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SARS-CoV-2 is a newly discovered virus which causes COVID-19 (coronavirus disease of 2019), initially documented as a human pathogen in 2019 in the city of Wuhan China, has now quickly spread across the globe with an urgency to develop effective treatments for the virus and emerging variants. Therefore, to identify potential therapeutics, an antiviral catalogue of compounds from the CAS registry, a division of the American Chemical Society was evaluated using a pharmacoinformatics approach. A total of 49,431 compounds were initially recovered. After a biological and chemical curation, only 23,575 remained. A machine learning approach was then used to identify potential compounds as inhibitors of SARS-CoV-2 based on a training dataset of molecular descriptors and fingerprints of known reported compounds to have favorable interactions with SARS-CoV-2. This approach identified 178 compounds, however, a molecular docking analysis revealed only 39 compounds with strong binding to active sites. Downstream molecular analysis of four of these compounds revealed various non-covalent interactions along with simultaneous modulation between ligand and protein active site pockets. The pharmacological profiles of these compounds showed potential drug-likeness properties. Our work provides a list of candidate anti-viral compounds that may be used as a guide for further investigation and therapeutic development against SARS-CoV-2.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers owing to a number of characteristics including difficulty in establishing early diagnosis and the absence of effective therapeutic regimens. A large number of genetic alterations have been ascribed to PDAC with mutations in the KRAS2 proto-oncogene thought to be an early event in the progression of disease. Recent lineage-tracing studies have shown that acinar cells expressing mutant Kras(G12D) are induced to transdifferentiate, generating duct-like cells through a process known as acinar-ductal metaplasia (ADM). ADM lesions then convert to precancerous pancreatic intraepithelial neoplasia (PanIN) that progresses to PDAC over time. Thus, understanding the earliest events involved in ADM/PanIN formation would provide much needed information on the molecular pathways that are instrumental in initiating this disease. As studying the transition of acinar cells to metaplastic ductal cells in vivo is complicated by analysis of the entire organ, an in vitro three dimensional (3D) culture system was used to model ADM outside the animal. Kras(G12D)-expressing acinar cells rapidly underwent ADM in 3D culture, forming ductal cysts that silenced acinar genes and activated duct genes, characteristics associated with in vivo ADM/PanIN lesions. Analysis of downstream KRAS signaling events established a critical importance for the Raf/MEK/ERK pathway in ADM induction. In addition, forced expression of the acinar-restricted transcription factor Mist1, which is critical to acinar cell organization, significantly attenuated Kras(G12D)-induced ADM/PanIN formation. These results suggest that maintaining MIST1 activity in Kras(G12D)-expressing acinar cells can partially mitigate the transformation activity of oncogenic KRAS. Future therapeutics that target both the MAPK pathway and Mist1 transcriptional networks may show promising efficacy in combating this deadly disease.

Previous work demonstrated that methyl palmitate possesses acaricidal activity against Tetranychus viennensis Boisduval (Acari: Tetranychidae) via an unknown mechanism. Here, the symptoms of methyl palmitate toxicity to T. viennensi were studied to determine the acaricidal mechanism of action of this fatty acid methyl ester. Methyl palmitate caused concentration-dependent mortality of T. viennensis, with a moderate concentration (5 mg/ml) eliciting excitement and premature oviposition without spinning shortly after exposure. Tremors of the appendages were subsequently observed, followed by quiescence after approximately 5 h. Mites developed dorsal fluid exosmosis at 15-20 h posttreatment with reduced egg production, followed shortly thereafter by death. Some typical neurotoxic symptoms such as excitement and convulsions were observed in methyl palmitate-exposed mites, suggesting that methyl palmitate may be a neurotoxin. Compared with other neurotoxic acaricides, methyl palmitate poisoning is a slow process in mites. Transmission electron microscopy revealed serious ultrastructural damage in response to 5 mg/ml methyl palmitate exposure. Autolysis of membranous structures was also observed, especially in the mitochondria, suggesting a novel mode of action for methyl palmitate-induced toxicity.

Pancreatic ductal adenocarcinoma (PDAC) is believed to arise through a multistep model comprised of putative precursor lesions known as pancreatic intraepithelial neoplasia (PanIN). Recent genetically engineered mouse models of PDAC demonstrate a comparable morphologic spectrum of murine PanIN (mPanIN) lesions. The histogenesis of PanIN and PDAC in both mice and men remains controversial. The most faithful genetic models activate an oncogenic Kras G12D knockin allele within the pdx1 - or ptf1a/p48 -expression domain of the entire pancreatic anlage during development, thus obscuring the putative cell(s)-of-origin from which subsequent mPanIN lesions arise. In our study, activation of this knockin Kras G12D allele in the Elastase - and Mist1 -expressing mature acinar compartment of adult mice resulted in the spontaneous induction of mPanIN lesions of all histological grades, although invasive carcinomas per se were not seen. We observed no requirement for concomitant chronic exocrine injury in the induction of mPanIN lesions from the mature acinar cell compartment. The acinar cell derivation of the mPanINs was established through lineage tracing in reporter mice, and by microdissection of lesional tissue demonstrating Cre -mediated recombination events. In contrast to the uniformly penetrant mPanIN phenotype observed following developmental activation of Kras G12D in the Pdx1 -expressing progenitor cells, the Pdx1 -expressing population in the mature pancreas (predominantly islet β cells) appears to be relatively resistant to the effects of oncogenic Kras . We conclude that in the appropriate genetic context, the differentiated acinar cell compartment in adult mice retains its susceptibility for spontaneous transformation into mPanIN lesions, a finding with potential relevance vis-à-vis the origins of PDAC.

A number of studies have shown that pancreatic ductal adenocarcinoma develops through precursor lesions termed pancreatic intraepithelial neoplasia (PanIN). PanINs are thought to initiate in the small ducts of the pancreas through activating mutations in the KRAS proto-oncogene. What remains unanswered is the identification of the individual cell type(s) that contributes to pancreatic ductal adenocarcinoma formation. To follow the cellular and molecular changes that occur in acinar and duct cell properties on Kras(G12D) expression, we took advantage of LSL-Kras(G12D/+)/p48(Cre/+) mice, which faithfully mimic the human disease. In young animals (4 weeks), the predominant cellular alteration in the exocrine pancreas was acinar metaplasia in which individual acini consisted of acinar cells and duct-like cells. Metaplastic acinar structures were highly proliferative, expressed Notch target genes, and exhibited mosaic expression patterns for epidermal growth factor receptor, ErbB2, and pErk. This expression pattern paralleled the expression pattern detected in mouse PanINs, suggesting that mouse PanINs and acinar-ductal metaplasia follow similar molecular pathways. Indeed, immunofluorescence studies confirmed the presence of acinar cells within mPanIN lesions, raising the possibility that Kras(G12D)-induced mPanINs develop from acinar cells that undergo acinar-ductal metaplasia. Identification of an acinar contribution to PanIN formation offers new directions for successful targeted therapeutic approaches to combat this disease.

The carmine spider mite, Tetranychus cinnabarinus (Acari: Tetranychidae), is an economically important and extremely polyphagous herbivorous pest, with the title of "resistance champion" among arthropods. Anticholinesterase insecticides such as organophosphate and carbamate account for more than one-third of global insecticide sales. The non-target toxicity and resistance problem of organophosphate and carbamate have become of growing concern, which may be due to the fact that they target the ubiquitous catalytic serine residue of acetylcholinesterase (AChE) in mammals, birds, and beneficial insects. In this study, the structural differences between T. cinnabarinus AChE and human AChE, at or near the catalytic pocket, were illustrated. From the SPECS chemical lead-compound database, 55 AChE inhibitor candidates were screened for high affinity for T. cinnabarinus AChE, but low affinity for human AChE, using the DOCK 6 and AutoDock Vina software. Three of the fifty-five candidates had inhibitory activity greater than that of the reversible AChE inhibitor eserine, with no observed inhibitory activities against human AChE. Two of the three had toxicity to T. cinnabarinus comparable to that of natural insecticidal pyrethrins. However, their potency is low compared with that of etoxazole, and further work is needed to optimize their potency. The selectivity of the three compounds over human and mite AChE may be due to their interaction with the mite-specific residues, as analyzed by Cyscore. The three compounds are potential lead compounds for development of novel acaricides against T. cinnabarinus with reduced toxicity to non-target species and a low propensity for resistance.

The study of microRNAs (miRNAs) became intertwined with cancer biology at an early stage. In one of the first publications reporting altered miRNA expression in cancer, Michael et al. found that loss of miR-143/145 is associated with colorectal cancer.1 This and other seminal studies opened the door to a new field of research, which over the past decade has revealed that miRNA dysregulation is involved in all aspects of malignancy including tumor initiation, progression and metastasis. As one of the most reproducible observations in this vast literature, loss of miR-143/145 has been reported in subsequent studies of colorectal cancer and other epithelial tumor types. Through repression of KRAS, MYC, ERK5 and other oncogenes, overexpression of miR-143/145 inhibits proliferation and survival of cancer cells in cell culture and xenografts.2,3 Therefore, miR-143/145 have been widely accepted as prototypical miRNA tumor suppressors. In a recent study,4 we attempted to complement these reports by examining the endogenous functions of miR-143/145 in mice. We found that loss of miR-143/145 does not lead to any discernable defects in intestinal development or homeostasis. This was not entirely unexpected, as a growing number of studies have reported that miRNAs are dispensable for development but perform important functions under stress conditions.5 Indeed, we found that miR-143/145 were essential for regeneration of epithelial cells following intestinal injury elicited by dextran sulfate sodium (DSS). Adjacent to DSS-induced ulcers, proliferation of wild-type epithelial cells switches from a hierarchical mode restricted to stem cells and transit-amplifying cells to a regenerative mode wherein all cells in the crypts multiply. However, in miR-143/145 null mice, epithelial cells failed to make this transition, resulting in inefficient epithelial repair, sustained inflammation and eventual death of the animals. In the course of investigating the cellular context of this pro-regenerative function, we were surprised to find that miR-143/145 expression is abundant in smooth muscle cells of muscularis mucosae and muscularis externa, myofibroblasts, and other cells of mesenchymal lineages, but completely absent in human and mouse intestinal epithelial cells (Fig. 1). This finding was further corroborated by genetic studies, as mice with mesenchymal deletion of miR-143/145 recapitulated the regenerative defect, while epithelial ablation of miR-143/145 had no discernable effect. Figure 1. miR-143/145 in intestinal regeneration and tumorigenesis. miR-143/145 are abundant in the mesenchymal cells of the intestine, including myofibroblasts and smooth muscle, but completely absent in the epithelium. Although dispensable for development and ... Smooth muscle cells in the GI tract form 2 distinct layers: muscularis mucosae and muscularis externa, which are classic histological structures that, by mechanical contraction, facilitate movement of food along the digestive tract. Previous studies have mainly focused on their mechanical and electrophysiology properties. Given the abundant expression of miR-143/145 in smooth muscle, we deleted the miRNAs specifically within this lineage to determine the contribution of this cell-type to the regenerative defect. Remarkably, smooth muscle-specific miR-143/145 deletion significantly impaired intestinal wound healing, uncovering a previously unrecognized signaling function for this lineage that provides instructive cues during epithelial regeneration. Paracrine signals may be elaborated by smooth muscle cells themselves (Fig. 1). In addition, we showed that within zones of injury, smooth muscle cells adopt a myofibroblast phenotype and migrate into the lamina propria, where they may functionally coordinate epithelial repair. This behavior is reminiscent of smooth muscle cells of the vasculature, which are known to switch between contractile and secretory phenotypes in injury settings.6 We demonstrated that miR-143/145-deficient myofibroblasts secrete an excess of the novel miR-143 target insulin-like growth factor binding protein 5 (IGFBP5), which sequesters IGF ligands and may thereby impair intestinal epithelial proliferation. Further studies are necessary to elucidate the broader signaling program that is dysregulated in miR-143/145-deficient smooth muscle and myofibroblasts. This approach is likely to yield important insights into the mechanisms that govern intestinal wound healing and may reveal new therapeutic strategies for ulcerative colitis, Crohn's disease, and other intestinal diseases. Our findings also have important implications for colorectal cancer. The absence of miR-143/145 in normal and cancerous epithelium clearly rules out a cell autonomous tumor suppressor function for these miRNAs. We suspect that the previously reported loss of miR-143/145 in colorectal cancer simply reflects the distinct cellular composition of normal and neoplastic intestinal tissue. Similar misinterpretations may have occurred in other cancer settings, underscoring the need for careful analyses to resolve the settings wherein these miRNAs may function in a cell autonomous manner to influence cancer cell behavior. Importantly, our findings do not invalidate the reported anti-tumorigenic activity of miR-143/145 in colorectal cancer cells or potential therapeutic strategies based upon delivery of these miRNAs. Nevertheless, the effects of such treatments on mesenchymal cells in target tissues have to be carefully evaluated. Finally, it is tempting to speculate that miR-143/145 could influence colorectal tumorigenesis through their ability to regulate mesenchymal-to-epithelial paracrine signaling (Fig. 1). It has been demonstrated that colorectal tumors exhibit degraded epithelial barrier function, which allows microbial penetration and tumor-promoting inflammation.7 Perhaps loss of miR-143/145 in the mesenchyme, which could impair a compensatory local epithelial regenerative response, exacerbates tumor-associated inflammation and thereby enhances malignant progression. Regardless, these new insights into the natural functions of miR-143/145 serve as yet another reminder that cancer cells do not live in isolation. Stromal components, such as myofibroblasts, vasculature, immune cells, and extracellular matrix, have to be taken into consideration to gain a full understanding of miRNA functions in tumorigenesis.

Down-regulation of miR-26 family members has been implicated in the pathogenesis of multiple malignancies. In some settings, including glioma, however, miR-26-mediated repression of PTEN promotes tumorigenesis. To investigate the contexts in which the tumor suppressor versus oncogenic activity of miR-26 predominates in vivo, we generated miR-26a transgenic mice. Despite measureable repression of Pten, elevated miR-26a levels were not associated with malignancy in transgenic animals. We documented reduced miR-26 expression in human colorectal cancer and, accordingly, showed that miR-26a expression potently suppressed intestinal adenoma formation in Apcmin/+ mice, a model known to be sensitive to Pten dosage. These studies reveal a tumor suppressor role for miR-26 in intestinal cancer that overrides putative oncogenic activity, highlighting the therapeutic potential of miR-26 delivery to this tumor type.

Pyruvate decarboxylase and alcohol dehydrogenase are efficient enzymes for ethanol production in Zymomonas mobilis. These two enzymes were over-expressed in Escherichia coli, a promising candidate for industrial ethanol production, resulting in high ethanol production in the engineered E. coli. To investigate the intracellular changes to the enzyme overexpression for homoethanol production, 2-DE and LC–MS/MS were performed. More than 1,000 protein spots were reproducibly detected in the gel by image analysis. Compared to the wild-type, 99 protein spots showed significant changes in abundance in the recombinant E. coli, in which 46 were down-regulated and 53 were up-regulated. Most proteins related to tricarboxylic acid cycle, glycerol metabolism and other energy metabolism were up-regulated, whereas proteins involved in glycolysis and glyoxylate pathway were down-regulated, indicating the rewired metabolism in the engineered E. coli. As glycolysis is the main pathway for ethanol production, and it was inhibited significantly in engineered E. coli, further efforts should be directed at minimizing the repression of glycolysis to optimize metabolism network for higher yields of ethanol production.

SummaryDownregulation of the miR-143/145 microRNA (miRNA) cluster has been repeatedly reported in colon cancer and other epithelial tumors. In addition, overexpression of these miRNAs inhibits tumorigenesis, leading to broad consensus that they function as cell-autonomous epithelial tumor suppressors. We generated mice with deletion of miR-143/145 to investigate the functions of these miRNAs in intestinal physiology and disease in vivo. Although intestinal development proceeded normally in the absence of these miRNAs, epithelial regeneration after injury was dramatically impaired. Surprisingly, we found that miR-143/145 are expressed and function exclusively within the mesenchymal compartment of intestine. Defective epithelial regeneration in miR-143/145-deficient mice resulted from the dysfunction of smooth muscle and myofibroblasts and was associated with derepression of the miR-143 target Igfbp5, which impaired IGF signaling after epithelial injury. These results provide important insights into the regulation of epithelial wound healing and argue against a cell-autonomous tumor suppressor role for miR-143/145 in colon cancer.

Background & Aims Invasive pancreatic ductal adenocarcinoma is thought to originate from duct-like lesions called pancreatic intraepithelial neoplasia (PanIN). PanINs progress from low grade (PanIN-1) to high grade (PanIN-3) as the cells attain molecular alterations to key regulatory genes, including activating mutations in the KRAS protooncogene. Despite a well-documented progression model, our knowledge of the initiator cells of PanINs and the transcriptional networks and signaling pathways that impact PanIN formation remains incomplete. Methods In this study, we examined the importance of the acinar-restricted transcription factor Mist1 to Kras G12D -induced mouse PanIN (mPanIN) formation in 3 different mouse models of pancreatic cancer. Results In the absence of Mist1 ( Mist1 KO ), Kras G12D -expressing mice exhibited severe exocrine pancreatic defects that were rescued by ectopic expression of Mist1 in acinar cells. mPanIN development was greatly accelerated in Mist1 KO / Kras G12D/+ pancreata, and in vitro assays revealed that Mist1 KO acinar cells were predisposed to convert to a ductal phenotype and activate epidermal growth factor receptor (EGFR) and Notch-signaling pathways. Conclusions We propose that convergence of EGFR, Notch, and Kras pathways in acinar cells lacking Mist1 leads to enhanced mPanIN formation.

Background & Aims Gastric cancer evolves in the setting of a pathologic mucosal milieu characterized by both loss of acid-secreting parietal cells and mucous cell metaplasias. Indeed, mucous cell metaplasia is considered the critical preneoplastic lesion for gastric cancer. Previous investigations have shown that infection of mice with Helicobacter felis or induction of acute parietal cell loss with the drug DMP-777 leads to the emergence of a type of metaplasia designated spasmolytic polypeptide-expressing metaplasia (SPEM). We have hypothesized that SPEM arises from proliferating cells in gland bases, either from a cryptic progenitor cell or by transdifferentiation of mature chief cells. Methods Taking advantage of the chief cell-restricted expression of Mist1-Cre-ER T2 , we used lineage mapping to examine whether SPEM lineages were derived from chief cells in 3 independent models of induction by DMP-777 treatment, L-635 treatment, or H felis infection. Results Treatment of mice with L-635 for 3 days led to rapid parietal cell loss, induction of a prominent inflammatory infiltrate, and emergence of SPEM. In all 3 models, SPEM developed, at least in part, from transdifferentiation of chief cells. We further found that acute parietal cell loss in the setting of inflammation (L-635 treatment) led to more rapid induction and expansion of SPEM derived from transdifferentiation of chief cells. Conclusions These studies provide direct evidence by lineage tracing that SPEM evolves from differentiated chief cells. Thus, mature gastric chief cells have the ability to act as cryptic progenitors and reacquire proliferative capacity within the context of mucosal injury and inflammation.

Our studies are focused on the identification and prevention of the initial transformation events associated with pancreatic ductal adenocarcinoma (PDA) development, an extremely lethal malignancy with poor prognosis and ineffective treatments. PDA initiates through activating mutations in the KRAS proto-oncogene (KrasG12D) and recent studies from our laboratory have determined that targeted expression of an endogenous KrasG12D allele in pancreatic acinar cells is sufficient to produce PDA precursor lesions (PanINs). These results suggest that PDA arises via acinar cells undergoing a process of acinar-to-ductal metaplasia (ADM) which is characterized by a loss of acinar cell properties with the acquisition of duct-like characteristics. Several studies indicate that the process of ADM requires inactivation of the acinar-specific transcription factor Mist1, since Mist1 null (Mist1KO) mice display accelerated ADM and PanIN formation upon KrasG12D expression. Additionally, acinar cell polarity is disrupted in Mist1KO mice with diffuse zymogen granule organization and mis-localization of nuclei. These results suggest that Mist1 has a key role in suppressing PDA formation by maintaining the acinar cell differentiation program. To identify the downstream genes affected by the absence of Mist1, we have generated an inducible Mist1 transgenic mouse line (LSL-Mist1myc) in which constitutive Mist1 expression is activated through Cre-mediated recombination. When LSL-Mist1myc mice are crossed to Mist1Cre-ER mice, Mist1myc expression can be induced in acinar cells upon Tamoxifen (TM) addition. Analysis of Mist1Cre-ER/Cre-ER; LSL-Mist1myc mice confirmed that exogenous Mist1myc expression is detected as early as 6 hours post-TM. Time course studies in a Mist1KO background revealed that nuclear and zymogen granule organization can be restored within 24 hours of Cre activation. We conclude that Mist1 actively controls pancreatic acinar cell organization and that induction of Mist1 in Mist1KO mice leads to complete reorganization of pancreatic tissues. Our current efforts are focused on establishing how Mist1 negatively regulates ADM and PanIN formation and to identify Mist1 targets that negatively regulate Kras-induced pancreas metaplasia and transformation. Citation Information: Cancer Res 2009;69(23 Suppl):A76.

Significance: Pancreatic ductal adenocarcinoma (PDA) is one of the most fatal malignancies, partially due to the lack of early detection methods. Background: Efforts to understand the early stages of PDA progression have led to identification of Kras mutations as the initiating event and pancreatic intraepithelial neoplasias (PanINs) as one of the cellular precursors of PDA. Recently, several labs have demonstrated that PanINs can be generated from pancreatic acinar cells upon expression of Kras mutants in mouse models. This dramatic shift from differentiated acinar cells to duct-like PanIN cells, generally termed acinar-ductal metaplasia (ADM), is accelerated by loss of Mist1, an acinar-restricted basic helix-loop-helix transcription factor. Despite this recent progress, the transcription and signaling networks that regulate ADM remain largely unknown. Results: To study these networks, we employed a collagen-based 3-dimensional (3D) culture model of ADM, where primary acinar cells can be transformed into duct-like cysts when treated with transforming growth factor-alpha (TGF-α). A similar acinar-to-ductal conversion was observed with primary acinar cells that express the KrasG12D mutant. Initial characterization of the ADM process revealed that the MAPK pathway is required in both settings. However, EGFR activity is required for TGF-α-induced conversion but not for KrasG12D-induced conversion. Consistent with previous data generated from mouse models, loss of Mist1 accelerates KrasG12D-induced conversion to ductal cysts in vitro. This accelerated ADM formation can be reversed by inhibition of EGFR, without affecting the basal level conversion induced by KrasG12D, suggesting that loss of Mist1 promotes transformation through an EGFR-dependent pathway. Further investigation has revealed that Mist1 directly activates transcription of the epidermal growth factor (EGF) gene, which encodes a classic ligand of EGFR. Conclusion: These data suggest that EGFR has a complex role in this ADM model. EGFR activity either promotes maintenance of acinar differentiation, or cooperates with KrasG12D-induced transformation, possibly depending on the functional specificity of different ligands. Citation Information: Cancer Res 2009;69(23 Suppl):A71.