Metformin in COVID-19: Is There a Role Beyond Glycemic Control?

Context: The coronavirus disease 2019 (COVID-19) pandemic is still a cause of worldwide health concern. Diabetes and its associated comorbidities are risk factors for mortality and morbidity in COVID-19. Selecting the right antidiabetic drug to achieve optimal glycemic control might mitigate some of the negative impacts of diabetes. Metformin continues to be the most widely administered antidiabetic agent. There is evidence of its beneficial outcome in COVID-19 independent of its glucose-lowering effect. Evidence Acquisition: A thorough literature search was conducted in PubMed, Google Scholar, Scopus, and Web of Science to identify studies investigating metformin in COVID-19. Result(s): Several overlapping mechanisms have been proposed to explain its antiviral properties. It could bring about conformational changes in the angiotensin-converting enzyme-2 receptor and decrease viral entry. The effects on the mammalian target of the rapamycin pathway and cellular pH have been proposed to reduce viral protein synthesis and replication. The immunomodulatory effects of metformin might counter the detrimental effects of hyperinflammation associated with COVID-19. Conclusion(s): These findings call for broader metformin usage to manage hyperglycemia in COVID-19.Copyright © 2023, International Journal of Endocrinology and Metabolism.

Beyond Articular Cartilage: A Systematic Review of the Extracellular Matrix in Other Components of the Osteoarthritic Joint

Background/Aims Traditionally viewed from the perspective of cartilage degeneration, osteoarthritis is increasingly seen as a disease of global joint dysfunction. Connective tissue extracellular matrix (ECM) is a crucial determinant of joint mechanobiology, providing cells with scaffolding, topographical cues, and a reservoir of soluble factors. While ECM dysregulation has been extensively studied in osteoarthritic cartilage, it remains poorly defined in other joint tissues. Here, we systematically review the composition, architecture, and remodelling of non-cartilage soft joint tissue ECM in human osteoarthritis and animal disease models. Methods A systematic search strategy was run through the MEDLINE, EMBASE and Scopus databases on 30 October 2020 and repeated on 1 October 2021. The search criteria included disease nomenclature, relevant tissues, as well as structural ECM components and architectural features. All papers were independently screened by two reviewers on the Covidence platform according to predefined eligibility criteria. Relevant clinical, demographic, and biological data were extracted from included studies, which were assessed for bias using the OHAT Risk of Bias Rating Tool for Human and Animal Studies. Results 148 of 8,156 identified studies met all eligibility criteria. 113 papers evaluated human osteoarthritis;of 35 animal studies, the most frequently used models involved surgical joint destabilisation in small mammals. ECM was best defined in menisci, ligaments, and synovium;fewer papers assessed skeletal muscles, tendons, and fat pads. Compared to the healthy joint, osteoarthritis is associated with qualitative and quantitative alterations in structural ECM components, most notably collagens and proteoglycans. In recent years, whole proteome sequencing has been employed to address these changes systematically. The mechanical properties of ECM change significantly in osteoarthritis in response to post-translational modifications, extensive calcification, and the marked loss of matrix organisation across the joint. Notably, some aspects of ECM remodelling in these tissues appear to precede discernible cartilage dysregulation. Similar ECM dysregulation is also observed in animal models, although intermodel variability in arthritogenic precipitant and the range of reported outcomes make comparisons difficult. Many studies are limited by significant bias, notably in the infrequent reporting of investigator blinding, and in the poor demographic matching of osteoarthritic and control patients. Encouragingly, the quality of methodology reporting and use of age-matched control populations have improved in recent years. Conclusion Current data provide compelling evidence of whole joint ECM changes in osteoarthritis and importantly suggest that these changes occur early in the disease process. How ECM dysfunction affects the behaviour of tissue-resident cells remains less well understood. Our work will support the design of disease-relevant biomaterials used to model osteoarthritis in vitro, helping to address this issue, by more accurately recreating the extracellular environment. Furthermore, the development of imaging modalities sensitive to connective tissue ECM changes warrants investigation from both diagnostic and prognostic perspectives.

A Review on Recent Monkey Pox Outbreak in 2022

Monkey pox, a zoonotic disease with clinical symptoms resembling smallpox, unexpectedly broke out and spread over the world after the outbreak of COVID-19, severely affecting several of the continents of the world. Monkey pox is currently a member of the genus otrhopox virus, which is a member of the sub family chorodoxvirinae. According to the available knowledge, small mammals and rodents have all been identified as potential sources of the monkey [ox virus]. The disease is characterized by a short febrile illness with lymphadenopathy followed by a rash which spreads centrifugally and passes through phases of macules, papules, vesicles, and pustules. Recovery occurs in most patients within 2-4 wk. Complications are more likely in children, pregnant women, and the immunocompromised. Specific diagnosis is by detection of viral DNA by PCR.Tecovirimat, brincidofovir, and cidofoviir are the medications used to treat monkey pox, immunoglobulin and new compounds are the vaccinations. This review will introduce a general overview of MPXV and describe the epidemiology, clinical features, evaluation, and treatment of monkey pox patients.Copyright © Journal of Global Trends in Pharmaceutical Sciences.

Genome editing technology and applications with the type I CRISPR system

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems, which are representative genome editing technologies, are classified into class 1 and class 2 in terms of evolutionary biology and are further classified into several subtypes. Class 2 CRISPR systems, including type II Cas9 and type V Cas12a, are the most commonly used for genome editing in eukaryotic cells, while type I CRISPR systems within Class 1 are also becoming available. Type I CRISPR recognizes longer target sequences than CRISPR-Cas9 and can induce large deletion mutations of several kilobases. These features demonstrate its potential as a novel and unique genome editing tool that can induce genetic disruption safely and reliably. Thus, it is expected to be utilized for gene therapy and industrial applications. Recently, the DNA cleavage mechanism of type I CRISPR has also revealed details from protein-complex analyses with X-ray crystallography, cryo-electron microscopy, and high-speed atomic force microscopy. The single-strand DNA trans-cleavage activity of type I CRISPR, called collateral activity, has broadened the potential application for CRISPR diagnostics, especially in the development of point-of-care testing methods for COVID-19. In this review, we present an overview of the type I CRISPR system, its application to genome editing, and genetic diagnosis using CRISPR-Cas3.Copyright © 2022

Administration of Exogenous Surfactant and Cytosolic Phospholipase A2alpha Inhibitors may Help COVID-19 Infected Patients with Chronic Diseases

Background: Coronavirus-19 (COVID-19) pandemic is a worldwide public health problem causing 347,070 deaths from December 25, 2019, till May 25, 2020. Phospholipids are structural components of mammalian cytoskeleton and cell membranes. Phosphatidylglycerol is an anionic lipid found in mammalian membranes in low amounts (1-2%) of the total phospholipids. Also, phosphatidylglycerol suppresses viral attachment to the plasma membrane and subsequent replication in lung cells. Phosphatidylglycerol depletion caused by over expression of cytosolic phos-pholipase A2alpha induces lipid accumulation in lung alveoli and promotes acute respiratory distress syndrome (ARDS). An exogenous-surfactant replacement has been successfully achieved in ARDS and improved oxygenation and lung mechanics. Inhibition of cytosolic phospholipase A2alpha impairs an early step of COVID-19 replication. Aim(s): The present study was carried out to explain the correlation between the administration of exogenous artificial surfactant as well as cytosolic phospholipase A2alpha inhibitors to improve oxygenation and lung mechanics and inhibit COVID-19 replication. Method(s): Database research was carried out on Medline, Embase, Cochrane Library, country-spe-cific journals, and following-up WHO reports published between December 25, 2019-May 25, 2020. Result(s): Till 25 May 2020, coronavirus cases were 5,307,298, with 347,070 deathsand 2,314,849 recovered cases. According to the WHO reports, most COVID-19 deaths seen are in people who suffered from other chronic diseases characterized by phospholipidosis and phosphatidylglycerol deficiency, including hypertension, liver, heart, and lung diseases and diabetes. Phospholipases A2 (PLA2) catalyze the cleavage of fatty acids esterified at the sn-2 position of glycerophospholipids leading to enhanced inflammation and lung damage. Also, cytosolic phospholipase A2alpha inhibitors may reduce the accumulation of viral proteins and RNA. In addition, administration of exogenous phospholipid surfactant may help COVID-19 infected patients with ARDS to remove inflammatory mediators. Conclusion(s): The present study showed a relation between phosphatidylglycerol deficiency in COVID-19 infected patients with ARDS and/or chronic diseases and their mortality. These findings also showed an important approach for the prevention and treatment of COVID-19 infections by using cytosolic phospholipase A2alpha inhibitors and exogenous administration of a specific phos-pholipid surfactant.Copyright © 2021 Bentham Science Publishers.

The Emergence of Novel Coronavirus Disease, Global Treatment Update and its Containment Strategies in Overpopulated Countries: A Review

The ongoing pandemic of the novel coronavirus SARS-CoV-2 (COVID-19) has created a major challenge for the public health worldwide. The reported cases indicate that the outbreak is more widespread than initially assumed. Around 18 million people have been infected with 689,000 reported deaths (August 2020;the number is increasing daily);with a high mutation rate, this virus poses an even more serious threat worldwide. The actual source of COVID-19 is still un-clear;even if the initial reports link it to the Chinese seafood wet market in Wuhan, other animals such as birds, snakes, and many small mammals including bats are also linked with this novel coro-navirus. The structure of the COVID-19 shows distinctive proteins among which spike proteins have a pivotal role in host cell attachment and virus-cell membrane fusion in order to facilitate virus infection. Currently, no specific antiviral treatment or vaccine is available. Various drug can-didates, including SARS-CoV and MERS-CoV protease inhibitors, neuraminidase inhibitors, RNA synthesis inhibitors, ACE2 inhibitors and lungs supportive therapy, are under trials. Cell-based therapy also appeared with remarkable treatment possibilities. In this article, we endeavored to succinctly cover the current and available treatment options, including pharmaceuticals, cell-based therapy, and traditional medicine. We also focused on the extent of damages by this novel coron-avirus in India, Pakistan, and Bangladesh;the strategies adopted and the research activities initiat-ed so far by these densely populated countries (neighboring China) are explained in this review.Copyright © 2021 Bentham Science Publishers.

Transcriptomic analyses unravel differential expression of genes involved in the N-glycosylation pathway of Phaeodactylum tricornutum ecotypes

Most of the biologics are glycoproteins. It is well-established that N-glycans harboured by proteins are involved in the protein half-life, bioactivity and immunogenicity. Currently, most of the biologics are produced in mammalian cells. However, microalgae emerged as a cheaper alternative biofactory. Among them, the diatom Phaeodactylum tricornutum benefits from numerous advantages and has been successfully used to produce biologics such as SARS-COV2 RBD and functional monoclonal antibodies (mAbs). These mAbs have been demonstrated to be glycosylated with oligomannosides that are similar to the mammalian ones and that result from processing steps occurring in the ER and the early Golgi apparatus. Surprisingly, these oligomannosides represent the major N-glycans population even if the diatom possesses glycoenzymes potentially involved in the biosynthesis of complex-type N-glycans in the Golgi apparatus. Therefore, it is essential to characterize the regulation of the P. tricornutum protein N-glycosylation pathway as well as the expression level of genes involved in the N-glycosylation of proteins. In the present work, we performed RNA-Seq analyses on different ecotypes of P. tricornutum and decode the differential expression of genes involved in the protein N-glycosylation pathway.

Description of the first case of SARS-CoV-2 reinfection in Galicia (Spain)

This study presents the first confirmed case of SARS-CoV-2 reinfection in Galicia, Spain, and a summary of the data on the first reinfections studied in the autonomous community. The first patient with a confirmed reinfection, aged 69, started clinical COVID-19 on 28/03/2020 with coughing and asthenia and oxygen saturation of 97%-98%.98%. He then complained of persistent weariness for several months. The clinical episode occurred while in quarantine on 03 March 2022 due to intimate interaction with her cohabitating daughter and a positive COVID-19 (secondary attack rate of 33%). The first positive result was achieved on 03 March 2020 (E gene CT<30, RdRP/S gene CT<30, N gene CT<30). Noserological study was done, and the patient was discharged on 04 March 2020 according to epidemiological guidelines. On 9 January 2021 he presented a second COVID-19 case after 289 days. In this second episode, the patient developed cough and general malaise on 09/01/2021 and was PCR positive on the same day (E gene CT<30, RdRP genes <30%). In both cases, the patient progressed well and did not require hospitalization. Between 1 April 2021, and 10 March 2021, there were 485 re-infections recorded in the Autonomous Community of Galicia, with 245 occurring in the province of A Corua (51%), 68 in Lugo (14%), 37 in Ourense (8%) and 135 in Pontevedra (28%). Because reinfection features and patterns may change, information must be collected throughout the pandemic.

Mutagenicity of N-Hydroxycytidine and Monlnupiravir in E. Coli and Mammalian Cell Cultures

Molnupiravir (MOV) has received FDA's Emergency Use Authorization for the treatment of COVID-19, which is caused by SARS-CoV-2 infection. MOV is a prodrug of the ribonucleoside analog, Nhydroxycytidine (NHC). Upon phosphorylation, NHC incorporates into nascent viral RNA during replication triggering “catastrophic” mutation of the viral genome. However, NHC can also enter the deoxy-ribonucleotide pool, become incorporated into DNA, and cause DNA mutations. In nonclinical safety assessments, MOV was positive (i.e., mutagenic) in the Ames assay but negative in regulatory in vitro and in vivo micronucleus assays. Multiple in vitro studies conducted in bacteriophages, bacteria, fungi, and mammalian cells have reported that NHC can induce DNA mutations, mainly A:T>G:C transitions. We used a recently developed error-corrected wholegenome sequencing technique for detecting mutations induced by MOV and NHC in cultures of E. coli, mouse L5178YTk+/-, and human TK6 cells. Treatment of bacterial and mammalian cultures (for 4 hours and 5 days, respectively) with either MOV or NHC increased mutation frequencies in a dose-dependent manner in all three models. The majority of induced mutations were A:T>G:C, consistent with the type of mutation caused by incorporation of dNHC opposite to dA in the first round of DNA replication and incorporation of dG opposite to dNHC in the subsequent round(s) of DNA replication. Trinucleotide mutational signatures in MOV/NHC-treated cells were similar in mouse and human cells and different from the background spontaneous mutational signatures in parental cell cultures. The specific mutational signature was evident in mammalian cells exposed to NHC concentrations comparable to those observed in the plasma of human subjects who received clinical doses of MOV. This data indicates more well-controlled rodent and clinical studies of MOV/NHC-induced mutagenicity should be done in the interest of public health safety.

The expression of SARS-CoV 2 co-receptors and glia reaction in eyes of normal and COVID-19 diseased patients

Purpose : With this research project we wanted to approach the question of whether SARS-CoV 2 can infect the eye. In order to infect ocular tissues, virus-specific receptors;coreceptors or proteases must be present in the eye tissue. SARS-CoV 2 uses the human angiotensin converting enzyme 2 (ACE2) receptor to enter cells. In addition, the mammalian serine protease TMPRSS2, the protease furin and the glycoprotein neuropilin are identified as relevant proteases for the interaction of the virus with ACE2. Last year, we were able to show that ACE2 is significantly more expressed in ocular tissue of covid patients. Here the expression level of the co-receptor and glia markers, as well as the present of virus was confirmed in this study. Methods : Seven eyes from donors without covid disease (COVID-) as well as ten fixed eyes from COVID-19 patients (COVID+) were analysed for their expression profile of ACE2, TMPRSS2, neuropilin and furin in the retina and cornea. The ocular tissues were examined for protein expression by immunohistochemical staining or for RNA expression by quantitative real-time PCR. In addition, viral spike protein was detected histologically in eyes, and expression profiles of GFAP and Iba-1 were assessed. Results : Similar to ACE2 and TMPRSS2, the two proteases neuropilin and furin were detected in the retina and cornea. Interestingly, the expression profile differed in terms of strength and localization, especially in the retina. The presence of the virus in both cornea and retina was also demonstrated by the detection of viral spike protein. In all COVID+ retinas, strong GFAP staining was observed as well as some Iba-1 positive cells, suggesting activation of macro- and microglia. Conclusions : Expression of ACE2, TMPRSS2, furin and neuropilin was demonstrated in COVID+ ocular tissues. In addition to the virus detection in retina and cornea, a glial reaction could also be observed. One can therefore assume an infection of the eye in these cases. However, in summary it can be said that an infection of the eye tissue is possible since all demanded receptors are present.

TRP ion channels and approaches in COVID-19

Living systems have hundreds of ion channels on their surfaces. The TRP protein family defined in Drosophila is one of the channels providing ion passage and is present in living systems from simple organisms to complex. TRP channels have various roles in sensory systems and are located in almost all cells. TRP cation channels of mammals consist of seven subfamilies and each of them has their own structure, location etc. (Clapham, 2003). Since they play a role in sensory transmission, sciencists think that they could be potential targets for relieving the symptoms of various diseases (Miller, 2006). COVID-19 disease caused by the SARS-Cov-2 virus occurs with symptoms such as headache, muscle pain, respiratory and digestive problems, and loss of taste and smell. TRP channels have been targeted in some approaches to reduce these symptoms. In some studies that blocked TRP channels, the symptoms were observed to disappear or to decrease significantly (Fernandes et al., 2012). The virus requires angiotensin converting enzyme 2 and transmembrane protease serine 2 proteins to enter host cells. These processes are mediated by endocytosis and by Ca+ flow. In this context, it has been observed that the blocking of TRP channels hinders the entry of the virus into the host. The aim of this review is to examine the severe symptoms, the potential roles of TRP ion channels in the spread and progression processes of the COVID-19 pandemic, which led to a worldwide crisis, as well as their therapeutic approaches.

A Comprehensive Review of Anti-Diabetic Drugs in COVID-19 Patients Co-morbidities with Diabetes

Diabetes is a chronic metabolic disorder emerging as a global burden. Diabetes serves as a risk factor for many complications inclusive of COVID-19. The SARS – 2 pathogens have led to Coronavirus disease. Coronaviruses are enveloped viruses with a single-stranded, positive-sense RNA genome recognized to cause respiratory infections in human beings. Diabetes patients being affected by coronavirus become more critical due to worsening hyperglycemia induces aggravation, endothelial dysfunction, and occlusion of blood vessels through the era of oxidative stress riding the down-regulation of glucose metabolism and hyperglycemia. Increased glucose level causes inflammation and tissue damage serves as a supportive factor for higher tissue damage in COVID patients. Sufferers with extreme COVID-19 have an exceptionally impaired interferon type 1 response with low IFN alpha activity in the blood, indicating excessive blood viral load, and an impaired inflammatory response. This can be alleviated by regular screening and appropriate therapy as like as metformin, camostat mesylate, chloroquine, and adjunctive therapy. Metformin is the desired preliminary drug to deal with T2DM. Camostat mesylate drug accelerated glycemia and insulin resistance and reduced fat buildup in mammalian models. Adjunctive treatment can be used to obviate the evolution of COVID-19.

The development of a novel SARS-CoV-2 virus neutralizing immunotoxin

For decades, scientific efforts were focused on the improvement of the effectiveness of the therapeutic antibodies, mainly in order reduce the dosage and thus lower the side-effects and costs. P4A1, a potent SARS-CoV-2 virus neutralizing antibody was already engineered to contain Fc fragment mutations, that dramatically increased the blood circulation time. In this work, we aimed to further enhance this neutralizing antibody efficacy by creating a next-generation virus neutralizing agent based on the P4A1 and conjugated with a highly processive Bacillus amyloliquefaciens RNase (barnase). Barnase itself is known to act as a mild toxin that drives the cells to apoptosis, and we propose that its RNase activity may enhance the protective effect through the hydrolysis of viral RNA in infected cells, and thereby additionally preventing pathogen replication. The main challenge in the assembly of such molecule is the intrinsic barnase toxicity in mammalian cells, what precludes the possibility to express it as a fusion protein. Further, we had shown that barnase, being a small (12.5 kDa) protein, contains very few surface reactive moieties that are available for conventional chemical crosslinking strategies. Therefore, the antibody-barnase fusion protein was obtained by enzymatic conjugation via the sortase A enzyme. The reaction conditions for bacterially expressed barnase and HEK293 derived P4A1 modified to contain heavy chain C-terminal sortase motif were thoroughly optimized and the reaction yield approached 80%. The immunotoxin RBD binding EC50 was not found to differ from the unconjugated P4A1 antibody and barnase activity was found to be 33% of the one for unmodified enzyme. Thus, we obtained the promising immunotoxin with a good yield, which had retained its RNase activity for the further in vitro virus neutralization studies.

Aromatic heptaene macrolides as an initial 'golden shot' towards systemic mycoses

Systemic mycoses are fungal diseases attacking the body through an internal organ. They are the cause of increasing morbidity rates, especially among the immunocompromised population (e.g. with HIV), but also exist as a co-infection in patients with COVID-19. Nowadays, the 'life-saving/last chance' drug (and a poison to humans), Amphotericin B (AmB), is the representative of nonaromatic heptaene macrolides. However, previously obtained data indicate significantly higher antifungal activity of the aromatic heptaene subgroup (AHM), which is unfortunately correlated with their high mammalian toxicity. The undertaken studies concern Partricin and Candicidin complexes as the main representatives of AHMs. Three aromatic heptaene macrolides: Partricin A, Partricin B and Candicidin D were isolated from fermentation broths of S. aureofaciens and S. griseus, respectively, by initial purification, followed by selected chromatographic techniques such as centrifugal partitioning chromatography (CPC) and prep-HPLC. Later on, they were subjected to controlled chromophore geometry change (cis-trans to all-trans) via photochemical isomerization, using UV light of k = 365 nm. The obtained all-trans isomers, regarded as aromatic analogues of AmB, were tested for in vitro selective toxicity indexes (STIs) towards C. albicans cells versus human erythrocyte model. Due to the diminished hemotoxicity of the all-trans isomers - which were maintaining most of their fungicidal power - the obtained STIs were very promising and far better than for AmB (previously published in: J.Górska et al. (2021) Int. J. Mol. Sci. 22). Hence, the all-trans AHMs bear a great potential to be weaponized on the antifungal battlefield. Nevertheless, since individual AHMs are commercially unavailable and difficult to obtain due to their complex nature, our studies will focus on facilitation of the isolation process and further improvement of their STIs, mainly through rational, structural modifications.

Zebrafish embryos to study BSL-3 viruses and antiviral molecules

Emerging viruses are currently a burden with the recent SARS-CoV-2 pandemic resulting in more than 6 million death worldwide. Other viruses such as Arboviruses, transmitted by mosquitoes, can also emerge easily and represent a threat for humans and animals. These viruses are often RNA enveloped viruses and require to be studied in a BSL-3 (Bio-Safety- Laboratory of level 3 security), underlying the need to develop simple and rapid antiviral screenings. In recent years, the zebrafish has become a powerful tool in the biomedical sector to study viral infection and immunity. The optical transparency of the zebrafish embryo offers a major advantage for real time imaging of the infection process, and study hostpathogen interactions at subcellular levels in living systems using fluorescently labelled pathogens. While zebrafish embryos have been shown to be a successful vertebrate model to study a large panel of human disease, this model has been very little explored to study BSL-3 pathogen infection and propagation. This project was created to develop the zebrafish infection model for emerging BSL-3 viruses, and evolve towards high-content screening methods for antiviral molecules. Thanks to the setup of a microinjection system under a laminar flow hood in the BSL-3, we were able to inject in zebrafish embryos several emerging viruses, including the Chikungunya virus (CHIKV), Dengue virus (DENV) and SARS-CoV-2. Using fluorescently tagged viruses, infection was monitored in real time in vivo and confirmed with classical virology methods (RT-qPCR, plaque assays, TCID50). We then tested several nucleoside analogues described for their antiviral activity in vitro for these different viruses and we were able to validate the antiviral effect of some of these molecules in infected embryos. Altogether, our zebrafish infection model will provide us a better understanding of in vivo infection and propagation of these emergent BSL-3 viruses, and will be used as an intermediate model between in vitro antiviral screens and in vivo screens in mammals.

Human impacts on mammals in and around a protected area before, during, and after COVID-19 lockdowns.

The dual mandate for many protected areas (PAs) to simultaneously promote recreation and conserve biodiversity may be hampered by negative effects of recreation on wildlife. However, reports of these effects are not consistent, presenting a knowledge gap that hinders evidence-based decision-making. We used camera traps to monitor human activity and terrestrial mammals in Golden Ears Provincial Park and the adjacent University of British Columbia Malcolm Knapp Research Forest near Vancouver, Canada, with the objective of discerning relative effects of various forms of recreation on cougars (Puma concolor), black bears (Ursus americanus), black-tailed deer (Odocoileus hemionus), snowshoe hares (Lepus americanus), coyotes (Canis latrans), and bobcats (Lynx rufus). Additionally, public closures of the study area associated with the COVD-19 pandemic offered an unprecedented period of human-exclusion through which to explore these effects. Using Bayesian generalized mixed-effects models, we detected negative effects of hikers (mean posterior estimate = -0.58, 95% credible interval [CI] -1.09 to -0.12) on weekly bobcat habitat use and negative effects of motorized vehicles (estimate = -0.28, 95% CI -0.61 to -0.05) on weekly black bear habitat use. We also found increased cougar detection rates in the PA during the COVID-19 closure (estimate = 0.007, 95% CI 0.005 to 0.009), but decreased cougar detection rates (estimate = -0.006, 95% CI -0.009 to -0.003) and increased black-tailed deer detection rates (estimate = 0.014, 95% CI 0.002 to 0.026) upon reopening of the PA. Our results emphasize that effects of human activity on wildlife habitat use and movement may be species- and/or activity-dependent, and that camera traps can be an invaluable tool for monitoring both wildlife and human activity, collecting data even when public access is barred. Further, we encourage PA managers seeking to promote both biodiversity conservation and recreation to explicitly assess trade-offs between these two goals in their PAs.

DESIGN of NOVEL and HIGHLY SELECTIVE SARS-CoV-2 MAIN PROTEASE INHIBITORS

Background: SARS-CoV-2 has caused a global pandemic, yet despite vaccine availability, it continues to inflict morbidity and mortality worldwide. The viral main protease (Mpro) is highly conserved across multiple coronaviruses and has a unique viral substrate specificity. Thus, highly selective Mpro inhibitors are expected to be safe, effective, and elude drug resistance for future coronaviruses. Methods: We used a conformationally restricted peptidomimetic to mimic the bioactive conformation of the Mpro-substrate complex to identify potent, selective Mpro inhibitors. We evaluated protease inhibition in biochemical assays, and cellular efficacy in Vero-E6 cells challenged with live virus representing parental (USA-WA1/2020), beta (B.1.351), and delta (B.1.617.2) variants by monitoring infection at day 2 post-infection measuring nucleocapsid-positive cells by high content imaging, and cytopathic effect (CPE) at day 4 post-infection using resazurin viability dye. Results were compared to reference compounds. Group differences were analyzed by two-sided, paired t-test. Results: AP-8-013 required a 2-hour incubation to achieve maximal dose-dependent Mpro inhibition with an IC50 = 230 ± 18 nM, reflecting its highly constrained conformation, compared to the more flexible Cpd 22 (AP-8-001;IC50 = 11 ± 0.7 nM) or GC-376 (IC50 = 18 ± 1.5 μM). Importantly, AP-8-013 showed exquisite selectivity for Mpro with no inhibition at key mammalian cysteine proteases, cathepsin B and L, or the serine protease thrombin, while Cpd 22 (Cat B IC50 = 24 ± 7.5 nM, Cat L IC50 = 1.8 ± 0.3 nM) or GC-376 (Cat B IC50 = 37 ± 1.5 nM, Cat L IC50 = < 1 nM) showed poor selectivity towards mammalian cysteine proteases. AP-8-013 was active in CPE cell-based assays with comparable potency to reference compounds, with EC50 = 4.7 μM compared to Cmp 22 (EC50 = 1.4 μM) or GC-376 (EC50 = 1.1 μM). Using intact SARS-CoV-2 infection-based assays, AP-8-013 significantly inhibited parental virus as well as beta and delta VOC (EC50s = 2.7, 2.5, and 6.0 μM, respectively). Finally, a 3:1 molar mixture of AP-8-013 and remdesivir significantly enhanced antiviral activity in CPE assays (EC50 = 1.3 μM;p < 0.05) when compared against either compound alone (EC50s = 4.7 and 3.3 μM, respectively). Conclusion: We have identified a novel drug-like Mpro inhibitor lead series which is highly selective over cysteine and serine proteases that can inhibit multiple SARS-CoV-2 VOC and increase the antiviral activity of remdesivir.

Serosurvey of Selected Zoonotic Pathogens in Polar Bears (Ursus maritimus Phipps, 1774) in the Russian Arctic

Antibodies to several pathogens were detected in the serum samples of nine polar bears (Ursus maritimus, Phipps, 1774) from areas of the Russian Arctic. Plasma was studied for antibodies to sixteen infectious and parasitic diseases using indirect Protein-A ELISA. It is known that when using ELISA, the interaction of antibodies with a heterologous antigen is possible due to immunological crossings between antigens. We investigated the plasma for the presence of antibodies to the major pathogens and for the presence of antibodies to pathogens, for which the cross-immunological reactions to these pathogens are described. For example, antibodies to the pathogens of opisthorchiasis, clonorchiasis, and ascariasis were found simultaneously in four polar bears. Antibodies to both anisakidosis and trichinellosis pathogens were found in six animals. The data obtained may also indicate a joint invasion by these pathogens. Unfortunately, due to the small number of animals sampled, it is impossible to carry out statistical processing of the data.