Increasing polio coverage with safer vaccines: a pressing need in Latin America

Given the actual risk of poliomyelitis outbreaks in the region due to poliovirus derived from the Sabin vaccine or the importation of wild poliovirus, the Latin American Society of Pediatric Infectious Diseases commissioned an ad hoc group of experts from the institution's Vaccines and Biologicals Committee, to draft an official position paper on the urgent need to increase immunization levels against the disease in the region and incorporate inactivated polio vaccine exclusive schedules in all national immunization programs. This publication discusses the main conclusions and recommendations generated as a result of such activity.Copyright © 2022, Sociedad Chilena de Infectologia. All rights reserved.

Influence of biochar on improving hydrological and nutrient status of two decomposed soils for yield of medicinal plant - Pinellia ternata

The root tuber of Pinellia ternata has been used as a traditional therapeutic herbal medicine. It is reported to impart beneficial attributes in recovering COVID-19 patients. To meet an increasing demand of P. ternata, this study is intended to investigate the effects of biochar on the soil hydrological and agronomic properties of two decomposed soils (i.e., completely decomposed granite (CDG) and lateritic soil) for the growth of P. ternata. The plant was grown in instrumented pots with different biochar application rate (0%, 3% and 5%) for a period of three months. Peanut shell biochar inclusion in both soils resulted in reduction of soil hydraulic conductivity and increase in soil water retention capacity. These alterations in hydrological properties were attributed to measured change in total porosity, biochar intra pore and hydrophilic functional groups. The macro-nutrient (i.e., N, P, K, Ca, and Mg) concentration of both soils increased substantially, while the pH and cation exchange capacity levels in the amended soils were altered to facilitate optimum growth of P. ternata. The tuber biomass in biochar amended CDG at all amendment rate increases by up to 70%. In case of lateritic soil, the tuber biomass increased by 23% at only 5% biochar application rate. All treatments satisfied the minimum succinic acid concentration required as per pharmacopoeia standard index. The lower tuber biomass exhibits a higher succinic acid concentration regardless of the soil type used to grow P. ternata. The biochar improved the yield and quality of P. ternata in both soils.

SARS-CoV-2 Nsp13 Catalytic Efficiency is Regulated by ATP: Mg2+ Stoichiometry and Functional Cooperativity Among Nsp13 Molecules

Coronavirus disease 19 (COVID-19) is a highly contagious and lethal disease caused by the SARS-CoV-2 positive-strand RNA virus. Nonstructural protein 13 (Nsp13) is the highly conserved ATPase/helicase required for replication of the SARS-CoV-2 genome which allows for the infection and transmission of COVID-19. We biochemically characterized the purified recombinant SARS-CoV-2 Nsp13 helicase protein expressed using a eukaryotic cell-based system and characterized its catalytic functions, focusing on optimization of its reaction conditions and assessment of functional cooperativity among Nsp13 molecules during unwinding of duplex RNA substrates. These studies allowed us to carefully determine the optimal reaction conditions for binding and unwinding various nucleic acid substrates. Previously, ATP concentration was suggested to be an important factor for optimal helicase activity by recombinant SARS-CoV-1 Nsp13. Apart from a single study conducted using fixed concentrations of ATP, the importance of the essential divalent cation for Nsp13 helicase activity had not been examined. Given the importance of the divalent metal ion cofactor for ATP hydrolysis and helicase activity, we assessed if the molar ratio of ATP to Mg2+ was important for optimal SARS-CoV-2 Nsp13 RNA helicase activity. We determined that Nsp13 RNA helicase activity was dependent on ATP and Mg2+ concentrations with an optimum of 1 mM Mg2+ and 2 mM ATP. Next, we examined Nsp13 helicase activity as a function of equimolar ATP:Mg2+ ratio and determined that helicase activity decreased as the equimolar concentration increased, especially above 5 mM. We determined that Nsp13 catalytic functions are sensitive to Mg2+ concentration suggesting a regulatory mechanism for ATP hydrolysis, duplex unwinding, and protein remodeling, processes that are implicated in SARS-CoV-2 replication and proofreading to ensure RNA synthesis fidelity. Evidence is presented that excess Mg2+ impairs Nsp13 helicase activity by dual mechanisms involving both allostery and ionic strength. In addition, using single-turnover reaction conditions, Nsp13 unwound partial duplex RNA substrates of increasing doublestranded regions (16-30 base pairs) with similar kinetic efficiency, suggesting the enzyme unwinds processively in this range under optimal reaction conditions. Furthermore, we determined that Nsp13 displayed sigmoidal behavior for helicase activity as a function of enzyme concentration, suggesting that functional cooperativity and oligomerization are important for optimal activity. The observed functional cooperativity of Nsp13 protomers suggests the essential coronavirus RNA helicase has roles in RNA processing events beyond its currently understood involvement in the SARS-CoV-2 replication-transcription complex (RTC), in which it was suggested that only one of the two Nsp13 subunits has a catalytic function, whereas the other has only a structural role in complex stability. Altogether, the intimate regulation of Nsp13 RNA helicase by divalent cation and protein oligomerization suggests drug targets for modulation of enzymatic activity that may prove useful for the development of novel anti-coronavirus therapeutic strategies. This work was supported by the Intramural Training Program, National Institute on Aging (NIA), NIH, and a Special COVID-19 Grant from the Office of the Scientific Director, NIA, NIH.Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.

Derivatization of sialylated glycopeptides plus based sialoglycopeptides enrichment using cation exchange media.

Glycosylation is one of the most important post-translational modifications. However, the characterizations of glycopeptides, especially the negatively charged sialoglycopeptides that are associated with various diseases, remain challenging, due to the co-existence with high abundant peptides and the low ionization efficiency of sialoglycopeptides resulting from the carboxyl groups. Therefore, it is essential to develop an efficient enrichment method for sialoglycopeptides. Here, we present a novel derivatization-based enrichment method that can (i) identify linkage isomers of sialic acids by generating mass difference, (ii) unify the net charge of peptides into zero, and (iii) introduce positive charges to sialoglycopeptides by conjugating quaternary ammonium with sialic acid. The derivatization, termed derivatization of sialylated glycopeptides plus (DOSG+), enables efficient enrichment through electrostatic interaction using weak cation exchange (WCX) media. DOSG+ -based WCX enrichment was validated and optimized with samples derived from bovine fetuin. Peptides were removed efficiently (recovery rate <1%). The signal intensity of a selected model sialoglycopeptide was increased by ∼30% (suggesting recovery rate >100%). The method was employed on human alpha-1 acid glycoprotein (AGP), and recombinant human erythropoietin (EPO), demonstrating the application of DOSG+ -based WCX enrichment on complexed N-linked and O-linked sialoglycopeptides. The method is simple, efficient, and targets small-scale sialoglycopeptide enrichment.

Development and Validation of a Novel Stability Indicating Reverse Phase High Performance Liquid Chromatographic Method for Related Substances and Assay Analysis of Molnupiravir Drug Substance and Drug Product

Molnupiravir, a broad-spectrum antiviral is an isopropyl ester prodrug of beta-D-N4-hydroxycytidine. Molnupiravir targets RNA-dependent RNA-polymerase enzyme of the viruses. A new stability-indicating HPLC-method was developed to determine related substances and assay of molnupiravir. Separation was achieved by using Shim-pack GWS C18 column. The method was validated according to current ICH requirements. The calibration plot gave a linear relationship for all known analytes over the concentration range from LOQ to 200%. LOD and LOQ for all known analytes were found in 0.05-0.08 microg mL-1 and 0.12-0.20 microg mL-1, respectively, the mean recovery was found to be 97.79-102.44 %. Study showed that the method, results of robustness, solution stability studies are precise and within the acceptable limits. Molnupiravir was found to degrade in acid, alkali, and oxidative conditions, and was stable in thermal, moisture, and photolytic degradation condition. The method is simple, accurate, precise, and reproducible for routine purity analysis of drug-samples.Copyright © 2022 Indian Drug Manufacturers' Association. All rights reserved.

Interaction of Masitinib with Organic Cation Transporters.

Tyrosine kinase inhibitors (TKI) such as Masitinib were reported to be useful as therapeutic options in malignant disorders and nonmalignant diseases, like coronavirus disease 2019 (COVID-19). Most kinases must be translocated into targeted cells by the action of specific transport proteins, as they are hydrophilic and not able to cross cell membranes freely. Accordingly, the efficacy of TKI in target cells is closely dependent on the expression of their transporters. Specifically, Masitinib is an organic cation and is expected to interact with organic cation transporters (OCT and Multidrug and Toxin Extrusion proteins-MATE-). The aim of this work was to characterize the interaction of Masitinib with different OCTs. Human embryonic kidney 293 cells stably transfected with murine or human OCT were used for the experiments. The interaction of Masitinib with OCTs was investigated using quenching experiments. The intracellular accumulation of this drug was quantified using high performance liquid chromatography. Our results identified interactions of Masitinib with almost all investigated mouse (m) and human (h) OCTs and hMATE1 and indicated OCT1 and hOCT2 to be especially potent Masitinib translocators across cell membranes. Interestingly, some important differences were observed for the interaction with murine and human OCTs. In the future, investigations concerning further in vitro and in vivo properties of Masitinib and its efficacy related to transporter-related uptake mechanisms under pathophysiological conditions should be performed. Clinical trials in humans and other animals with Masitinib have already shown promising results. However, further research is necessary to understand the disease specific transport mechanisms of Masitinib to contribute to a successful and responsible therapy employment.

Landscape Analysis of Quercetin: A Potential Candidate Against SARS-CoV-2

Fruit, vegetables, and green tea contain quercetin (a flavonoid). Some of the diet's most signifi-cant sources of quercetin are apples, onions, tomatoes, broccoli, and green tea. Antioxidant, anticancer, anti-inflammatory, antimicrobial, antibacterial, and anti-viral effects have been studied of quercetin. The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) virus, ribonucleic acid (RNA) polymer-ase, and other essential viral life-cycle enzymes are all prevented from entering the body by quercetin. Despite extensive in vitro and in vivo investigations on the immune-modulating effects of quercetin and vitamin C treatment. 3-methyl-quercetin has been shown to bind to essential proteins necessary to convert minus-strand RNA into positive-strand RNAs, preventing the replication of viral RNA in the cytoplasm. Quercetin has been identified as a potential SARS-CoV-2 3C-like protease (3CLpro) suppressor in recent molecular docking studies and in silico assessment of herbal medicines. It has been demonstrated that quercetin increases the expression of heme oxygenase-1 through the nuclear factor erythroid-related factor 2 (Nrf2) signal network. Inhibition of heme oxygenase-1 may increase bilirubin synthesis, an endoge-nous antioxidant that defends cells. When human gingival fibroblast (HGF) cells were exposed to lipo-polysaccharide (LPS), inflammatory cytokine production was inhibited. The magnesium (Mg+2) cation complexation improves quercetin free radical scavenging capacity, preventing oxidant loss and cell death. The main objective of this paper is to provide an overview of the pharmacological effects of quercetin, its protective role against SARS-CoV-2 infection, and any potential molecular processes.Copyright © 2022 Bentham Science Publishers.

MULTISCALE KINETIC TRANSPORT MODELS FOR THE SPREAD OF EPIDEMICS WITH UNCERTAIN DATA

Most epidemiological models are rooted in the pioneering work proposed by Ker- mack and McKendrick and are based on systems of deterministic ODEs, which describe the temporal evolution of the spread of an infectious disease assuming population and territorial homogeneity. Generally, the concept of the average behavior of a population is su cient to have a rst reliable description of an epidemic development, but the inclusion of the spatial compo-nent becomes crucial when it is necessary to consider spatially heterogeneous interventions, as in the case of the COVID-19 pandemic. Moreover, any realistic data-driven model must take into account the large uncertainty in the values reported by o cial sources such as the amount of infectious individuals. In this work, drawing inspiration from kinetic theory, recent advances on the development of stochastic multiscale kinetic transport models for the spread of epidemics under uncertain data are presented. The propagation of the infectious disease is described by the spatial movement and interactions of individuals divided into commuters moving in the ter-ritory on a wide scale and non-commuters acting only on urban scales. The resulting models are solved numerically through a suitable stochastic Asymptotic-Preserving IMEX Runge-Kutta Finite Volume Collocation Method, which ensures a consistent treatment of the system of equa-tions, without loss of accuracy when entering in the sti, di usive regime. Application studies concerning the spread of the COVID-19 pandemic in Italy assess the validity of the proposed methodology. © 2022, Scipedia S.L. All rights reserved.

Kawasaki disease in the time of COVID-19-the international kawasaki disease registry

Background and Aim: The considerable overlap in case definition and clinical features between patients with COVID-19 associated Multisystem Inflammatory Syndrome in Children (MIS-C) and Kawasaki disease (KD) suggests shared pathogenesis. We sought to compare demographic, clinical presentation, management and outcomes of patients by COVID-19 status. Method(s): The International KD Registry (IKDR) began enrolling patients with clinical features of either acute MIS-C or KD or fever with hyperinflammation beginning in January 2020. The IKDR is unique regarding broad patient selection and includes sites from North, Central and South America, Europe, Asia and the Middle East. Patient groups stratified by COVID-19 status were compared. Result(s): As of October 6, 2021, 1330 patients were registered from 31 sites. COVID status was POSITIVE for 59% (confirmed household COVID-19 contact and/or positive SARS-CoV-2 PCR or serology), POSSIBLE for 4% (suggestive clinical features but some negative tests or absent exposure), NEGATIVE for 23%, and UNKNOWN (no known exposure and testing not com-pleted) for 14% (TABLE). Most of the UNKNOWN patients were from early in the COVID-19 pandemic before MIS-C was defined and before COVID-19 serologic testing was widely used. POSITIVE and POSSIBLE patients were older, had fewer KD clinical criteria, greater gastrointestinal symptoms, were more likely to present with shock and require ICU admission and inotropic support. POSSIBLE patients had greater days from symptom onset to first immune modulation treatment, with no differences between groups regarding days from admission to first treatment. Most patients in each group received intravenous immune globu-lin, with POSITIVE and POSSIBLE patients more likely to have received steroids and anakinra. NEGATIVE and UNKNOWN patients had higher maximal coronary artery Z scores, with a trend to having higher categories of aneurysm involvement. Conclusion(s): While there was considerable overlap in presentation, management and outcomes between COVID-19 POSITIVE/POSSIBLE (presumed MIS-C) and COVID NEGATIVE/UNKNOWN patients (presumed KD), COVID-19 POSITIVE/POSSIBLE patients had more severe presentations and required more intensive management, although coronary artery outcomes trended to be less severe. Patient recruitment con-tinues, and in-depth comparison of laboratory features and appli-cation of machine learning approaches to patient differentiation and prediction of optimal management pathways are forthcoming.

An Unusual Clinical Evolution in Anticontactin- 1 Positive CIDP

Specific clinical, electrophysiological and serological features are used to recognise a phenotype fitting the atypical chronic inflammatory demyelinating (CIDP) variant spectrum. We report a 28-year-old male patient, without any significant history apart from a recent asymptomatic COVID-19 infection, presenting at first with bilateral facial nerve palsy, subsequently -three months later- developing an subacute onset symmetric sensory ataxia and arefl exia, and thirdly experiencing diffuse rapidly progressive motor deficits. Additional investigations suggested an autoimmune polyneuropathy: Liquor analysis showed cytoalbuminologic dissociation. Cerebrospinal fluid protein elevation was remarkable: 631 mg/dL. Nerve conduction studies showed prominent distal latencies prolongation and dispersion of the potentials, meeting the electrodiagnostic criteria of the European Federation of Neurological Societies/Peripheral Nerve Society for CIDP (2021). Full spine magnetic resonance imaging depicted pathological thickening and enhancement of the roots of the cauda equina as seen in radiculitis. There was no or poor response to conventional treatment, i.e. immunoglobulins (IVIG), corticosteroids and even plasmapheresis. Muscle weakness deteriorated. Presence of serum IgG4 anti- contactin-1 (CNTN1) antibodies was found by ELISA identifi- cation and titration, and the patient improved substantially after rituximab treatment. While contributing to the expanding confidence in nodal and paranodal antibodies as valuable biomarkers in clinical practice, our case entails several peculiarities: 1/ SARS-CoV2 positivity as a possible trigger of this auto-immune polyneuropathy 2/ A considerably younger age of onset than in the patients already described (range 33-76 years). 3/ The clinical course progressed in an atypical manner even for atypical CIDP: Initial presentation with bilateral asymmetric facial palsy, followed by sensory ataxia, which prompted the initial diagnosis of Miller-Fisher syndrome, and later development of severe motor impairment. 4/ Proteinorachy was so pronounced that we considered neuroborreliosis as a potential associated disorder. Borrelia seroconversion occurred after the first IVIG-treatment, and could be false positive. However, the patient was treated with intravenous ceftriaxone, which had no effect on the clinic. 5/ Antibodies against CNTN1 were undetectable after 2 months of rituximab. Emphasising the both diagnostic and therapeutic importance of recognising a phenotype compatible with atypical CIDP, an underrecognized and consequently undertreated disease where early diagnosis and prevention of axonal damage is crucial in.

Recent Advances in Metal Chalcogenide Quantum Dots: From Material Design to Biomedical Applications

With the rapid progress in nanomaterials and biochemistry, there has been an explosion of interest in biomolecule-modified quantum dots (QDs) for biomedical applications. Metal chalcogenide quantum dots (MCQDs), as the most widely studied QDs, have attracted tremendous attention in the biomedical field on account of their unique and excellent optical properties and the ease of biomolecular modifications. Herein, important advances in MCQDs over recent years are reviewed, from materials design to biomedical applications. Especially, this review focuses on the challenges encountered in the applications of MCQDs in biomedical fields and how these problems can be solved by rational design of synthesis methods and modifications, which have opened a universal route to develop the functionalized MCQDs. Moreover, recent processes in bioimaging, biosensing, and cancer therapy based on MCQDs are examined, including the rapid detection and diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This review provides broad insights into MCQDs in the biomedical field and will inspire material researchers to develop MCQDs in the future.

Molecular Docking and Pharmacokinetic Studies of Aquillochin and Grewin as SARS-CoV-2 Mpro Inhibitors

Background: The COVID-19 pandemic emerged at the end of 2019 in China and spread rapidly all over the world. Scientists strive to find virus-specific antivirals against COVID-19 disease. This study aimed to assess bioactive coumarinolignans (Aquillochin, Grewin) as potential SARS-CoV-2 main protease (SARS-CoV-2 Mpro) inhibitors using a molecular docking study. Methods: The detailed interactions between coumarinolignans and SARS-CoV-2 Mpro were determined as hydrophobic bonds, hydrogen bonds, electronic bonds, inhibition activity, ligand efficiency, bonding type, and distance using Autodock 4.2 software. SARS-CoV-2 Mpro was docked with Aquillochin and Grewin, and the docking results were analyzed by Autodock 4.2 and Biovia Discovery Studio 4.5. Nelfinavir and Lopinavir were used as standards for comparison. Results: The binding energies of the SARS-CoV-2 Mpro-coumarinolignan’s complexes were identified from the molecular docking of SARS-CoV-2 Mpro. Aquillochin and Grewin were found to be-7.5 and-8.4 kcal/mol, respectively. The binding sites of the coumarinolignans to SARS-CoV-2 Mpro were identified with the main interactions being π-alkyl, alkyl, π-cation, π-π T-Shaped, and hydrogen bonding. Furthermore, SwissADME web tools were used to evaluate ADMET properties and pharmacokinetic parameters of Aquillochin and Grewin. The results of ADMET and pharmacokinetic results of the Aquillochin and Grewin showed that these coumarinolignans were consonant with the many accepted rules and the criteria of drug-likeness. Conclusion: Aquillochin and Grewin obey Lipinski’s rule of five. According to the results obtained from molecular docking studies and ADMET predictions, Aquillochin and Grewin have shown weak efficacy as drug candidates against COVID-19 disease.

Responses of regional environmental factors to the reference value of neutrophil-lymphocyte ratio in healthy adults

Objective: This paper screened the factors that may influence the spatial differentiation of Neutrophil-to-lymphocyte ratio (NLR) reference values in healthy adults in China and explored the trend of NLR reference values in China. Methods: For this research, we collected the NLR of 162 681 healthy adults from 62 cities in China. Spearman regression analysis was used to analyze the correlation between NLR and 25 geography secondary indexes. We extracted 9 indexes with significant correlation, built a random forest (RF) model, and predicted the country's urban healthy adults' NLR reference value. By using the disjunctive Kriging method, we obtained the geographical distribution of NLR reference value of healthy adults in China. Results: The reference value of NLR of healthy adults in China was significantly correlated with the 9 secondary indexes, namely, altitude, sunshine duration, annual average temperature, annual average relative humidity, annual temperature range, annual average wind speed, content of organic matter in topsoil, cation exchange capacity in topsoil (clay), and total amount of CaSO4 in soil. The geographical distribution of NLR values of healthy adults in China showed a trend of being higher in Southeast China and lower in Northwest China, higher in coastal areas and lower in inland areas. Conclusion: This study lays a foundation for further research on the mechanism of different influencing factors on the reference value of NLR index. A random forest model composed of significant influencing factors has been established to provide the basis for formulating reference criteria for the prognostic factors of the novel coronavirus using NLR reference values in different regions.

Comparative Analysis of Results of RT-PCR and Truenat in diagnosis of Covid 19

Background: Almost a year has passed since the start for Covid 19 pandemic, however with no human immunity and no vaccine for prevention early diagnosis remains the mainstay to contain the infection and prevent the spread of the vi- rus. RT-PCR is said to be the most sensitive test currently. However, Truenat is also widely used being approved by ICMR, hence a comparative study of RT-PCR & Truenat was taken up in this study. Methods: A total of 200 samples were taken from patients having signs and symptoms (clinically suspected) of COVID 19. Samples obtained via oropharyngeal and nasopharyngeal swabs were analyzed on both RT-PCR and Truenat. Viral load in samples were evaluated using Ct value of targeted genes by both the techniques. Results: Out of 200 samples, 184 showed similar results via RT-PCR and Truenat i.e., 61 positive and 123 negatives. 16 samples showed discordant results. Out of 16 samples, 5 were positive and 11 were negative by RT -PCR. However, by Truenat 11 were positive and 5 were negative. The Ct values of targeted genes range between 13-30 for E-gene and 16-32 for RdRp gene. Conclusions: The detection of SARS COV-2 patients with mild form of disease (which were persistently negative by RT- PCR) was higher by Truenat. P value being 0.077 which is significant at 90% level of significance. Hence though identifi- cation of viral RNA by RT-PCR is the gold standard, its sensitivity is lower compared to Truenat. Hence, we can suggest that Truenat is a diagnostic method with higher sensitivity, closed system hence lower chances of contamination and at the same time providing faster results at low cost, easy to perform as a point of care test, portable and requiring lower expertise to operate compared to RT-PCR

An efficient blockchain-based hierarchical data sharing for Healthcare Internet of Things

With the assistance of Internet of Things (IoT), the fast developing Healthcare Internet of Things (H-IOT) have promoted the healthcare ecosystem into the era of Health 5.0 and enables many promising medical applications, such as remote healthcare that is crucial in pandemic (e.g, COVID-19). Healthcare participants can make accurate diagnosis, treatment and research based on the shared Personal Health Records (PHRs) sensed from remote H-IOT devices. However, current H- IOT systems fall short of a secure and trustworthy PHR sharing service in remote healthcare, which is able to prevent user privacy leakage and PHR violation together with high efficiency in key distribution apart from supporting efficient data retrieval and fine-grained access control. In response, we present a blockchain- based hierarchical data sharing framework (BHDSF) to provide fine-grained access control and efficient retrieval over encrypted PHRs with low consumed hierarchical key distribution and key leakage resistance. Compared with existing solutions, BHDSF takes untrusted cloud and malicious auditor into consideration simultaneously, and achieves trustworthy PHR integrity auditing and metadata verification by leveraging blockchain technique. Besides, BHDSF enables efficiently aggregative authentication for source records from H-IoT devices, which is lacked in most of existing data sharing frameworks. Finally, we demonstrate the feasibility of BHDSF by conducting extensive empirical tests over real-world dataset. IEEE

Sustainable tourism versus tourism depredation. Balearic Islands, Spain

The present research tries to explain the reality of the double model of tourism existing in the Islas Baleares (Spain): on the one hand, sustainable tourism, ecotourism, cultural and historical tourism;on the other, massified tourism, tourism based on nightlife that generates a large number of problems and negative side effects, having Mallorca town of Calvià as epicenter. Bearing in mind that the Islas Baleares tourism sector is facing a period of great uncertainty and crisis as a result of the effects of the COVID-19 pandemic and the confinement decreed by the Spanish government for approximately three months, the challenges, problems, and issues of the activity at present place the sector at a true turning point. © 2021, Universidad de Los Andes. All rights reserved.

Functional Interaction Between Drosophila Olfactory Sensory Neurons and Their Support Cells.

Insects detect volatile chemicals using antennae, which house a vast variety of olfactory sensory neurons (OSNs) that innervate hair-like structures called sensilla where odor detection takes place. In addition to OSNs, the antenna also hosts various support cell types. These include the triad of trichogen, tormogen, and thecogen support cells that lie adjacent to their respective OSNs. The arrangement of OSN supporting cells occurs stereotypically for all sensilla and is widely conserved in evolution. While insect chemosensory neurons have received considerable attention, little is known about the functional significance of the cells that support them. For instance, it remains unknown whether support cells play an active role in odor detection, or only passively contribute to homeostasis, e.g., by maintaining sensillum lymph composition. To investigate the functional interaction between OSNs and support cells, we used optical and electrophysiological approaches in Drosophila. First, we characterized the distribution of various supporting cells using genetic markers. By means of an ex vivo antennal preparation and genetically-encoded Ca2+ and K+ indicators, we then studied the activation of these auxiliary cells during odor presentation in adult flies. We observed acute responses and distinct differences in Ca2+ and K+ fluxes between support cell types. Finally, we observed alterations in OSN responses upon thecogen cell ablation in mature adults. Upon inducible ablation of thecogen cells, we notice a gain in mechanical responsiveness to mechanical stimulations during single-sensillum recording, but a lack of change to the neuronal resting activity. Taken together, these results demonstrate that support cells play a more active and responsive role during odor processing than previously thought. Our observations thus reveal that support cells functionally interact with OSNs and may be important for the extraordinary ability of insect olfactory systems to dynamically and sensitively discriminate between odors in the turbulent sensory landscape of insect flight.

SARS-CoV-2 Ion Channel ORF3a Enables TMEM16F-Dependent Phosphatidylserine Externalization to Augment Procoagulant Activity of the Tenase and Prothrombinase Complexes

Severe SARS-CoV-2 infection is complicated by dysregulation of the blood coagulation system and high rates of thrombosis, but virus-intrinsic mechanisms underlying this phenomenon are poorly understood. Increased intracellular calcium concentrations promote externalization of phosphatidylserine (PS), the membrane anionic phospholipid required for assembly and activation of the tenase and prothrombinase complexes to drive blood coagulation. TMEM16F is a ubiquitous phospholipid scramblase that mediates externalization of PS in a calcium-dependent manner. As SARS-CoV-2 ORF3a encodes a presumed cation channel with the ability to transport calcium, we hypothesized that ORF3a expression by infected host cells perturbs the cellular calcium rheostat, driving TMEM16F-dependent externalization of PS and enhancing procoagulant activity. Using a doxycycline-inducible system, synchronized expression of ORF3a in A549 pulmonary epithelial cells resulted in a time-dependent augmentation of tissue factor (TF) procoagulant activity exceeding 9-fold by 48 hours (p < 0.0001), with no change in TF cell-surface expression. This enhancement was dependent upon PS as determined by inhibition with the PS-binding protein lactadherin. Over 2-fold enhancement of prothrombinase activity (p < 0.0001) was also observed by 48 hours. ORF3a increased intracellular calcium levels by 18-fold at 48 hours (p < 0.0001), as determined by the intracellular calcium indicator fluo-4. After 16 hours of ORF3a expression, more than 60% of cells had externalized PS (p < 0.001) without increased cell death, as quantified by flow cytometry following annexin V binding. Immunofluorescence microscopy staining for ORF3a, annexin V, and nuclei confirmed ORF3a expression within internal and cell surface membranes and increased PS externalization. PS externalization was insensitive to the pan-caspase inhibitor z-VAD-FMK, and there was no evidence of apoptotic activation as determined by caspase-3 cleavage. By contrast, ORF3a expression did not augment coagulation in cells deficient in the calcium-dependent phospholipid scramblase TMEM16F. Similarly, ORF3a-enhanced TF procoagulant activity (p < 0.01) and prothrombinase activity (p<0.05) was completely abrogated using TMEM16 inhibitors, including the uricosuric agent benzbromarone that has been registered for human use in over 20 countries. Live SARS-CoV-2 infection of A549-ACE2 cells increased cell surface factor Xa generation at MOI 0.1 (p < 0.01) but not MOI 0.01 or following heat inactivation of the virus, and RNA sequencing confirmed ORF3a induction without increased F3 expression. RNA sequencing of human SARS-CoV-2 infected lung autopsy and control tissue (n= 53) confirmed these findings in vivo. Immunofluorescence staining for ORF3a and KRT8/18 and CD31 in SARS-CoV-2 infected human lung autopsy specimens demonstrated ORF3a expression in pulmonary epithelium and endothelial cells, highlighting the potential pathologic relevance of this mechanism. Here we demonstrate that expression of the SARS-CoV-2 accessory protein ORF3a increases the intracellular calcium concentration and TMEM16F-dependent PS scrambling to augment procoagulant activity of the tenase and prothrombinase complexes. Our studies of human cells and tissues infected with SARS-CoV-2 support the pathologic relevance of this mechanism. We highlight the therapeutic potential to target the ORF3a-TMEM16F axis as with benzbromarone to mitigate dysregulation of coagulation and thrombosis during severe SARS-CoV-2 infection. Disclosures: Schwartz: Miromatrix Inc: Membership on an entity's Board of Directors or advisory committees;Alnylam Inc.: Consultancy, Speakers Bureau. Schulman: CSL Behring: Consultancy, Research Funding.

Earth Materials and Environmental Applications 2020

The road performance, dynamic properties, and environment impact tests on mechanical property and environmental safety of an industrial residue waste subgrade material were evaluated. The effect of calcium carbonate on the mechanical properties and microstructure of red clay was studied by adding precipitated calcium carbonate to red clay at ratios of 0%, 5%, 10%, 15%, and 20%, and then, shear tests were conducted. In the study of sorptive removal of color dye safranin O (SO) by fibrous clay minerals and zeolites, it was found that the cation exchange capacity (CEC) of the minerals played a key role in SO+ removal and the sorbed SO+ cations were limited to the external surfaces of the minerals due to limited channel size of the fibrous minerals.

E484K and N501Y SARS-CoV 2 spike mutants Increase ACE2 recognition but reduce affinity for neutralizing antibody.

SARS-CoV2 mutants B.1.1.7, B.1.351, and P.1 contain a key mutation N501Y. B.1.135 and P.1 lineages have another mutation, E484K. Here, we decode the effect of these two mutations on the host receptor, ACE2, and neutralizing antibody (B38) recognition. The N501Y RBD mutant binds to ACE2 with higher affinity due to improved π-π stacking and π-cation interactions. The higher binding affinity of the E484K mutant is caused due to the formation of additional hydrogen bond and salt-bridge interactions with ACE2. Both the mutants bind to the B38 antibody with reduced affinity due to the loss of several hydrogen-bonding interactions. The insights obtained from the study are crucial to interpret the increased transmissibility and reduced neutralization efficacy of rapidly emerging SARS-CoV2 VOCs.