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The cell entry of SARS-CoV-2 has emerged as an attractive drug repurposing target for COVID-19. Here we combine genetics and chemical perturbation to demonstrate that ACE2-mediated entry of SARS-Cov and CoV-2 requires the cell surface heparan sulfate (HS) as an assisting cofactor: ablation of genes involved in HS biosynthesis or incubating cells with a HS mimetic both inhibit Spike-mediated viral entry. We show that heparin/HS binds to Spike directly, and facilitates the attachment of Spike-bearing viral particles to the cell surface to promote viral entry. We screened approved drugs and identified two classes of inhibitors that act via distinct mechanisms to target this entry pathway. Among the drugs characterized, Mitoxantrone is a potent HS inhibitor, while Sunitinib and BNTX disrupt the actin network to indirectly abrogate HS-assisted viral entry. We further show that drugs of the two classes can be combined to generate a synergized activity against SARS-CoV-2-induced cytopathic effect. Altogether, our study establishes HS as an attachment factor that assists SARS coronavirus cell entry and reveals drugs capable of targeting this important step in the viral life cycle.

The ability to model human neurological tissues in vitro has been a major hurdle to effective drug development for neurological disorders. iPSC-derived brain organoids have emerged as a compelling solution to this problem as they have the potential to relevantly model the protein expression pattern and physiology of specific brain regions. Although many protocols now exist for the production of brain organoids, few attempts have been made to do an in-depth kinetic evaluation of expression of mature regiospecific markers of brain organoids. To address this, we differentiated midbrain-specific brain organoids from iPSC-lines derived from three apparently healthy individuals using a matrix-free, bioreactor method. We monitored the expression of midbrain-specific neuronal markers from 7 to 90-days using immunofluorescence and immunohistology. The organoids were further characterized using electron microscopy and RNA-seq. In addition to serving as a potential benchmark for the future evaluation of other differentiation protocols, the markers observed in this study can be useful as control parameters to identify and evaluate the disease phenotypes in midbrain organoid derived from patient iPSC-lines with genetic neurological disorders.

NGLY1 deficiency is an ultra-rare, autosomal recessive genetic disease caused by mutations in the NGLY1 gene encoding N-glycanase one that removes N-linked glycan. Patients with pathogenic mutations in NGLY1 have complex clinical symptoms including global developmental delay, motor disorder and liver dysfunction. To better understand the disease pathogenesis and the neurological symptoms of the NGLY1 deficiency we generated and characterized midbrain organoids using patient-derived iPSCs from two patients with distinct disease-causing mutations–one homozygous for p. Q208X, the other compound heterozygous for p. L318P and p. R390P and CRISPR generated NGLY1 knockout iPSCs. We demonstrate that NGLY1 deficient midbrain organoids show altered neuronal development compared to one wild type (WT) organoid. Both neuronal (TUJ1) and astrocytic glial fibrillary acid protein markers were reduced in NGLY1 patient-derived midbrain organoids along with neurotransmitter GABA. Interestingly, staining for dopaminergic neuronal marker, tyrosine hydroxylase, revealed a significant reduction in patient iPSC derived organoids. These results provide a relevant NGLY1 disease model to investigate disease mechanisms and evaluate therapeutics for treatments of NGLY1 deficiency.

Abstract BACKGROUND & AIMS Glutamate carboxypeptidase II (GCPII) is elevated in active inflamed biopsies of both Crohn’s disease and ulcerative colitis patients and is a promising therapeutic target for the treatment of IBD. We have previously presented the development and characterization of a first-in-class, gut-restricted, and orally active GCPII inhibitor, (S)-IBD3540, and have shown that oral (S)-IBD3540 attenuates disease severity in multiple murine colitis models, including acute dextran sulfate sodium (DSS)-induced colitis. Here, we extend our knowledge regarding (S)-IBD3540’s anti-inflammatory effects in spontaneously occurring colitis of interleukin 10 (IL10-/-) mice by performing longitudinal measurement of inflammatory biomarker fecal lipocalin 2 (LCN2) and terminal colon cytokine and chemokine measurements. METHODS Female BALB/cAnNTac-Il10em7Tac mice (Taconic, Germantown, NY) were acquired at 4 weeks of age and monitored three times weekly for the onset of clinical signs, including alterations in stool consistency. Following onset of symptoms, groups were randomized and treatment was initiated with vehicle or 100 mg/kg eq. (S)-IBD3540 once daily by oral gavage. Prior to treatment, and biweekly thereafter, fecal LCN2 was quantified using a mouse Lipocalin-2/NGAL DuoSet ELISA (R&D Systems). Upon study termination, proximal colon homogenates were prepared and analyzed by a mouse Cytokine/Chemokine 31-Plex Discovery Assay Array (Eve Technologies). RESULTS Impressively, despite initiating treatment in highly symptomatic mice 100 mg/kg oral (S)-IBD3540 was found to reverse abnormalities in stool consistency. Additionally, (S)-IBD3540 had potent anti-inflammatory effects. As expected, fecal LCN2 was observed to increase throughout the study period in vehicle treated mice, however, fecal LCN2 was significantly decreased in (S)-IBD3540 treated mice (p<0.05). Similarly, colon multiplex analysis identified significant reductions in multiple cytokines and chemokines implicated in IL10 colitis pathobiology, with notable reductions in cytokines TNFa, IL-12p40, and IL-17, and chemokines IP-10 and MIP-2. CONCLUSION (S)-IBD3540 is a novel, gut-restricted, GCPII inhibitor that has robust anti-inflammatory activity in IL10-/- colitis. Encouragingly, we have now found that in this model (S)-IBD3540 reduces pro-inflammatory cytokines/chemokines that are the targets of existing IBD medications. Studies to compare efficacy to one of these agents, anti-IL-12p40, are in progress.

Neutralizing antibodies targeting the SARS-CoV-2 spike protein have shown a great preventative/therapeutic potential. Here, we report a rapid and efficient strategy for the development and design of SARS-CoV-2 neutralizing humanized nanobody constructs with sub-nanomolar affinities and nanomolar potencies. CryoEM-based structural analysis of the nanobodies in complex with spike revealed two distinct binding modes. The most potent nanobody, RBD-1-2G(NCATS-BL8125), tolerates the N501Y RBD mutation and remains capable of neutralizing the B.1.1.7 (Alpha) variant. Molecular dynamics simulations provide a structural basis for understanding the neutralization process of nanobodies exclusively focused on the spike-ACE2 interface with and without the N501Y mutation on RBD. A primary human airway air-lung interface (ALI) ex vivo model showed that RBD-1-2G-Fc antibody treatment was effective at reducing viral burden following WA1 and B.1.1.7 SARS-CoV-2 infections. Therefore, this presented strategy will serve as a tool to mitigate the threat of emerging SARS-CoV-2 variants.

Neurological complications are common in patients with COVID-19. While SARS-CoV-2, the causal pathogen of COVID-19, has been detected in some patient brains, its ability to infect brain cells and impact their function are not well understood, and experimental models using human brain cells are urgently needed. Here we investigated the susceptibility of human induced pluripotent stem cell (hiPSC)-derived monolayer brain cells and region-specific brain organoids to SARS-CoV-2 infection. We found modest numbers of infected neurons and astrocytes, but greater infection of choroid plexus epithelial cells. We optimized a protocol to generate choroid plexus organoids from hiPSCs, which revealed productive SARS-CoV-2 infection that leads to increased cell death and transcriptional dysregulation indicative of an inflammatory response and cellular function deficits. Together, our results provide evidence for SARS-CoV-2 neurotropism and support use of hiPSC-derived brain organoids as a platform to investigate the cellular susceptibility, disease mechanisms, and treatment strategies for SARS-CoV-2 infection. Competing Interest Statement The authors have declared no competing interest.

Background: This study aimed to assess the level of patient satisfaction with the preoperative anesthetic evaluation provided in a tertiary level and identify factors leading to dissatisfaction that could be addressed to improve patient care and experience. A proper pre-anesthetic consultation has been linked to improved patient compliance and satisfaction with the care they received. Method: The study was a cross-sectional questionnaire-based study conducted from November 2022 to January 2023 at Patan Hospital, Patan Academy of Health Sciences, Nepal, with a sample size of 370 adult patients above 18 years old undergoing elective surgeries. The questionnaire used in the study was divided into three sections. The first section consisted of sociodemographic data, the second section had yes/no questions related to pre-anesthetic assessment, and the third section assessed patients' satisfaction using a three-point Likert scale with Dissatisfied, Not sure, and Satisfied. Not sure and dissatisfied were classified as dissatisfied during tabulation. Result: The results of the study showed that the majority of patients were happy with the amount of time spent and the explanations provided during the pre-anesthetic checkup. After the preanesthetic consultation, the majority of patients claimed to feel less worried, and overall satisfaction was also very high. Among the 370 participants, 165 (44.6%) were male, and 205 (55.4%) were female, and the mean age of the participants was 38.11 (±15.796). Most of the patients, 228 (61.6%), were in the age group of >18 to 39. A total of 309 patients were satisfied with the explanations given to them during the preanesthetic consultation, among which 198 (64.08%) were from >18 to 39 age group. Conclusion: The study suggests that preoperative anesthesia assessment is an important interaction between the patient and the anesthetist that allows the anesthetist to properly assess the patient's medical condition and also helps the patient get a clear understanding of the planned anesthesia and the complications that might arise in the perioperative period. It also suggests that providing adequate information about anesthesia and surgery can decrease preoperative and postoperative anxiety.

Effective small molecule therapies to combat the SARS-CoV-2 infection are still lacking as the COVID-19 pandemic continues globally. High throughput screening assays are needed for lead discovery and optimization of small molecule SARS-CoV-2 inhibitors. In this work, we have applied viral pseudotyping to establish a cell-based SARS-CoV-2 entry assay. Here, the pseudotyped particles (PP) contain SARS-CoV-2 spike in a membrane enveloping both the murine leukemia virus (MLV) gag-pol polyprotein and luciferase reporter RNA. Upon addition of PP to HEK293-ACE2 cells, the SARS-CoV-2 spike protein binds to the ACE2 receptor on the cell surface, resulting in priming by host proteases to trigger endocytosis of these particles, and membrane fusion between the particle envelope and the cell membrane. The internalized luciferase reporter gene is then expressed in cells, resulting in a luminescent readout as a surrogate for spike-mediated entry into cells. This SARS-CoV-2 PP entry assay can be executed in a biosafety level 2 containment lab for high throughput screening. From a collection of 5,158 approved drugs and drug candidates, our screening efforts identified 7 active compounds that inhibited the SARS-CoV-2-S PP entry. Of these seven, six compounds were active against live replicating SARS-CoV-2 virus in a cytopathic effect assay. Our results demonstrated the utility of this assay in the discovery and development of SARS-CoV-2 entry inhibitors as well as the mechanistic study of anti-SARS-CoV-2 compounds. Additionally, particles pseudotyped with spike proteins from SARS-CoV-2 B.1.1.7 and B.1.351 variants were prepared and used to evaluate the therapeutic effects of viral entry inhibitors. Competing Interest Statement The authors have declared no competing interest. Footnotes * http://opendata.ncats.nih.gov

The National Center for Advancing Translational Sciences (NCATS) has developed an online open science platform – named the NCATS OpenData Portal (ODP) – for quickly and freely sharing complete NCATS translational datasets via an open-access, user-friendly interface. This paper describes the establishment of the ODP, initially deployed during the COVID-19 crisis, and provides a detailed analysis of COVID-19 drug repurposing screening datasets that served as the first large-scale use case for the platform. Over 10,000 compounds were tested across 17 quantitative high-throughput assays, covering a wide spectrum of the SARS-CoV-2 life cycle. In total, over 87,000 concentration-response curves and 426,000 data points were made publicly available on ODP in near real-time, enabling immediate access to complete datasets. The resource is flexible in accommodating various types and structures of data, and it has already expanded since its launch to host additional datasets for COVID-19, other viruses of pandemic potential, and beyond. The OpenData Portal has been designed as a scalable platform for real-time data sharing across drug discovery campaigns, regardless of disease area, with the overarching goal of accelerating discovery at NCATS, the NIH, and the greater scientific community.