High-content screening of Thai medicinal plants reveals Boesenbergia rotunda extract and its component Panduratin A as anti-SARS-CoV-2 agents.

Since December 2019, the emergence of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has caused severe pneumonia, a disease named COVID-19, that became pandemic and created an acute threat to public health. The effective therapeutics are in urgent need. Here, we developed a high-content screening for the antiviral candidates using fluorescence-based SARS-CoV-2 nucleoprotein detection in Vero E6 cells coupled with plaque reduction assay. Among 122 Thai natural products, we found that Boesenbergia rotunda extract and its phytochemical compound, panduratin A, exhibited the potent anti-SARS-CoV-2 activity. Treatment with B. rotunda extract and panduratin A after viral infection drastically suppressed SARS-CoV-2 infectivity in Vero E6 cells with IC50 of 3.62 µg/mL (CC50 = 28.06 µg/mL) and 0.81 µΜ (CC50 = 14.71 µM), respectively. Also, the treatment of panduratin A at the pre-entry phase inhibited SARS-CoV-2 infection with IC50 of 5.30 µM (CC50 = 43.47 µM). Our study demonstrated, for the first time, that panduratin A exerts the inhibitory effect against SARS-CoV-2 infection at both pre-entry and post-infection phases. Apart from Vero E6 cells, treatment with this compound was able to suppress viral infectivity in human airway epithelial cells. This result confirmed the potential of panduratin A as the anti-SARS-CoV-2 agent in the major target cells in human. Since B. rotunda is a culinary herb generally grown in China and Southeast Asia, its extract and the purified panduratin A may serve as the promising candidates for therapeutic purposes with economic advantage during COVID-19 situation.

A novel potent autophagy inhibitor ECDD-S27 targets vacuolar ATPase and inhibits cancer cell survival.

Autophagy is a conserved lysosomal-dependent cellular degradation process and its dysregulation has been linked to numerous diseases including neurodegeneration, infectious diseases, and cancer. Modulation of autophagy is therefore considered as an attractive target for disease intervention. We carried out a high-content image analysis screen of natural product-derived compounds to discover novel autophagy modulating molecules. Our screen identified ECDD-S27 as the most effective compound for increasing the number of autophagic vacuoles inside cells. The structure of ECDD-S27 revealed that it is a derivative of cleistanthin A, a natural arylnaphthalene lignan glycoside found in plants. ECDD-S27 increases the number of autophagic vacuoles by inhibiting the autophagic flux and is able to restrict the survival of different cancer cells at low nanomolar concentrations. Molecular docking and SERS analysis showed that ECDD-S27 may potentially target the V-ATPase. Upon treatment of various cancer cells with ECDD-S27, the V-ATPase activity is potently inhibited thereby resulting in the loss of lysosomal acidification. Taken together, these data indicated that ECDD-S27 retards the autophagy pathway by targeting the V-ATPase and inhibits cancer cell survival. The observed antitumor activity without cytotoxicity to normal cells suggests the therapeutic potential warranting further studies on lead optimization of the compound for cancer treatment.

Gene expression profiling of articular cartilage reveals functional pathways and networks of candidate genes for osteochondrosis in pigs.

Osteochondrosis (OC) is a joint disorder that frequently causes leg weakness in growing pigs, resulting in welfare problems and economic losses. We aimed to detect molecular pathways relevant to the emergence of the disease and to identify candidate genes for the liability to the disorder. Therefore, we compared microarray-based expression patterns of articular cartilage with (n=11) and without (n=11) histologically diagnosed OC lesions obtained from discordant sib-pairs. A total of 1,564 genes were found with different transcript abundance [differentially expressed (DE) genes] at q≤0.05. To further identify candidate genes, we integrated data from quantitative trait loci (QTL) and genome-wide association (GWA) studies with the expression analysis. We detected 317 DE genes within the QTL confidence intervals, of which 26 DE genes also overlapped GWA regions. Ingenuity Pathway Analysis suggests a pathogenic role of immune response, angiogenesis, and synthesis of extracellular matrix pathways for OC. These processes could facilitate the emergence of defects. But they may also promote the degradation of articular cartilage and the worsening of the disease. A functional network was derived that comprised genes with functional and positional clues of their role in bone and cartilage metabolisms and development, including extracellular matrix genes (e.g., LOX, OGN, and ASPN), angiogenesis genes (e.g., ANGPTL4 and PDGFA), and immune response genes (e.g., ICAM1, AZGP1, C1QB, C1QC, PDE4B, and CDA). The study identified molecular processes linked to OC and several genes with positional, genetic-statistical, and functional evidence for their role in the emergence of articular cartilage lesions and the liability to OC.

Genome-Wide Association Identifies TBX5 as Candidate Gene for Osteochondrosis Providing a Functional Link to Cartilage Perfusion as Initial Factor.

Osteochondrosis (OC) is an orthopedic syndrome of the joints that occurs in children and adolescents and domestic animals, particularly pigs, horses, and dogs. OC is the most frequent cause of leg weakness in rapidly growing pigs causing animal welfare issues and economic losses. In this study, a genome-wide association study (GWAS) was performed using the Porcine 60k SNPChip in animals of the breed Large White (n = 298) to identify chromosome regions and candidate genes associated with OC lesion scores. A total of 19 SNPs on chromosomes (SSC) 3, 5, 8, 10, 14, and 18 were significantly associated with OC lesion scores (p-values ≤ 10(-5)). The SNPs MARC0098684, MARC00840086, MARC0093124, and ASGA0062794 at SSC14 36.1-38.2 Mb encompass a region of six linkage disequilibrium (LD) blocks. The most significant SNP ASGA0062794 is located in a LD block spanning 465 kb and covering the gene encoding T-box transcription factor 5 (TBX5). A SNP (c.54T > C) identified in TBX5 was significantly associated with OC lesion scores in a single-marker analysis. TBX5 c.54T > C showed highest LD with ASGA00627974 (r (2) = 0.96) and superior association with OC lesion scores over other SNPs when included in the genome scan, whereas its treatment as an additional fixed effect in the GWAS statistical model led to a drop of significance of nearby markers. Moreover, real-time PCR showed different transcript abundance of TBX5 in healthy and defect cartilage. The results imply that the association signal obtained on SCC14 is largely attributable to TBX5 c.54T > C likely to be in LD with a regulatory polymorphism of TBX5. The transcription factor TBX5 interacts with GJA5 and MEF2C both being involved in vascularization. This study provides evidence for epistatic interaction of TBX5 and MEF2C, thus supporting deficiency of blood supply to growth cartilage as being fundamental for the initiation of OC.

KRT8, FAF1 and PTH1R gene polymorphisms are associated with leg weakness traits in pigs.

The liability to lesions of dysfunctions of bone and joints in pigs, summarized as leg weakness and mostly expressed as osteochondrosis, is an animal welfare and economic issue in pig production. The objective of this study was to identify polymorphisms in the functional and positional candidate genes keratin 8 (KRT8), Fas-associated factor 1 (FAF1) and parathyroid hormone type I receptor (PTH1R) and to evaluate their association with leg weakness traits. Therefore, osteochondrosis lesions were scored in animals of a Duroc × Pietrain F2 population (DuPi; n = 310) and commercial herds of the breed Large White (n = 298). In addition, bone mineralization traits were observed in DuPi population. SNPs were identified in genes KRT8 (g.8,039G > A), FAF1 (g.380,914T > C) and PTH1R (c.1,672C > T). KRT8 showed significant association with bone mineral density and content (P ≤ 0.05). FAF1 was association with OC lesions score of all joints inspected (P ≤ 0.05). PTH1R showed significant dominance effects on OC lesion scores of the distal femur articular cartilage (P = 0.01) and epiphysis of the distal ulna (P = 0.05) as well as sums of scores of all joints (OCsum, P = 0.04) and assignment to groups of either severely or gently affected animals (OCcat, P = 0.01). This study reveals clear genetic-statistical evidence for a link of KRT8, FAF1 and PTH1R with some of leg weakness related traits in pigs.

Association and expression study of MMP3, TGFß1 and COL10A1 as candidate genes for leg weakness-related traits in pigs.

The present study was aimed to determine the association between metalloproteinase 3 (MMP3), transforming growth factor beta 1 (TGFß1) and collagen type X alpha I (COL10A1) gene polymorphisms with traits related to leg weakness in pigs. Three hundred Duroc × Pietrain cross breds (DuPi) and 299 pigs of a commercial population (CP) were used for the experiment. DuPi animals were examined for 10 different traits describing leg and feet structure, osteochondrosis (OC) scores and bone density status. Data of OC score at condylus medialis humeri, condylus medialis femoris and distal epiphysis ulna regions of CP were used for association analysis. Significant association (P < 0.05) was found for MMP3 SNP (g.158 C>T) with OC at head of femur and bone mineral density in the DuPi population. Association (P < 0.05) was found between SNP of TGFß1 (g.180 G>A) with rear leg score and the principle component denoting both OC and feet and leg scores in the DuPi population. No association was found between COL10A1 (g.72 C>T) and leg weakness related traits. The associations of SNPs with OC traits could not be confirmed in the commercial population. Expression analysis of the three candidate genes was performed to compare between healthy and OC. TGFß1 was found to be highly expressed (P < 0.05) in the OC compared to healthy cartilages, but no significant different expressions were observed for MMP3 and COL10A1 genes. The present finding suggested that TGFß1 and MMP3 genes variants have an effect on some of the leg weakness related traits.