Open-source milligram-scale, four channel, automated protein purification system (preprint)

Liquid chromatography purification of multiple recombinant proteins, in parallel, could catalyze research and discovery if the processes are fast and approach the robustness of traditional, "one-protein-at-a-time" purification. Here, we report an automated, four channel chromatography platform that we have designed and validated for parallelized protein purification at milligram scales. The device can purify up to four proteins (each with its own single column), has inputs for up to eight buffers or solvents that can be directed to any of the four columns via a network of software-driven valves, and includes an automated fraction collector with ten positions for 1.5 or 5.0 mL collection tubes and four positions for 50 mL collection tubes for each column output. The control software can be accessed either via Python scripting, giving users full access to all steps of the purification process, or via a simple-to-navigate touch screen graphical user interface that does not require knowledge of the command line or any programming language. Using our instrument, we report milligram-scale, parallelized, single-column purification of a panel of mammalian cell expressed coronavirus (SARS-CoV-2, HCoV-229E, HCoV-OC43, HCoV-229E) trimeric Spike and monomeric Receptor Binding Domain (RBD) antigens, and monoclonal antibodies targeting SARS-CoV-2 Spike (S) and Influenza Hemagglutinin (HA). We include a detailed hardware build guide, and have made the controlling software open source, to allow others to build and customize their own protein purifier systems.

Public Health Preparedness in the Era of Weather Modification and Climate Engineering.

The use of technological and chemical means aiming to achieve favorable weather conditions or reduce the risk of weather extremes is known as Weather Modification (WM). The United States of America, the People's Republic of China, Thailand, the United Arab Emirates, and Europe have employed WM in an effort to prevent hurricanes and storms, control precipitations, mitigate deforestation and drought, and enhance agriculture. Recently, the use of WM has been expanded toward decreasing air pollution and creating favorable weather conditions for major political and athletic events. The increasing significance and use of WM call for consideration upon its positive and negative effects on human health, close collaboration among health experts and WM decision makers, and relevant public health emergency contingency planning.

Palliative care and COVID-19: a bibliometric analysis.

OBJECTIVE: To assess the impact of COVID-19 on the palliative care (PC) publication trend in the last 10 years and the collaboration between countries and main topics that were discussed in the papers. METHODS: We used Scopus to identify publications on PC between 2012 and 2021 and publications about PC and COVID-19 between 2020 and 2021. We used VOSviewer to assess the main topics using the keywords from the papers and to assess country collaboration. RESULTS: 1937 publications resulted. An increase in publications about PC was observed during the pandemic, only partially explained by OVID-19-related publications. Cancer-related PC publications were the ones with the most marked increase. We identified six clusters in the distribution of the keywords: bioethics, cancer, nursing home/telemedicine, public health, caring and PC following the WHO definition. The countries with higher number of publications were the United States and England. CONCLUSION: We showed an increase in the number of PC publications in the last 2 years that was only partially explained by COVID-19-related publications. Most of the publications increase was due to cancer-related publications, since, during the time of the pandemic, publications on cancer and PC increased markedly, while those on heart failure, lung disease and dementia, remained constant.

SARS-CoV-2 Envelope-mediated Golgi pH dysregulation interferes with ERAAP retention in cells (preprint)

Endoplasmic reticulum (ER) aminopeptidase associated with antigen processing (ERAAP) trims peptide precursors in the ER for presentation by major histocompatibility (MHC)-I molecules to surveying CD8+T-cells. This function allows ERAAP to regulate the nature and quality of the peptide repertoire and, accordingly, the resulting immune responses. We recently showed that infection with murine cytomegalovirus leads to a dramatic loss of ERAAP levels in infected cells. In mice, this loss is associated with the activation of QFL T-cells, a subset of T-cells that monitor ERAAP integrity and eliminate cells experiencing ERAAP dysfunction. In this study, we aimed to identify host factors that regulate ERAAP expression level and determine whether these could be manipulated during viral infections. We performed a CRISPR knockout screen and identified ERp44 as a factor promoting ERAAP retention in the ER. ERp44's interaction with ERAAP is dependent on the pH gradient between the ER and Golgi. We hypothesized that viruses that disrupt the pH of the secretory pathway interfere with ERAAP retention. Here, we demonstrate that expression of the Envelope (E) protein from Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) leads to Golgi pH neutralization and consequently decrease of ERAAP intracellular levels. Furthermore, SARS-CoV-2-induced ERAAP loss correlates with its release into the extracellular environment. ERAAP's reliance on ERp44 and a functioning ER/Golgi pH gradient for proper localization and function led us to propose that ERAAP serves as a sensor of disturbances in the secretory pathway during infection and disease.

Summarising data and factors associated with COVID-19 related conspiracy theories in the first year of the pandemic: a systematic review and narrative synthesis.

Conspiracy theories can have particularly harmful effects by negatively shaping health-related behaviours. A significant number of COVID-19 specific conspiracy theories emerged in the immediate aftermath of the pandemic outbreak. The aim of this study was to systematically review the literature on conspiracy theories related to COVID-19 during the first year of the pandemic (2020), to identify their prevalence, their determinants and their public health consequences. A comprehensive literature search was carried out in PubMed and PsycINFO to detect all studies examining any conspiracy theory related to COVID-19 between January 1st 2020, and January 10th 2021. Forty-three studies were included with a total of 61,809 participants. Between 0.4 and 82.7% of participants agreed with at least one conspiracy belief. Certain sociodemographic factors (young age, female gender, being non-white, lower socioeconomic status), psychological aspects (pessimism, blaming others, anger) and other qualities (political conservatism, religiosity, mistrust in science and using social media as source of information) were associated with increased acceptance of conspiracy theories. Conspiracy beliefs led to harmful health-related behaviours and posed a serious public health threat. Large-scale collaborations between governments and healthcare organizations are needed to curb the spread of conspiracy theories and their adverse consequences.

The human disease gene LYSET is essential for lysosomal enzyme transport and viral infection.

Lysosomes are key degradative compartments of the cell. Transport to lysosomes relies on GlcNAc-1-phosphotransferase-mediated tagging of soluble enzymes with mannose 6-phosphate (M6P). GlcNAc-1-phosphotransferase deficiency leads to the severe lysosomal storage disorder mucolipidosis II (MLII). Several viruses require lysosomal cathepsins to cleave structural proteins and thus depend on functional GlcNAc-1-phosphotransferase. We used genome-scale CRISPR screens to identify lysosomal enzyme trafficking factor (LYSET, also named TMEM251) as essential for infection by cathepsin-dependent viruses including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). LYSET deficiency resulted in global loss of M6P tagging and mislocalization of GlcNAc-1-phosphotransferase from the Golgi complex to lysosomes. Lyset knockout mice exhibited MLII-like phenotypes, and human pathogenic LYSET alleles failed to restore lysosomal sorting defects. Thus, LYSET is required for correct functioning of the M6P trafficking machinery and mutations in LYSET can explain the phenotype of the associated disorder.

Characterization of pathogen-inactivated COVID-19 convalescent plasma and responses in transfused patients.

BACKGROUND: Efficacy of donated COVID-19 convalescent plasma (dCCP) is uncertain and may depend on antibody titers, neutralizing capacity, timing of administration, and patient characteristics. STUDY DESIGN AND METHODS: In a single-center hypothesis-generating prospective case-control study with 1:2 matched dCCP recipients to controls according to disease severity at day 1, hospitalized adults with COVID-19 pneumonia received 2 × 200 ml pathogen-reduced treated dCCP from 2 different donors. We evaluated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies in COVID-19 convalescent plasma donors and recipients using multiple antibody assays including a Coronavirus antigen microarray (COVAM), and binding and neutralizing antibody assays. Outcomes were dCCP characteristics, antibody responses, 28-day mortality, and dCCP -related adverse events in recipients. RESULTS: Eleven of 13 dCCPs (85%) contained neutralizing antibodies (nAb). PRT did not affect dCCP antibody activity. Fifteen CCP recipients and 30 controls (median age 64 and 65 years, respectively) were enrolled. dCCP recipients received 2 dCCPs from 2 different donors after a median of one hospital day and 11 days after symptom onset. One dCCP recipient (6.7%) and 6 controls (20%) died (p = 0.233). We observed no dCCP-related adverse events. Transfusion of unselected dCCP led to heterogeneous SARS CoV-2 antibody responses. COVAM clustered dCCPs in 4 distinct groups and showed endogenous immune responses to SARS-CoV-2 antigens over 14-21 days post dCCP in all except 4 immunosuppressed recipients. DISCUSSION: PRT did not impact dCCP anti-virus neutralizing activity. Transfusion of unselected dCCP did not impact survival and had no adverse effects. Variable dCCP antibodies and post-transfusion antibody responses indicate the need for controlled trials using well-characterized dCCP with informative assays.

Genome-wide bidirectional CRISPR screens identify mucins as host factors modulating SARS-CoV-2 infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes a range of symptoms in infected individuals, from mild respiratory illness to acute respiratory distress syndrome. A systematic understanding of host factors influencing viral infection is critical to elucidate SARS-CoV-2-host interactions and the progression of Coronavirus disease 2019 (COVID-19). Here, we conducted genome-wide CRISPR knockout and activation screens in human lung epithelial cells with endogenous expression of the SARS-CoV-2 entry factors ACE2 and TMPRSS2. We uncovered proviral and antiviral factors across highly interconnected host pathways, including clathrin transport, inflammatory signaling, cell-cycle regulation, and transcriptional and epigenetic regulation. We further identified mucins, a family of high molecular weight glycoproteins, as a prominent viral restriction network that inhibits SARS-CoV-2 infection in vitro and in murine models. These mucins also inhibit infection of diverse respiratory viruses. This functional landscape of SARS-CoV-2 host factors provides a physiologically relevant starting point for new host-directed therapeutics and highlights airway mucins as a host defense mechanism.

The impact of the COVID-19 pandemic on language deserves more attention: Evidence from university students in Greece (preprint)

The COVID-19 pandemic has had a dire effect on young people. Recent studies have reported its impact on several mental health domains in this population group. However little has been discussed about the influence of disrupted education and socialization on the linguistic ability of young people. We report early observations from a cross - sectional study conducted among university students in Thessaloniki, Greece. Approximately 3 out of 4 participants observed foreign terms to be introduced in their everyday language, and most of them reported that they have used terms coming from at least 3 different languages. More than half of the participants agreed that the current changes to their vocabulary may have an impact ontheir future linguistic competence, and about one third of them expressed fears that nationallanguages will be negatively impacted by the situation. Nevertheless, approximately two thirds of them disagreed with the latter and opted for enhancing biology and health literacy rather than language in educational curricula. Overall, university students in Greece consider that the pandemic has diversified their vocabulary and potentially affected their linguistic competence. Integrating this knowledge in the current understanding of youth's mental health amidst COVID-19 can help researchers and healthcare practitioners meet the needs of this population group.

C-reactive Protein Elevation Is Associated With QTc Interval Prolongation in Patients Hospitalized With COVID-19.

Background: The relationship between inflammation and corrected QT (QTc) interval prolongation is currently not well defined in patients with COVID-19. Objective: This study aimed to assess the effect of marked interval changes in the inflammatory marker C-reactive protein (CRP) on QTc interval in patients hospitalized with COVID-19. Methods: In this retrospective cohort study of hospitalized adult patients admitted with COVID-19 infection, we identified 85 patients who had markedly elevated CRP levels and serial measurements of an ECG and CRP during the same admission. We compared mean QTc interval duration, and other clinical and ECG characteristics between times when CRP values were high and low. We performed mixed-effects linear regression analysis to identify associations between CRP levels and QTc interval in univariable and adjusted models. Results: Mean age was 58 ± 16 years, of which 39% were women, 41% were Black, and 25% were White. On average, the QTc interval calculated via the Bazett formula was 15 ms higher when the CRP values were "high" vs. "low" [447 ms (IQR 427-472 ms) and 432 ms (IQR 412-452 ms), respectively]. A 100 mg/L increase in CRP was associated with a 1.5 ms increase in QTc interval [ß coefficient 0.15, 95% CI (0.06-0.24). In a fully adjusted model for sociodemographic, ECG, and clinical factors, the association remained significant (ß coefficient 0.14, 95% CI 0.05-0.23). Conclusion: An interval QTc interval prolongation is observed with a marked elevation in CRP levels in patients with COVID-19.

Systematic functional interrogation of SARS-CoV-2 host factors using Perturb-seq (preprint)

Numerous host factors of SARS-CoV-2 have been identified by screening approaches, but delineating their molecular roles during infection and whether they can be targeted for antiviral intervention remains a challenge. Here we use Perturb-seq, a single-cell CRISPR screening approach, to investigate how CRISPR interference of host factors changes the course of SARS-CoV-2 infection and the host response in human lung epithelial cells. Our data reveal two classes of host factors with pronounced phenotypes: factors required for the response to interferon and factors required for entry or early infection. Among the latter, we have characterized the NF-{kappa}B inhibitor I{kappa}B (NFKBIA), as well as the translation factors EIF4E2 and EIF4H as strong host dependency factors acting early in infection. Overall, our study provides high-throughput functional validation of host factors of SARS-CoV-2 and describes their roles during viral infection in both infected and bystander cells.

Migration and newborn screening: time to build on the European Asylum, Integration and Migration Fund?

PURPOSE: The authors discuss the need for newborn screening in the context of the migration policy of the European Union, and particularly, the European Asylum, Migration and Integration Fund. METHODS: The authors searched scholarly databases (Pubmed, Scopus, Google scholar) and grey literature (LexEuropa, Policy reports) to identify original peer-reviewed research examining the migration to the European Union and the provision of healthcare to infants born to refugees and immigrant mothers. Resources in language different from English, French, German and Greek were not taken into consideration. RESULTS: Every year, a large number of refugees and immigrants from sub-Saharan Africa and Middle East countries travel to and enter in Europe. It has been estimated that two thirds of those seeking asylum are women and children. Many of these children have been born on the way to Europe or in migrant camps. Essential newborns' health screening is not accessible in most cases. Congenital conditions such as hypothyroidism and phenylketonuria may remain untreated, and once these infants are diagnosed, the organic damage could be irreversible. Prolonged necessary hospitalisation might be out of consideration at a time when clinics and hospitals are overstrained with COVID-19 patients. CONCLUSIONS: It is essential to ensure that newborn screening will be performed in a timely and evidence-based manner as well as that the information will be communicated between hospitals and within countries' health networks. In order to achieve these goals interdisciplinary and international technical and logistical collaboration are required.

COMPARISON OF THE TREATMENT OF SICKLE CELL VASO-OCCLUSIVE CRISIS PRIOR TO AND DURING COVID 19 PANDEMIC

Purpose of Study Vaso-occlusive crisis (VOC) is one of the hallmark signs of Sickle Cell Disease (SCD) and a leading cause of emergency department (ED) visits. There are multiple guidelines in place on the management of pain in VOC. Large ED patient volumes and inability to obtain IV access are major causes of delay to medications. The use of individual pain plans and standardized approaches attempt to reduce the time to pain management, but it is unclear how much these help. This study aims to evaluate if there is a difference in the treatment of VOC prior to and during the COVID-19 pandemic. Methods Used This is a single center retrospective chart review. Patients 6mo - 25yo who presented to the pediatric ED between March 15th to December 15th, 2019 or between March 15th to December 15th, 2020 with SCD in VOC were included. Data collected included age, gender, encounter date and time, disposition, pain scores, nursing and physician assignment time, time of IV access, first pain medication, number and timing of pain medications, arrivals/hour in the ED, ED LOS, presence of acute chest, pain plans, or fevers. Summary of Results Between March and December 2019 the ED saw 119 patients with VOC, 68% male with an average age of 13.9 years, compared to the 103 patients, 61% male with an average age of 15.2 years seen in the first 10 months of the pandemic. There was no significant difference in the length of stay in the ED between time periods in this patient population. The percentage of pain plans increased significantly in 2020, with 98% of patients having an individual pain plan compared to 88% prior. Overall patients presenting with VOC received IV medications as first line therapy 90% of the time, with ketorolac, morphine and hydromorphone as the top three first medications ordered. On average, in both time periods, patients received their first pain medications within 65 minutes of ED arrival (p = 0.47). There was a significant difference in pain medication administration. In 2019 an average of 2.9 pain medications (0.68 medications/hour) were given, and an average of 1.9 opioid medications were given per ED visit compared to 3.7 pain medications (0.82 medications/hour) and 2.65 opioid medications were given per ED visit during the pandemic (p < 0.05). The presence or history of fever significantly reduced the amount pain medications administered in both time periods. Conclusions During the first 10 months of the pandemic time to initial care in the ED did not significantly change for patients with VOC, though subsequent care with the number of pain medications increased during pandemic times. This may be secondary to lower ED patient volumes or increased individual pain plans for these patients. The presence of a fever in patients with SCD at or prior to presentation decreased the number of total pain medications given to patients arriving in VOC. (Table Presented).