Geospatial forest fire risk assessment and zoning by integrating MaxEnt in Gorkha District, Nepal.

Forest fires are an imminent danger to natural forest ecosystems, and carrying out zoning studies and forest fire risk assessments are of great practical significance in steering fire prevention, minimizing fire incidents, and limiting the environmental consequences of fire. Using the Gorkha district of Nepal as a case study, this study used remotely sensed high-temperature fire data as the forest fire sample. Nine parameters related to topography, climatic conditions, vegetation, and human intervention were used as environmental variables affecting fire occurrence. Next, a MaxEnt forest fire risk assessment model was generated with GIS and R, which analysed the contribution, significance, and responses of environmental variables to the forest fire in Gorkha District. The findings demonstrate that (1) following a test of sample locations for forest fires, the MaxEnt model has excellent relevance and practicality when applied to fire risk assessment; (2) Out of 2747 fire points in the forest, only 110 Spatio-temporally independent fire points were used for the model building having high and normal confidence level. Regarding Area Under Curve (AUC) values, the training data yielded results of 0.875, while the test data produced acceptable results of 0.861 with a standard deviation of 0.0322; (3) the importance of climatic and Land Use Land Cover (LULC) variables to forest fire are 56.2 % and 32.9 %, respectively, and their contribution to forest fire are 32 % and 47.6 %, respectively. (4) There are numerous and intricate ways that environmental factors influence forest fires. The forest fire response curves to the nine chosen environmental variables are complex and nonlinear rather than linear; Maximum temperature of the warmest month (bio_5), Isothermality (bio_3), Precipitation of Driest Quarter (bio_17) and mean Diurnal Range (bio_2) bear a nonlinear positive link with the possibility of forest fires. In contrast, elevation, slope, temperature seasonality (bio_4), distance from the settlement, and LULC have a favorable stimulating response to the possibility of forest fires within an appropriate interval. (5) In Gorkha, there are geographical differences in the risk of forest fires. Only 12.83 % of the whole area is made up of areas at significantly high risk or above, compared to 87.17 % for high-risk and below.

SRX3177, a CDK4/6-PI3K-BET inhibitor, in combination with an RdRp inhibitor, Molnupiravir, or an entry inhibitor MU-UNMC-2, has potent antiviral activity against the Omicron variant of SARS-CoV-2.

Despite considerable progress in developing vaccines and antivirals to combat COVID-19, the rapid mutations of the SARS-CoV-2 genome have limited the durability and efficacy of the current vaccines and therapeutic interventions. Hence, it necessitates the development of novel therapeutic approaches or repurposing existing drugs that target either viral life cycle, host factors, or both. Here, we report that SRX3177, a potent triple-activity CDK4/6-PI3K-BET inhibitor, blocks replication of the SARS-CoV-2 Omicron variant with IC50 values at sub-micromolar concentrations without any impact on the cell proliferation of Calu-3 cells at and below its IC50 concentration. When SRX3177 is combined with EIDD-1931 (active moiety of a small-molecule prodrug Molnupiravir) or MU-UNMC-2 (a SARS-CoV-2 entry inhibitor) at a fixed doses matrix, a synergistic effect was observed, leading to the significant reduction in the dose of the individual compounds to achieve similar inhibition of SARS-CoV-2 replication. Herein, we report that the combination of SRX3177/MPV or SRX3177/UM-UNMC-2 has the potential for further development as a combinational therapy against SARS-CoV-2 and in any future outbreak of beta coronavirus.

Efficient trapping and destruction of SARS-CoV-2 using PECO-assisted Molekule air purifiers in the laboratory and real-world settings.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is transmitted human-to-human via aerosols and air-borne droplets. Therefore, capturing and destroying viruses from indoor premises are essential to reduce the probability of human exposure and virus transmission. While the heating, ventilation, and air conditioning (HVAC) systems help in reducing the indoor viral load, a targeted approach is required to effectively remove SARS-CoV-2 from indoor air to address human exposure concerns. The present study demonstrates efficient trapping and destruction of SARS-CoV-2 via nano-enabled filter technology using the UV-A-stimulated photoelectrochemical oxidation (PECO) process. Aerosols containing SARS-CoV-2 were generated by nebulization inside an air-controlled test chamber where an air purifier (Air Mini+) was placed. The study demonstrated the efficient removal of SARS-CoV-2 (99.98 %) from the test chamber in less than two minutes and PECO-assisted destruction (over 99%) on the filtration media in 1 h. Furthermore, in a real-world scenario, the Molekule Air-Pro air purifier removed SARS-CoV-2 (a negative RT-qPCR result post-running the filter device) from the circulating air in a COVID-19 testing facility. Overall, the ability of two FDA-approved class II medical devices, Molekule Air-Mini+ and Air-Pro air purifiers, to remove and destroy SARS-CoV-2 in indoor settings was successfully demonstrated. The study indicates that as the "tripledemic" of COVID-19, influenza, and respiratory syncytial virus (RSV) overwhelm the healthcare facilities in the USA, the use of a portable air filtration device will help contain the spread of the viruses in close door facilities, such as in schools and daycare facilities.

Burden of Care among Mothers Having Children with Down Syndrome.

BACKGROUND: Down syndrome is the most common chromosomal disorder associated with mental retardation. Parents who are the primary caregivers of a child with a disability face numerous challenges in their day-to-day life. The objective of the study was to find out the burden of care among mothers having children with down syndrome. METHODS: A descriptive cross-sectional study was carried out among 96 mothers having down syndrome children enrolled in the Down Syndrome Society, Nepal. Purposive Sampling technique was adopted for data collection. The Modified Caregiver Strain Index tool was used to collect data through interview. Data were collected from June 14, 2021 to August 1, 2021, which was analyzed by using descriptive and inferential statistics. RESULTS: Findings revealed that majority of the mothers (77.1%) had high level of burden of care. Majority (89.6%) of the mothers involved in the study were always overwhelmed about their child's conditions. More than half (55.2%) of the mothers were always financially strained in care giving, 57.3% had always done work adjustments and 60.4% of mothers always had emotional adjustments to be made. Similarly, 53.1% participants always felt that care giving was a physical strain. Burden of care was significantly associated with the age of delivery (p value= 0.008). CONCLUSIONS: The study concludes that mothers having children with down syndrome tend to have high level of burden of care and it is associated with the age at delivery. Therefore, health care providers including concerned authority are recommended to conduct different programs to support the caregivers in order to reduce their burden as well as to raise awareness program related to preventive measures of down syndrome in community.

Engineering an ACE2-Derived Fragment as a Decoy for Novel SARS-CoV-2 Virus.

Entry inhibitors are an important resource in the response against emerging pathogens like the novel SARS-CoV-2, which enters human cells via interaction between the surface spike glycoprotein and the cellular membrane receptor angiotensin-converting enzyme 2 (ACE2). Using a combination of comparative structural analyses of the binding surface of the spike to ACE2, docking experiments, and molecular dynamics simulations, we identified a stable fragment of ACE2 that binds to the spike, is soluble, and is not predicted to bind to its physiological ligand angiotensin II. From this fragment we computationally designed and experimentally validated a smaller, stable peptide that disrupts ACE2-spike interaction at nanomolar concentrations, suggesting its potential use as a decoy that could interfere with viral binding by competition.

Systems biology analyses reveal enhanced chronic morphine distortion of gut-brain interrelationships in simian human immunodeficiency virus infected rhesus macaques.

Background: Commonly used opioids, such as morphine have been implicated in augmented SIV/HIV persistence within the central nervous system (CNS). However, the extent of myeloid cell polarization and viral persistence in different brain regions remains unclear. Additionally, the additive effects of morphine on SIV/HIV dysregulation of gut-brain crosstalk remain underexplored. Therefore, studies focused on understanding how drugs of abuse such as morphine affect immune dynamics, viral persistence and gut-brain interrelationships are warranted. Materials and methods: For a total of 9 weeks, rhesus macaques were ramped-up, and twice daily injections of either morphine (n = 4) or saline (n = 4) administered. This was later followed with infection with SHIVAD8EO variants. At necropsy, mononuclear cells were isolated from diverse brain [frontal lobe, cerebellum, medulla, putamen, hippocampus (HIP) and subventricular zone (SVZ)] and gut [lamina propria (LP) and muscularis (MUSC) of ascending colon, duodenum, and ileum] regions. Multiparametric flow cytometry was used to were profile for myeloid cell polarity/activation and results corroborated with indirect immunofluorescence assays. Simian human immunodeficiency virus (SHIV) DNA levels were measured with aid of the digital droplet polymerase chain reaction (PCR) assay. Luminex assays were then used to evaluate soluble plasma/CSF biomarker levels. Finally, changes in the fecal microbiome were evaluated using 16S rRNA on the Illumina NovaSeq platform. Results: Flow Cytometry-based semi-supervised analysis revealed that morphine exposure led to exacerbated M1 (CD14/CD16)/M2 (CD163/CD206) polarization in activated microglia that spanned across diverse brain regions. This was accompanied by elevated SHIV DNA within the sites of neurogenesis-HIP and SVZ. HIP/SVZ CD16+ activated microglia positively correlated with SHIV DNA levels in the brain (r = 0.548, p = 0.042). Simultaneously, morphine dependence depleted butyrate-producing bacteria, including Ruminococcus (p = 0.05), Lachnospira (p = 0.068) genera and Roseburia_sp_831b (p = 0.068). Finally, morphine also altered the regulation of CNS inflammation by reducing the levels of IL1 Receptor antagonist (IL1Ra). Conclusion: These findings are suggestive that morphine promotes CNS inflammation by altering receptor modulation, increasing myeloid brain activation, distorting gut-brain crosstalk, and causing selective enhancement of SHIV persistence in sites of neurogenesis.

Modular nanoarray vaccine for SARS-CoV-2.

The current vaccine development strategies for the COVID-19 pandemic utilize whole inactive or attenuated viruses, virus-like particles, recombinant proteins, and antigen-coding DNA and mRNA with various delivery strategies. While highly effective, these vaccine development strategies are time-consuming and often do not provide reliable protection for immunocompromised individuals, young children, and pregnant women. Here, we propose a novel modular vaccine platform to address these shortcomings using chemically synthesized peptides identified based on the validated bioinformatic data about the target. The vaccine is based on the rational design of an immunogen containing two defined B-cell epitopes from the spike glycoprotein of SARS-CoV-2 and the universal T-helper epitope PADRE. The epitopes were conjugated to short DNA probes and combined with a complementary scaffold strand, resulting in sequence-specific self-assembly. The immunogens were then formulated by conjugation to gold nanoparticles by three methods or by co-crystallization with epsilon inulin. BALB/C mice were immunized with each formulation, and the IgG immune responses and virus neutralizing titers were compared. The results demonstrate that this assembly is immunogenic and generates neutralizing antibodies against wildtype SARS-CoV-2 and the Delta variant.

Immunomodulatory LncRNA on antisense strand of ICAM-1 augments SARS-CoV-2 infection-associated airway mucoinflammatory phenotype.

Noncoding RNAs are important regulators of mucoinflammatory response, but little is known about the contribution of airway long noncoding RNAs (lncRNAs) in COVID-19. RNA-seq analysis showed a more than 4-fold increased expression of IL-6, ICAM-1, CXCL-8, and SCGB1A1 inflammatory factors; MUC5AC and MUC5B mucins; and SPDEF, FOXA3, and FOXJ1 transcription factors in COVID-19 patient nasal samples compared with uninfected controls. A lncRNA on antisense strand to ICAM-1 or LASI was induced 2-fold in COVID-19 patients, and its expression was directly correlated with viral loads. A SARS-CoV-2-infected 3D-airway model largely recapitulated these clinical findings. RNA microscopy and molecular modeling indicated a possible interaction between viral RNA and LASI lncRNA. Notably, blocking LASI lncRNA reduced the SARS-CoV-2 replication and suppressed MUC5AC mucin levels and associated inflammation, and select LASI-dependent miRNAs (e.g., let-7b-5p and miR-200a-5p) were implicated. Thus, LASI lncRNA represents an essential facilitator of SARS-CoV-2 infection and associated airway mucoinflammatory response.

Binding of the SARS-CoV-2 envelope E protein to human BRD4 is essential for infection.

Emerging new variants of SARS-CoV-2 and inevitable acquired drug resistance call for the continued search of new pharmacological targets to fight the potentially fatal infection. Here, we describe the mechanisms by which the E protein of SARS-CoV-2 hijacks the human transcriptional regulator BRD4. We found that SARS-CoV-2 E is acetylated in vivo and co-immunoprecipitates with BRD4 in human cells. Bromodomains (BDs) of BRD4 bind to the C-terminus of the E protein, acetylated by human acetyltransferase p300, whereas the ET domain of BRD4 recognizes the unmodified motif of the E protein. Inhibitors of BRD4 BDs, JQ1 or OTX015, decrease SARS-CoV-2 infectivity in lung bronchial epithelial cells, indicating that the acetyllysine binding function of BDs is necessary for the virus fitness and that BRD4 represents a potential anti-COVID-19 target. Our findings provide insight into molecular mechanisms that contribute to SARS-CoV-2 pathogenesis and shed light on a new strategy to block SARS-CoV-2 infection.

Peptide Nanoarray Scaffold Vaccine for SARS-COV-2 and Its Variants of Concerns.

The current vaccine development strategies for the COVID-19 pandemic utilize whole inactive or attenuated viruses, virus-like particles, recombinant proteins, and antigen-coding DNA and mRNA with various delivery strategies. While highly effective, these vaccine development strategies are time-consuming and often do not provide reliable protection for immunocompromised individuals, young children, and pregnant women. Here, we propose a novel modular vaccine platform to address these shortcomings using chemically synthesized peptides and identified based on the validated bioinformatic data about the target. The vaccine is based on the rational design of an immunogen containing two defined B-cell epitopes from the spike protein of SARS-Co-V2 and a universal T-helper epitope PADRE assembled on the DNA scaffold. The results demonstrate that this assembly is immunogenic and generates neutralizing antibodies against SARS-CoV-2 wild type and its variants of concerns (VOC). This newly designed peptide nanoarray scaffold vaccine is useful in controlling virus transmission in immunocompromised individuals, as well as individuals who are prone to vaccine-induced adverse reactions. Given that the immunogen is modular, epitopes or immunomodulatory ligands can be easily introduced in order to tailor the vaccine to the recipient. This also allows the already developed vaccine to be modified rapidly according to the identified mutations of the virus.

Diminished Peripheral CD29hi Cytotoxic CD4+ T Cells Are Associated With Deleterious Effects During SIV Infection.

Cytotoxic CD4+ T cells (CD4+ CTLs) limit HIV pathogenesis, as evidenced in elite controllers (a subset of individuals who suppress the virus without the need for therapy). CD4+ CTLs have also been shown to kill HIV-infected macrophages. However, little is known about their contribution towards HIV persistence, how they are affected following exposure to immune modulators like morphine, and what factors maintain their frequencies and function. Further, the lack of robust markers to identify CD4+ CTLs in various animal models limits understanding of their role in HIV pathogenesis. We utilized various PBMC samples obtained from SIV infected and cART treated rhesus macaques exposed to morphine or saline and subjected to flow cytometry evaluations. Thereafter, we compared and correlated the expression of CD4+ CTL-specific markers to viral load and viral reservoir estimations in total CD4+ T cells. We found that CD29 could be reliably used as a marker to identify CD4+ CTLs in rhesus macaques since CD29hi CD4+ T cells secrete higher cytotoxic and proinflammatory cytokines following PMA/ionomycin or gag stimulation. In addition, this immune cell subset was depleted during untreated SIV infection. Strikingly, we also observed that early initiation of cART reconstitutes depleted CD29hi CD4+ T cells and restores their function. Furthermore, we noted that morphine exposure reduced the secretion of proinflammatory cytokines/cytotoxic molecules in CD29hi CD4+ T cells. Lastly, increased functionality of CD29hi CD4+ T cells as depicted by elevated levels of either IL-21 or granzyme B hi T Bet+ gag specific responses were linked to limiting the size of the replication-competent reservoir during cART treatment. Collectively, our data suggest that CD4+ CTLs are crucial in limiting SIV pathogenesis and persistence.

Defining the Innate Immune Responses for SARS-CoV-2-Human Macrophage Interactions.

Host innate immune response follows severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, and it is the driver of the acute respiratory distress syndrome (ARDS) amongst other inflammatory end-organ morbidities. Such life-threatening coronavirus disease 2019 (COVID-19) is heralded by virus-induced activation of mononuclear phagocytes (MPs; monocytes, macrophages, and dendritic cells). MPs play substantial roles in aberrant immune secretory activities affecting profound systemic inflammation and end-organ malfunctions. All follow the presence of persistent viral components and virions without evidence of viral replication. To elucidate SARS-CoV-2-MP interactions we investigated transcriptomic and proteomic profiles of human monocyte-derived macrophages. While expression of the SARS-CoV-2 receptor, the angiotensin-converting enzyme 2, paralleled monocyte-macrophage differentiation, it failed to affect productive viral infection. In contrast, simple macrophage viral exposure led to robust pro-inflammatory cytokine and chemokine expression but attenuated type I interferon (IFN) activity. Both paralleled dysregulation of innate immune signaling pathways, specifically those linked to IFN. We conclude that the SARS-CoV-2-infected host mounts a robust innate immune response characterized by a pro-inflammatory storm heralding end-organ tissue damage.

Discovery and Evaluation of Entry Inhibitors for SARS-CoV-2 and Its Emerging Variants.

The outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the coronavirus disease 19 (COVID-19) pandemic. Despite unprecedented research and developmental efforts, SARS-CoV-2-specific antivirals are still unavailable for the treatment of COVID-19. In most instances, SARS-CoV-2 infection initiates with the binding of Spike glycoprotein to the host cell ACE2 receptor. Utilizing the crystal structure of the ACE2/Spike receptor-binding domain (S-RBD) complex (PDB file 6M0J) in a computer-aided drug design approach, we identified and validated five potential inhibitors of S-RBD and ACE-2 interaction. Two of the five compounds, MU-UNMC-1 and MU-UNMC-2, blocked the entry of pseudovirus particles expressing SARS-CoV-2 Spike glycoprotein. In live SARS-CoV-2 infection assays, both compounds showed antiviral activity with IC50 values in the micromolar range (MU-UNMC-1: IC50 = 0.67 µM and MU-UNMC-2: IC50 = 1.72 µM) in human bronchial epithelial cells. Furthermore, MU-UNMC-1 and MU-UNMC-2 effectively blocked the replication of rapidly transmitting variants of concern: South African variant B.1.351 (IC50 = 9.27 and 3.00 µM) and Scotland variant B.1.222 (IC50 = 2.64 and 1.39 µM), respectively. Following these assays, we conducted "induced-fit (flexible) docking" to understand the binding mode of MU-UNMC-1/MU-UNMC-2 at the S-RBD/ACE2 interface. Our data showed that mutation N501Y (present in B.1.351 variant) alters the binding mode of MU-UNMC-2 such that it is partially exposed to the solvent and has reduced polar contacts. Finally, MU-UNMC-2 displayed high synergy with remdesivir, the only approved drug for treating hospitalized COVID-19 patients. IMPORTANCE The ongoing coronavirus infectious disease 2019 (COVID-19) pandemic is caused by a novel coronavirus named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). More than 207 million people have been infected globally, and 4.3 million have died due to this viral outbreak. While a few vaccines have been deployed, a SARS-CoV-2-specific antiviral for the treatment of COVID-19 is yet to be approved. As the interaction of SARS-CoV-2 Spike protein with ACE2 is critical for cellular entry, using a combination of a computer-aided drug design (CADD) approach and cell-based in vitro assays, we report the identification of five potential SARS-CoV-2 entry inhibitors. Out of the five, two compounds (MU-UNMC-1 and MU-UNMC-2) have antiviral activity against ancestral SARS-CoV-2 and emerging variants from South Africa and Scotland. Furthermore, MU-UNMC-2 acts synergistically with remdesivir (RDV), suggesting that RDV and MU-UNMC-2 can be developed as a combination therapy to treat COVID-19 patients.

Mental Health Issues During and After COVID-19 Vaccine Era.

The COVID-19 pandemic has persisted for more than a year, and post-COVID-19 sequelae of neurological complications, including direct and indirect effects on the central nervous system (CNS), have been recognized. There is a plethora of evidence for neurological, cognitive, and emotional deficits in COVID-19 patients. Acute neurological symptoms like neuroinflammation, cognitive impairment, loss of smell, and brain stroke are common direct effects among SARS-CoV-2 infected individuals. Work-associated stress, lockdowns, social distancing, and quarantine in response to contain SARS-CoV-2 have also affected the mental health of large populations, regardless of age. Public health emergencies have affected individuals and communities, resulting in emotional reactions and unhealthy behaviors. Although vaccines have been widely distributed and administered among large populations, vaccine hesitancy still exists and may be due to apprehension about vaccine efficacy, preliminary trials, and associated side effects. This review highlights the impact of COVID-19 on the CNS by outlining direct and indirect effects and factors contributing to the decline in people's mental health throughout the COVID-19 pandemic both during and after vaccine administration. Furthermore, we also discuss reasons for vaccine hesitancy and why some groups of people are deprived of vaccines. Finally, we touched upon the social determinants of mental health and their impact on disadvantaged populations during times of crisis which may help policymakers set up some action plans to mitigate the COVID-19 mental health turmoil during this ongoing pandemic.

Therapeutic implications of SARS-CoV-2 dysregulation of the gut-brain-lung axis.

The emergence and rapid spread of novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused over 180 million confirmed cases resulting in over 4 million deaths worldwide with no clear end in sight for the coronavirus disease 19 (COVID-19) pandemic. Most SARS-CoV-2 exposed individuals experience mild to moderate symptoms, including fever, cough, fatigue, and loss of smell and taste. However, many individuals develop pneumonia, acute respiratory distress syndrome, septic shock, and multiorgan dysfunction. In addition to these primarily respiratory symptoms, SARS-CoV-2 can also infiltrate the central nervous system, which may damage the blood-brain barrier and the neuron's synapses. Resultant inflammation and neurodegeneration in the brain stem can further prevent efferent signaling to cranial nerves, leading to the loss of anti-inflammatory signaling and normal respiratory and gastrointestinal functions. Additionally, SARS-CoV-2 can infect enterocytes resulting in gut damage followed by microbial dysbiosis and translocation of bacteria and their byproducts across the damaged epithelial barrier. As a result, this exacerbates pro-inflammatory responses both locally and systemically, resulting in impaired clinical outcomes. Recent evidence has highlighted the complex interactions that mutually modulate respiratory, neurological, and gastrointestinal function. In this review, we discuss the ways SARS-CoV-2 potentially disrupts the gut-brain-lung axis. We further highlight targeting specific responses to SARS-CoV-2 for the development of novel, urgently needed therapeutic interventions. Finally, we propose a prospective related to the individuals from Low- and Middle-Income countries. Here, the underlying propensity for heightened gut damage/microbial translocation is likely to result in worse clinical outcomes during this COVID-19 pandemic.

Defining the Immune Responses for SARS-CoV-2-Human Macrophage Interactions.

Host innate immune response follows severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, and it is the driver of the acute respiratory distress syndrome (ARDS) amongst other inflammatory end-organ morbidities. Such life-threatening coronavirus disease 2019 (COVID-19) is heralded by virus-induced activation of mononuclear phagocytes (MPs; monocytes, macrophages, and dendritic cells). MPs play substantial roles in aberrant immune secretory activities affecting profound systemic inflammation and end organ malfunctions. All follow an abortive viral infection. To elucidate SARS-CoV-2-MP interactions we investigated transcriptomic and proteomic profiles of human monocyte-derived macrophages. While expression of the SARS-CoV-2 receptor, the angiotensin-converting enzyme 2, paralleled monocyte-macrophage differentiation it failed to affect productive viral infection. In contrast, simple macrophage viral exposure led to robust pro-inflammatory cytokine and chemokine expression but attenuated type I interferon (IFN) activity. Both paralleled dysregulation of innate immune signaling pathways specifically those linked to IFN. We conclude that the SARS-CoV-2-infected host mounts a robust innate immune response characterized by a pro-inflammatory storm heralding consequent end-organ tissue damage.

Distinct Mucoinflammatory Phenotype and the Immunomodulatory Long Noncoding Transcripts Associated with SARS-CoV-2 Airway Infection.

Respiratory epithelial cells are the primary target for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We investigated the 3D human airway tissue model to evaluate innate epithelial cell responses to SARS-CoV-2 infection. A SARS-CoV-2 clinical isolate productively infected the 3D-airway model with a time-dependent increase in viral load (VL) and concurrent upregulation of airway immunomodulatory factors ( IL-6, ICAM-1 , and SCGB1A1 ) and respiratory mucins ( MUC5AC, MUC5B, MUC2 , and MUC4) , and differential modulation of select long noncoding RNAs (lncRNAs i.e., LASI, TOSL, NEAT1 , and MALAT1 ). Next, we examined these immunomodulators in the COVID-19 patient nasopharyngeal swab samples collected from subjects with high- or low-VLs (∻100-fold difference). As compared to low-VL, high-VL patients had prominent mucoinflammatory signature with elevated expression of IL-6, ICAM-1, SCGB1A1, SPDEF, MUC5AC, MUC5B , and MUC4 . Interestingly, LASI, TOSL , and NEAT1 lncRNA expressions were also markedly elevated in high-VL patients with no change in MALAT1 expression. In addition, dual-staining of LASI and SARS-CoV-2 nucleocapsid N1 RNA showed predominantly nuclear/perinuclear localization at 24 hpi in 3D-airway model as well as in high-VL COVID-19 patient nasopharyngeal cells, which exhibited high MUC5AC immunopositivity. Collectively, these findings suggest SARS-CoV-2 induced lncRNAs may play a role in acute mucoinflammatory response observed in symptomatic COVID-19 patients.

Discovery and in-vitro evaluation of potent SARS-CoV-2 entry inhibitors.

SARS-CoV-2 infection initiates with the attachment of spike protein to the ACE2 receptor. While vaccines have been developed, no SARS-CoV-2 specific small molecule inhibitors have been approved. Herein, utilizing the crystal structure of the ACE2/Spike receptor binding domain (S-RBD) complex in computer-aided drug design (CADD) approach, we docked ∼8 million compounds within the pockets residing at S-RBD/ACE2 interface. Five best hits depending on the docking score, were selected and tested for their in vitro efficacy to block SARS-CoV-2 replication. Of these, two compounds (MU-UNMC-1 and MU-UNMC-2) blocked SARS-CoV-2 replication at sub-micromolar IC 50 in human bronchial epithelial cells (UNCN1T) and Vero cells. Furthermore, MU-UNMC-2 was highly potent in blocking the virus entry by using pseudoviral particles expressing SARS-CoV-2 spike. Finally, we found that MU-UNMC-2 is highly synergistic with remdesivir (RDV), suggesting that minimal amounts are needed when used in combination with RDV, and has the potential to develop as a potential entry inhibitor for COVID-19.

Blockade of SARS-CoV-2 infection in vitro by highly potent PI3K-α/mTOR/BRD4 inhibitor.

Pathogenic viruses like SARS-CoV-2 and HIV hijack the host molecular machinery to establish infection and survival in infected cells. This has led the scientific community to explore the molecular mechanisms by which SARS-CoV-2 infects host cells, establishes productive infection, and causes life-threatening pathophysiology. Very few targeted therapeutics for COVID-19 currently exist, such as remdesivir. Recently, a proteomic approach explored the interactions of 26 of 29 SARS-CoV-2 proteins with cellular targets in human cells and identified 67 interactions as potential targets for drug development. Two of the critical targets, the bromodomain and extra-terminal domain proteins (BETs): BRD2/BRD4 and mTOR, are inhibited by the dual inhibitory small molecule SF2523 at nanomolar potency. SF2523 is the only known mTOR PI3K-α/(BRD2/BRD4) inhibitor with potential to block two orthogonal pathways necessary for SARS-CoV-2 pathogenesis in human cells. Our results demonstrate that SF2523 effectively blocks SARS-CoV-2 replication in lung bronchial epithelial cells in vitro , showing an IC 50 value of 1.5 µM, comparable to IC 50 value of remdesivir (1.1 µM). Further, we demonstrated that the combination of doses of SF2523 and remdesivir is highly synergistic: it allows for the reduction of doses of SF2523 and remdesivir by 25-fold and 4-fold, respectively, to achieve the same potency observed for a single inhibitor. Because SF2523 inhibits two SARS-CoV-2 driven pathogenesis mechanisms involving BRD2/BRD4 and mTOR signaling, our data suggest that SF2523 alone or in combination with remdesivir could be a novel and efficient therapeutic strategy to block SARS-CoV-2 infection and hence be beneficial in preventing severe COVID-19 disease evolution. ONE SENTENCE SUMMARY: Evidence of in silico designed chemotype (SF2523) targeting PI3K-α/mTOR/BRD4 inhibits SARS-CoV-2 infection and is highly synergistic with remdesivir.