taught straight from the internet (face to face). There is an average gap of 101.79 between the experimental class's 100 and 92 interest scores, which is the widest and the narrowest. For the control group, the highest score was 95 (88.65) and the lowest score was 70. According to these findings, using Google Meet as a learning tool has a significant influence on students' motivation to study. Since it is so simple to set up, it may be used whenever and wherever a student needs it to be. Copyright © 2022, Anka Publishers. All rights reserved.

Early Prediction of COVID-19 Patient Survival by Targeted Plasma Multi-Omics and Machine Learning.

The recent surge of coronavirus disease 2019 (COVID-19) hospitalizations severely challenges healthcare systems around the globe and has increased the demand for reliable tests predictive of disease severity and mortality. Using multiplexed targeted mass spectrometry assays on a robust triple quadrupole MS setup which is available in many clinical laboratories, we determined the precise concentrations of hundreds of proteins and metabolites in plasma from hospitalized COVID-19 patients. We observed a clear distinction between COVID-19 patients and controls and, strikingly, a significant difference between survivors and nonsurvivors. With increasing length of hospitalization, the survivors' samples showed a trend toward normal concentrations, indicating a potential sensitive readout of treatment success. Building a machine learning multi-omic model that considers the concentrations of 10 proteins and five metabolites, we could predict patient survival with 92% accuracy (area under the receiver operating characteristic curve: 0.97) on the day of hospitalization. Hence, our standardized assays represent a unique opportunity for the early stratification of hospitalized COVID-19 patients.

Semi-virtual patch testing -a review of the clinical impact, practicalities, patient experience and environmental impact in a tertiary dermatology centre

Background: The COVID-19 pandemic has contributed to changes in NHS services. From July 2020 we introduced semi-virtual patch testing, where suitable patients remove patches at home on day 2 (D2) and take photos, which are reviewed during the D4 face to face appointment. Objectives: Our aim was to assess the continued efficacy and practicality of this service, as well as the photo quality, patient satisfaction and environmental impact. Methods: Questionnaire data were collected from 181 patients/ carers attending their patch testing from July 2020 -December 2021. A clinician (consultant/ clinical fellow) concurrently completed a questionnaire on photo quality. Results: Data were available for 181 patients aged 0-89, 74.8% were females and 25.2% were male. 92.9% (130/140) removed patches at home on D2. Only 3.9% (5/129) would have preferred to attend hospital. Almost all patients (127/136) felt fairly confident/ confident removing the patches at home and reported good overall experience (131/135). 85.0% (108/127) reported saving >1 hour. 73.6% (95/129) would have had to take time off work/ caring commitments to attend on D2. 79.8% (67/84) came by car/ taxi. The mean one-way mileage was 13.3 (range 0.2-46.5). 96.7% (89/92) used a smart phone device for D2 photos. Overall presentation of the D2 reading was clear/ adequate in 97.8% (89/91). Conclusions: This method is an excellent option for most patients. There were high levels of patient satisfaction, reduced travel, and reduced work absence. Most patients can produce high quality images with good focus and colour exposure via smart phones.

Online Hydrophilic Interaction Chromatography (HILIC) Enhanced Top-Down Mass Spectrometry Characterization of the SARS-CoV-2 Spike Receptor-Binding Domain.

SARS-CoV-2 cellular infection is mediated by the heavily glycosylated spike protein. Recombinant versions of the spike protein and the receptor-binding domain (RBD) are necessary for seropositivity assays and can potentially serve as vaccines against viral infection. RBD plays key roles in the spike protein's structure and function, and thus, comprehensive characterization of recombinant RBD is critically important for biopharmaceutical applications. Liquid chromatography coupled to mass spectrometry has been widely used to characterize post-translational modifications in proteins, including glycosylation. Most studies of RBDs were performed at the proteolytic peptide (bottom-up proteomics) or released glycan level because of the technical challenges in resolving highly heterogeneous glycans at the intact protein level. Herein, we evaluated several online separation techniques: (1) C2 reverse-phase liquid chromatography (RPLC), (2) capillary zone electrophoresis (CZE), and (3) acrylamide-based monolithic hydrophilic interaction chromatography (HILIC) to separate intact recombinant RBDs with varying combinations of glycosylations (glycoforms) for top-down mass spectrometry (MS). Within the conditions we explored, the HILIC method was superior to RPLC and CZE at separating RBD glycoforms, which differ significantly in neutral glycan groups. In addition, our top-down analysis readily captured unexpected modifications (e.g., cysteinylation and N-terminal sequence variation) and low abundance, heavily glycosylated proteoforms that may be missed by using glycopeptide data alone. The HILIC top-down MS platform holds great potential in resolving heterogeneous glycoproteins for facile comparison of biosimilars in quality control applications.

Multi-omics Characterization of Neutrophil Extracellular Trap Formation in Severe and Mild COVID-19 Infections (preprint)

The detailed mechanisms of COVID-19 infection pathology remain poorly understood. To improve our understanding of SARS-CoV-2 pathology, we performed a multi-omics analysis of an immunologically naive SARS-CoV-2 clinical cohort from the plasma of uninfected controls, mild, and severe infections. A comparison of healthy controls and patient samples showed activation of neutrophil degranulation pathways and formation of neutrophil extracellular trap (NET) complexes that were activated in a subset of the mild infections and more prevalent in severe infections (containing multiple NET proteins in individual patient samples). As a potential mechanism to suppress NET formation, multiple redox enzymes were elevated in the mild and severe symptom population. Analysis of metabolites from the same cohort showed a 24- and 60-fold elevation in plasma L-cystine, the oxidized form of cysteine, which is a substrate of the powerful antioxidant glutathione, in mild and severe patients, respectively. Unique to patients with mild infections, the carnosine dipeptidase modifying enzyme (CNDP1) was up-regulated. The strong protein and metabolite oxidation signatures suggest multiple compensatory pathways working to suppress oxidation and NET formation in SARS-CoV-2 infections.

Contrail coverage over the United States before and during the COVID-19 pandemic

Contrails are potentially the largest contributor to aviation-attributable climate change, but estimates of their coverage are highly uncertain. No study has provided observation-based continental-scale estimates of the diurnal, seasonal, and regional variability in contrail coverage. We present contrail coverage estimates for the years 2018, 2019 and 2020 for the contiguous United States, derived by developing and applying a deep learning algorithm to over 100 000 satellite images. We estimate that contrails covered an area the size of Massachusetts and Connecticut combined in the years 2018 and 2019. Comparing 2019 and 2020, we quantify a 35.8% reduction in distance flown above 8 km altitude and an associated reduction in contrail coverage of 22.3%. We also find that the diurnal pattern in contrail coverage aligns with that of flight traffic, but that the amount of contrail coverage per distance flown decreases in the afternoon.

Allergic reactions to coronavirus disease 2019 vaccines and addressing vaccine hesitancy: Northwell Health experience.

BACKGROUND: Allergic and nonallergic adverse reactions have been reported with global coronavirus disease 2019 (COVID-19) vaccination. It was previously hypothesized that polyethylene glycol (PEG) may be responsible for anaphylactic reactions to messenger RNA (mRNA) COVID-19 vaccines. OBJECTIVE: To report the workflow established at our institution, types, and frequency of adverse reactions to mRNA COVID-19 vaccines in patients presenting for allergy evaluation. METHODS: A COVID-19 vaccine adverse reaction registry was established. We used PEG prick skin testing, followed by PEG challenges in selected cases, to ensure PEG tolerance and encourage completion of COVID-19 vaccination series. RESULTS: A total of 113 patients were included. Most vaccine reactions (86.7%) occurred in women. Anaphylaxis occurred only in women, all of which had a history of allergic disease and two-thirds had asthma. Anaphylaxis rate was 40.6 cases per million. None of the anaphylactic cases developed hypotension, required intubation, or required hospital admission. Systemic allergic symptoms, not fulfilling anaphylaxis criteria, were significantly more common in Pfizer-BioNTech than Moderna-vaccinated patients (P = .02). We observed a higher incidence of dermatologic nonurticarial reactions in men (P = .004). Among first-dose reactors, 86.7% received and tolerated the second dose. We observed a high rate of false-positive intradermal skin test results and frequent subjective symptoms with oral PEG challenge. CONCLUSION: Intradermal PEG testing has limited utility in evaluating anaphylaxis to mRNA vaccines. Most severe postvaccination allergic symptoms are not caused by hypersensitivity to PEG. Most people with reaction to the initial mRNA vaccine can be safely revaccinated. Patients with anaphylaxis to COVID-19 vaccines benefit from physician-observed vaccination.

Capitalizing on Missed Opportunities for Sexual Health Workforce Development by Adoption of a Sexual Health Paradigm

STIs remain a major cause of morbidity, dis-proportionately affecting younger persons and having lifelong consequences.1,2 Reportable STI rates have increased since 1997, and the latest data reflectan all-time high.3 Estimates suggest that approximately one in five people in the United States had an STI on any given day in 20184 Although the STI burden is increasing across all population groups, adolescents and young adults, women, men who have sex with men, and other groups underserved by mainstream health and public health systems remain disproportionately affected. [...]the dire need for increased public health attention and resources for addressing the "hidden epidemic" of STIs persists today.1,2 The lack of progress in STI prevention and control is owing to longstanding underinvestment in the broader public health system and its workforce, as highlighted during the COVID-19 pandemic. [...]recognition of sexual health as an integral component of broader health and well-being creates opportunities for using additional resources and partnerships (e.g., in education, family services, community health) to supplement STI-specific funding and infrastructure and a STIspecific workforce. [...]additional practitioners across clinical health care and public health, most notably primary care providers, can be used if a well-being-focused sexual health paradigm is adopted and applied to workforce development. [...]the committee identifies a wide range of professionals and stakeholders as part of the sexual health workforce. The committee therefore recommends that clinical practice guidelines and training curricula for health care generalists define a minimum set of sexual health competencies, more heavily emphasizing the importance of the consistent delivery of recommended sexual health services, such as sexual histories, STI screening, and vaccination.2 As first-line providers trained to deliver most aspects of sexual health promotion, STI prevention, and STI management and as the largest segment of the health care workforce, nurses are particularly well positioned to increase the reach of sexual health services.2 The committee encourages a broader scope of nursing practice in sexual health services as meaningful for strengthening the sexual health workforce and reducing STI disparities- guidance aligned with the vision outlined in the National Academy of Medicine's The Future of Nursing 2020-2030 report to use nurses for addressing social determinants of health and population health in the United States.8 Given that about 90% of the US population lives within two miles of a community pharmacy, pharmacists can serve as convenient entry points into the health care system, including for sexual health services.2 The committee therefore highlights the utility of incorporating pharmacists into the sexual health workforce, particularly for STI testing using point-of-care tests.

Using Wastewater Surveillance Data to Support the COVID-19 Response - United States, 2020-2021.

Wastewater surveillance, the measurement of pathogen levels in wastewater, is used to evaluate community-level infection trends, augment traditional surveillance that leverages clinical tests and services (e.g., case reporting), and monitor public health interventions (1). Approximately 40% of persons infected with SARS-CoV-2, the virus that causes COVID-19, shed virus RNA in their stool (2); therefore, community-level trends in SARS-CoV-2 infections, both symptomatic and asymptomatic (2) can be tracked through wastewater testing (3-6). CDC launched the National Wastewater Surveillance System (NWSS) in September 2020 to coordinate wastewater surveillance programs implemented by state, tribal, local, and territorial health departments to support the COVID-19 pandemic response. In the United States, wastewater surveillance was not previously implemented at the national level. As of August 2021, NWSS includes 37 states, four cities, and two territories. This report summarizes NWSS activities and describes innovative applications of wastewater surveillance data by two states, which have included generating alerts to local jurisdictions, allocating mobile testing resources, evaluating irregularities in traditional surveillance, refining health messaging, and forecasting clinical resource needs. NWSS complements traditional surveillance and enables health departments to intervene earlier with focused support in communities experiencing increasing concentrations of SARS-CoV-2 in wastewater. The ability to conduct wastewater surveillance is not affected by access to health care or the clinical testing capacity in the community. Robust, sustainable implementation of wastewater surveillance requires public health capacity for wastewater testing, analysis, and interpretation. Partnerships between wastewater utilities and public health departments are needed to leverage wastewater surveillance data for the COVID-19 response for rapid assessment of emerging threats and preparedness for future pandemics.

The brighter side of home schooling for children with special needs: Learning from COVID-19 lockdown

COVID-19 has resulted in widespread social isolation, quarantines, and suspended academic activity. Children with special needs are socially, financially, and educationally impacted by dramatic changes to laws and restrictions put in place to curtail this devastating global pandemic. This study addresses the beneficial improvements that occurred in the lives of children with special needs during the quarantine period. It led to improved skills, better family dynamics, and an enhancement in technological expertise. Here, we use the case study approach. Twelve parents of children with special needs were selected from the Kozhikode district of Kerala, India. Convenient sampling methods were used for the selection of respondents. The data obtained from the twelve participants were scrutinized. The verbatims were coded with the QDA-Miner Software. Our findings indicate that the lockdown has reinforced family bonds;opened up spaces for homeschooling and digital learning;strengthened relationships with parents, communities, and teachers;and paved the way for technological adaptation. During lockdown, children with special needs continued to learn and build skills in a nurturing familial environment. © Authors.

Evaluating current chest imaging practices of pediatric patients with COVID-19: A retrospective analysis.

PURPOSE: This retrospective analysis aims to examine the effectiveness of the current chest imaging guidelines regarding COVID-19 positive pediatric patients on our study group of patients aged 0 to 18. MATERIALS AND METHODS: We examined clinical and imaging data of 178 pediatric COVID-19 positive patients confirmed by PCR admitted to the Children's Hospital of Los Angeles between March 6, 2020 and June 23, 2020. RESULTS: Of 178 patients, only 46 (27%) patients underwent any form of chest imaging. Thirteen (28%) of 46 imaged patients had positive chest X-rays (CXR) or computed tomography (CT) chest findings, with 8 (62%) of the 13 patients suggesting pneumonia or multifocal pneumonia, 3 (23%) patients having acute respiratory distress syndrome, and 2 (15%) patients demonstrating left sided pleural effusions thought to be the result of ruptured appendicitis unrelated to their COVID-19 diagnosis. All but one patient had significant prior medical histories with an associated comorbid medical condition. Of the 46 imaged patients, 17 (37%) patients had a negative chest X-ray, and 15 (33%) patients had suggestive findings of viral etiology. 132 patients were not imaged. CONCLUSION: Our study population corroborated current chest imaging guidelines in pediatric patients. Chest imaging modalities such as CXR and CT should be reserved for patients who are severely symptomatic and/or possess prior comorbidities such as immunosuppression, diabetes, asthma, obesity, or where other differential etiologies must be entertained.

Spread of COVID-19 in Zambia: An assessment of environmental and socioeconomic factors using a classification tree approach.

The global pandemic emergent from SARS-COV-2 (COVID-19) has continued to cause both health and socio-economic challenges worldwide. However, there is limited information on the factors affecting the dynamics of COVID-19, especially in developing countries, including African countries. In this study, we have focused on understanding the association of COVID-19 cases with environmental and socioeconomic factors in Zambia - a sub-Saharan African country. We used Zambia's district-level COVID-19 data, covering 18 March 2020 (i.e., from first reported cases) to 17 July 2020. Geospatial approaches were used to organize, extract and establish the dataset, while a classification tree (CT) technique was employed to analyze the factors associated with the COVID-19 cases. The analyses were conducted in two stages: (1) the binary analysis of occurrences of COVID-19 (i.e., COVID-19 or No COVID-19), and (2) a risk level analysis which grouped the number of cases into four risk levels (high, moderate, low and very low). The results showed that the distribution of COVID-19 cases in Zambia was significantly influenced by the socioeconomic factors compared to environmental factors. More specifically, the binary model showed that distance to the airport, population density and distance to the town centres were the most combination influential factors, while the risk level analysis indicated that areas with high rates of human immuno-deficient virus (HIV) infection had relatively high chances of having many COVID-19 cases compared to areas with low HIV rates. The districts that are far from major urban establishments and that experience higher temperatures have lower chances of having COVID-19 cases. This study makes two major contributions towards the understanding of COVID-19 dynamics: (1) the methodology presented here can be effectively applied in other areas to understand the association of environmental and socioeconomic factors with COVID-19 cases, and (2), the findings from this study present the empirical evidence of the relationship between COVID-19 cases and their associated environmental and socioeconomic factors. Further studies are needed to understand the relationship of this disease and the associated factors in different cultural settings, seasons and age groups, especially as the COVID-19 cases increase and spread in many countries.

2021 Model Aquatic Health Code (4th Edition)

The Centers for Disease Control and Prevention (CDC) will be releasing the 4th edition of the Model Aquatic Health Code (MAHC, www.cdc.gov/mahc) in the coming months. The MAHC represents a collaboration among local, state, and federal public health officials, particularly environmental health practitioners, and representatives of the aquatics sector to optimize prevention of illness and injury associated with public aquatic venues (e.g., pools, hot tubs, and water playgrounds). Cyanuric acid (CYA) binds to chlorine to prevent it from being degraded by UV light from the sun. Consequently, CYA increases the amount of time it takes for chlorine to inactivate pathogens. CYA is sold as a stand-alone product or as chlorinated isocyanurates (chlorine and CYA, commonly known as dichlor or trichlor). In 2015, CMAHC established a CYA ad hoc committee that included representatives from across the CYA industry and researchers but did not include state or local public health officials. CMAHC charged the committee to develop guidance on CYA concentrations.

Direct and Precise Measurement of Bevacizumab Levels in Human Plasma Based on Controlled Methionine Oxidation and Multiple Reaction Monitoring.

Bevacizumab is a monoclonal antibody which targets vascular endothelial growth factor A (VEGF-A) and is used to treat various cancers and recently COVID-19. The dosage recommendations for bevacizumab are determined on the basis of body weight, and the drug is administered after defined time intervals, when it is presumed to still be above its minimum effective serum concentration. Interindividual and disease-stage-related variations in bevacizumab catabolism, however, can affect the proper dosing of patients, resulting in plasma concentrations which may not be within the optimal therapeutic window for the drug. Therapeutic drug monitoring (TDM) enables the assessment of patients' serum concentrations and allows personalized dosing which has the potential to improve efficacy and reduce side effects. While TMD is often performed using ligand-based assays, mass spectrometry (MS)-based TDM offers improved specificity. Here, we present a robust multiple reaction monitoring (MRM)-MS-based TDM method for the precise quantification of bevacizumab plasma concentrations, based on the controlled oxidation of the methionine-containing peptide, STAYLQMNSLR. The assay shows good linearity (r 2 = 0.9951), robustness, and precision (CVs < 20%) for the quantification of bevacizumab, with a lower limit of quantification (S/N > 10) of 1.8 µg/mL of plasma, without the need for enrichment and requiring less than 1 µL of plasma and less than 6 h from sampling to result.

Genome-wide CRISPR Screens Reveal Host Factors Critical for SARS-CoV-2 Infection.

Identification of host genes essential for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection may reveal novel therapeutic targets and inform our understanding of coronavirus disease 2019 (COVID-19) pathogenesis. Here we performed genome-wide CRISPR screens in Vero-E6 cells with SARS-CoV-2, Middle East respiratory syndrome CoV (MERS-CoV), bat CoV HKU5 expressing the SARS-CoV-1 spike, and vesicular stomatitis virus (VSV) expressing the SARS-CoV-2 spike. We identified known SARS-CoV-2 host factors, including the receptor ACE2 and protease Cathepsin L. We additionally discovered pro-viral genes and pathways, including HMGB1 and the SWI/SNF chromatin remodeling complex, that are SARS lineage and pan-coronavirus specific, respectively. We show that HMGB1 regulates ACE2 expression and is critical for entry of SARS-CoV-2, SARS-CoV-1, and NL63. We also show that small-molecule antagonists of identified gene products inhibited SARS-CoV-2 infection in monkey and human cells, demonstrating the conserved role of these genetic hits across species. This identifies potential therapeutic targets for SARS-CoV-2 and reveals SARS lineage-specific and pan-CoV host factors that regulate susceptibility to highly pathogenic CoVs.