SARS-CoV-2 Early Infection Signature Identified Potential Key Infection Mechanisms and Drug Targets

Background The ongoing COVID-19 outbreak has caused devastating mortality and posed a significant threat to public health worldwide. Despite the severity of this illness and 2.3 million worldwide deaths, the disease mechanism is mostly unknown. Previous studies that characterized differential gene expression due to SARS-CoV-2 infection lacked robust validation. Although vaccines are now available, effective treatment options are still out of reach. Results To characterize the transcriptional activity of SARS-CoV-2 infection, a gene signature consisting of 25 genes was generated using a publicly available RNA-Sequencing (RNA-Seq) dataset of cultured cells infected with SARS-CoV-2. The signature estimated infection level accurately in bronchoalveolar lavage fluid (BALF) cells and peripheral blood mononuclear cells (PBMCs) from healthy and infected patients (mean 0.001 vs. 0.958; P \u3c 0.0001). These signature genes were investigated in their ability to distinguish the severity of SARS-CoV-2 infection in a single-cell RNA-Sequencing dataset. TNFAIP3, PPP1R15A, NFKBIA, and IFIT2 had shown bimodal gene expression in various immune cells from severely infected patients compared to healthy or moderate infection cases. Finally, this signature was assessed using the publicly available ConnectivityMap database to identify potential disease mechanisms and drug repurposing candidates. Pharmacological classes of tricyclic antidepressants, SRC-inhibitors, HDAC inhibitors, MEK inhibitors, and drugs such as atorvastatin, ibuprofen, and ketoconazole showed strong negative associations (connectivity score \u3c − 90), highlighting the need for further evaluation of these candidates for their efficacy in treating SARS-CoV-2 infection. Conclusions Thus, using the 25-gene SARS-CoV-2 infection signature, the SARS-CoV-2 infection status was captured in BALF cells, PBMCs and postmortem lung biopsies. In addition, candidate SARS-CoV-2 therapies with known safety profiles were identified. The signature genes could potentially also be used to characterize the COVID-19 disease severity in patients’ expression profiles of BALF cells

American Association of Colleges of Pharmacy (AACP) Co-Curriculum Toolkit

The co-curriculum is intended to run parallel to the formal curriculum and support the development of students’ professional knowledge, skills, abilities, behaviors, and attitudes, including scientific foundation, knowledge application, and practice competencies. Additionally, the co-curriculum can also support proficiency in skills and achievement of competencies that are concurrently taught within the formal curriculum. ACPE evaluates the way programs assess students’ acquisition of knowledge and application of knowledge to practice within co-curricular experiences and especially how they advance the professional development of students within affective domain areas of learning

Increased Expression of TGF-β Signaling Components in a Mouse Model of Fibrosis Induced by Submandibular Gland Duct Ligation.

Transforming growth factor-β (TGF-β) is a multi-functional cytokine with a well-described role in the regulation of tissue fibrosis and regeneration in the liver, kidney and lung. Submandibular gland (SMG) duct ligation and subsequent deligation in rodents is a classical model for studying salivary gland damage and regeneration. While previous studies suggest that TGF-β may contribute to salivary gland fibrosis, the expression of TGF-β signaling components has not been investigated in relation to mouse SMG duct ligation-induced fibrosis and regeneration following ductal deligation. Following a 7 day SMG duct ligation, TGF-β1 and TGF-β3 were significantly upregulated in the SMG, as were TGF-β receptor 1 and downstream Smad family transcription factors in salivary acinar cells, but not in ductal cells. In acinar cells, duct ligation also led to upregulation of snail, a Smad-activated E-cadherin repressor and regulator of epithelial-mesenchymal transition, whereas in ductal cells upregulation of E-cadherin was observed while snail expression was unchanged. Upregulation of these TGF-β signaling components correlated with upregulation of fibrosis markers collagen 1 and fibronectin, responses that were inhibited by administration of the TGF-β receptor 1 inhibitors SB431542 or GW788388. After SMG regeneration following a 28 day duct deligation, TGF-β signaling components and epithelial-mesenchymal transition markers returned to levels similar to non-ligated controls. The results from this study indicate that increased TGF-β signaling contributes to duct ligation-induced changes in salivary epithelium that correlate with glandular fibrosis. Furthermore, the reversibility of enhanced TGF-β signaling in acinar cells of duct-ligated mouse SMG after deligation indicates that this is an ideal model for studying TGF-β signaling mechanisms in salivary epithelium as well as mechanisms of fibrosis initiation and their resolution

P2Y2 Nucleotide Receptor Prompts Human Cardiac Progenitor Cell Activation by Modulating Hippo Signaling

RationaleAutologous stem cell therapy using human c-Kit+ cardiac progenitor cells (hCPCs) is a promising therapeutic approach for treatment of heart failure (HF). However, hCPCs derived from aged patients with HF with genetic predispositions and comorbidities of chronic diseases exhibit poor proliferative and migratory capabilities, which impair overall reparative potential for injured myocardium. Therefore, empowering functionally compromised hCPCs with proregenerative molecules ex vivo is crucial for improving the therapeutic outcome in patients with HF.ObjectiveTo improve hCPC proliferation and migration responses that are critical for regeneration by targeting proregenerative P2Y2 nucleotide receptor (P2Y2R) activated by extracellular ATP and UTP molecules released following injury/stress.Methods and resultsc-Kit+ hCPCs were isolated from cardiac tissue of patients with HF undergoing left ventricular assist device implantation surgery. Correlations between P2 nucleotide receptor expression and hCPC growth kinetics revealed downregulation of select P2 receptors, including P2Y2R, in slow-growing hCPCs compared with fast growers. hCPC proliferation and migration significantly improved by overexpressing or stimulating P2Y2R. Mechanistically, P2Y2R-induced proliferation and migration were dependent on activation of YAP (yes-associated protein)-the downstream effector of Hippo signaling pathway.ConclusionsProliferation and migration of functionally impaired hCPCs are enhanced by P2Y2R-mediated YAP activation, revealing a novel link between extracellular nucleotides released during injury/stress and Hippo signaling-a central regulator of cardiac regeneration. Functional correlations exist between hCPC phenotypic properties and P2 purinergic receptor expression. Lack of P2Y2R and other crucial purinergic stress detectors could compromise hCPC responsiveness to presence of extracellular stress signals. These findings set the stage for subsequent studies to assess purinergic signaling modulation as a potential strategy to improve therapeutic outcome for use of hCPCs in patients with HF

P2Y 2

RationaleAutologous stem cell therapy using human c-Kit+ cardiac progenitor cells (hCPCs) is a promising therapeutic approach for treatment of heart failure (HF). However, hCPCs derived from aged patients with HF with genetic predispositions and comorbidities of chronic diseases exhibit poor proliferative and migratory capabilities, which impair overall reparative potential for injured myocardium. Therefore, empowering functionally compromised hCPCs with proregenerative molecules ex vivo is crucial for improving the therapeutic outcome in patients with HF.ObjectiveTo improve hCPC proliferation and migration responses that are critical for regeneration by targeting proregenerative P2Y2 nucleotide receptor (P2Y2R) activated by extracellular ATP and UTP molecules released following injury/stress.Methods and resultsc-Kit+ hCPCs were isolated from cardiac tissue of patients with HF undergoing left ventricular assist device implantation surgery. Correlations between P2 nucleotide receptor expression and hCPC growth kinetics revealed downregulation of select P2 receptors, including P2Y2R, in slow-growing hCPCs compared with fast growers. hCPC proliferation and migration significantly improved by overexpressing or stimulating P2Y2R. Mechanistically, P2Y2R-induced proliferation and migration were dependent on activation of YAP (yes-associated protein)-the downstream effector of Hippo signaling pathway.ConclusionsProliferation and migration of functionally impaired hCPCs are enhanced by P2Y2R-mediated YAP activation, revealing a novel link between extracellular nucleotides released during injury/stress and Hippo signaling-a central regulator of cardiac regeneration. Functional correlations exist between hCPC phenotypic properties and P2 purinergic receptor expression. Lack of P2Y2R and other crucial purinergic stress detectors could compromise hCPC responsiveness to presence of extracellular stress signals. These findings set the stage for subsequent studies to assess purinergic signaling modulation as a potential strategy to improve therapeutic outcome for use of hCPCs in patients with HF

Submandibular gland ductal ligation followed by deligation results in reversible acinar cell atrophy, immune cell infiltration and glandular fibrosis.

<p>(A, D, G) Control SMG, (B, E, H) 7 day duct-ligated SMG and (C, F, I) 7 day duct-ligated SMG followed by deligation and recovery for 28 days were subjected to immunofluorescence staining with (A-C) antibodies to the acinar cell marker AQP5 (green) and Hoechst nuclear stain (blue), (D-F) antibodies to the pan-immune cell marker CD45 (red) and Hoechst nuclear stain (blue) and (G-I) hematoxylin and eosin. Results indicate that ligation of the main SMG excretory duct induces (B) loss of acinar cells, (E) substantial immune cell infiltration and (H) atrophy of acinar cells (white arrow) and loss of secretory granules (pink) within ductal cells (black arrow) that is reversed by subsequent deligation (C, F, I). Images are representative of results from at least 3 independent experiments and scale bar = 20 μm.</p

Upregulation of Snail and Slug in response to SMG duct ligation is reversible upon duct deligation.

<p>RT-PCR analysis of cDNA prepared from whole SMGs shows significant upregulation of (A) Snail and (B) Slug mRNA after a 7 day duct ligation, which is reversed to control levels after deligation and a 28 day recovery. Data represent means ± S.E.M. (n = 4 for control, n = 8 for 7 day ligation, n = 5 for 7 day ligation, deligation and a 28 day recovery), where ***<i>P</i><0.001 and *<i>P</i><0.05 indicate significant differences in mRNA expression, as compared to control SMG. (C) Immunofluorescence analysis reveals that Snail (red) expression is primarily upregulated in acinar cells after a 7 day duct ligation, and returns to control levels after deligation and recovery. (D) Dual-immunofluorescence staining confirmed the colocalization of Snail (red) with the acinar marker AQP5 (green) following a 7 day duct ligation. Hoechst nuclear stain in blue and scale bar = 20 μm. Images are representative of results from at least 3 independent experiments.</p

Upregulation of TGF-β1, TGF-β3 and TGF-β R1 in response to SMG duct ligation is reversible upon deligation.

<p>(A) Western analysis of whole gland lysates shows upregulation of pro-TGF-β1/2/3 expression (top) in response to 7 day SMG duct ligation that is reversible following deligation and recovery for 28 days. Ponceau S staining (bottom) shows equal amounts of total protein in each well. (B) RT-PCR analysis of cDNA prepared from whole SMGs shows significant upregulation of TGF-β1 and TGF-β3, but not TGF-β2, mRNA expression after 7 days of SMG excretory duct ligation (grey bars), as compared to contralateral control glands (white bars). When ducts were ligated for 7 days then deligated for 28 days (black bars), expression levels of TGF-β1 and TGF-β3 mRNA return to control levels, whereas TGF-β2 mRNA levels remain unchanged. Data represent means ± S.E.M. (n = 7 for control and 7 day ligation, n = 5 for 7 day ligation, deligation and a 28 day recovery), where *<i>P</i><0.05 and **<i>P</i><0.01 indicate significant differences in mRNA expression, as compared to control. (C) Dual-immunofluorescence analysis of 8 μm frozen SMG sections for control, 7 day ligation and 7 day ligation followed by deligation and recovery for 28 days reveals that TGF-β R1 (red) expression is upregulated primarily in acinar cells (marked by residual AQP5 expression; green) after a 7 day SMG duct ligation, whereas little staining is visible in ductal cells. After a 7 day duct ligation followed by deligation and a 28 day recovery, TGF-β R1 expression levels are similar to control. Hoechst nuclear stain in blue and scale bar = 20 μm. Images are representative of results from at least 3 independent experiments.</p

TGF-β R1 inhibitors SB431542 and GW788388 attenuate duct ligation-induced upregulation of fibrosis markers.

<p>RT-PCR analysis of 7 day ligated and contralateral unligated control SMGs shows significant attenuation of 7 day duct ligation-induced collagen 1 and fibronectin mRNA upregulation in mice treated with either SB431542 (20 mg/kg mouse weight) or GW788388 (2 mg/kg mouse weight), as compared to DMSO-treated controls. Data represent means ± S.E.M. (n = 6 for DMSO, n = 6 for SB431542, n = 5 for GW788388), where *<i>P</i><0.05 and ***<i>P</i><0.001 indicate significant differences in mRNA expression, as compared to DMSO-treated controls.</p