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The coronavirus disease 2019 (COVID-19) pandemic has underscored the lack of approved drugs against acute viral diseases. Plants are considered inexhaustible sources of drugs for several diseases and clinical conditions, but plant-derived compounds have seen little success in the field of antivirals. Here, we present the case for the use of compounds from vascular plants, including alkaloids, flavonoids, polyphenols, and tannins, as antivirals, particularly for the treatment of COVID-19. We review current evidence for the use of these phytochemicals against SARS-CoV-2 infection and present their potential targets in the SARS-CoV-2 replication cycle.

Enzymes of the ten-eleven translocation family are considered to play an important role in the regulation of DNA methylation patterns by converting 5-methylcytosine to 5-hydroxymethylcytosine. Known as a maternal transcript enriched in mature oocytes, ten-eleven translocation-3 (TET3) has been suggested to initiate DNA demethylation of the paternal genome in zygotes. Previous studies in mouse cells indicate that the N-terminal CXXC domain of TET3 is important in catalyzing the oxidation of 5-methylcytosine through its potential DNA binding ability; however, it is not clear whether the DNA binding capacity of CXXC domain is required for the 5-hydroxymethylcytosine conversion in mammalian embryos. Here, we identified TET3 isoforms in porcine oocytes and investigated the role of the oocyte specific TET3 isoform (pTET3L) in controlling postfertilization demethylation in porcine embryos. The pTET3L possessed sequences representing a known DNA binding domain, the CXXC, and injection of the TET3 CXXC fused with GFP into mature porcine oocytes resulted in exclusive localization of the GFP-CXXC in the pronuclei. The CXXC overexpression reduced the 5-methylcytosine level in zygotes and enhanced the DNA demethylation of the NANOG promoter in 2-cell stage embryos. Furthermore, there was an increase in the transcript abundance of NANOG and ESRRB in blastocysts developed from GFP-CXXC injected oocytes. Targeted knockdown of pTET3L resulted in the downregulation of pluripotency genes in subsequently developed blastocysts. The findings indicate that the CXXC domain of TET3 serves as a critical component for the postfertilization demethylation of porcine embryos and coordinates proper expression of pluripotency related genes in blastocysts.

New therapies to treat pancreatic cancer are direly needed. However, efficacious interventions lack a strong preclinical model that can recapitulate patients’ anatomy and physiology. Likewise, the availability of human primary malignant tissue for ex vivo studies is limited. These are significant limitations in the biomedical device field. We have developed RAG2/IL2RG deficient pigs using CRISPR/Cas9 as a large animal model with the novel application of cancer xenograft studies of human pancreatic adenocarcinoma. In this proof-of-concept study, these pigs were successfully generated using on-demand genetic modifications in embryos, circumventing the need for breeding and husbandry. Human Panc01 cells injected subcutaneously into the ears of RAG2/IL2RG deficient pigs demonstrated 100% engraftment with growth rates similar to those typically observed in mouse models. Histopathology revealed no immune cell infiltration and tumor morphology was highly consistent with the mouse models. The electrical properties and response to irreversible electroporation of the tumor tissue were found to be similar to excised human pancreatic cancer tumors. The ample tumor tissue produced enabled improved accuracy and modeling of the electrical properties of tumor tissue. Together, this suggests that this model will be useful and capable of bridging the gap of translating therapies from the bench to clinical application.

SummaryThe worldwide production of in vitro-produced embryos in livestock species continues to grow. The current gold standard for selecting quality oocytes and embryos is morphologic assessment, yet this method is subjective and varies based on experience. There is a need for a non-invasive, objective method of selecting viable oocytes and embryos. The aim of this study was to determine if ooplasm area, diameter including zona pellucida (ZP), and ZP thickness of artificially activated oocytes and in vitro fertilized (IVF) zygotes are indicative of development success in vitro and correlated with embryo quality, as assessed by total blastomere number. Diameter affected the probability of development to the blastocyst stage in activated oocytes on day 7 (P < 0.01) and day 8 (P < 0.001), and had a tendency to affect IVF zygotes on day 8 (P = 0.08). Zona pellucida thickness affected the probability of development on day 7 (P < 0.01) and day 8 (P < 0.001) in activated oocytes, and day 8 for IVF zygotes (P < 0.05). An interaction between ZP thickness and diameter was observed on days 7 and 8 (P < 0.05) in IVF zygotes. Area did not significantly affect the probability of development, but was positively correlated with blastomere number on day 8 for IVF zygotes (P = 0.01, conditional R2 = 0.09). Physical parameters of bovine zygotes have the potential for use as a non-invasive, objective selection method. Upon further development, methods used in this study could be integrated into embryo production systems to improve IVF success.

Staphylococcus aureus is a leading cause of hospital- and community-acquired infections. Despite current advances in antimicrobial chemotherapy, the infections caused by S. aureus remain challenging due to their ability to readily develop resistance. Indeed, antibiotic resistance, exemplified by methicillin-resistant S. aureus (MRSA) is a top threat to global health security. Furthermore, the current rate of antibiotic discovery is much slower than the rate of antibiotic-resistance development. It seems evident that the conventional in vitro bacterial growth-based screening strategies can no longer effectively supply new antibiotics at the rate needed to combat bacterial antibiotic-resistance. To overcome this antibiotic resistance crisis, screening assays based on host-pathogen interactions have been developed. In particular, the free-living nematode Caenorhabditis elegans has been used for drug screening against MRSA. In this review, we will discuss the general principles of the C. elegans-based screening platform and will highlight its unique strengths by comparing it with conventional antibiotic screening platforms. We will outline major hits from high-throughput screens of more than 100,000 small molecules using the C. elegans-MRSA infection assay and will review the mode-of-action of the identified hit compounds. Lastly, we will discuss the potential of a C. elegans-based screening strategy as a paradigm shift screening platform.

Immunocompromised mice are commonly utilized to study pancreatic cancer and other malignancies. The ability to xenograft tumors in either subcutaneous or orthotopic locations provides a robust model to study diverse biological features of human malignancies. However, there is a dire need for large animal models that better recapitulate human anatomy in terms of size and physiology. These models will be critical for biomedical device development, surgical optimization, and drug discovery. Here, we describe the generation and application of immunocompromised pigs lacking RAG2 and IL2RG as a novel model for human xenograft studies. These SCID-like pigs closely resemble NOD scid gamma mice and are receptive to human tumor tissue, cell lines, and organoid xenografts. However, due to their immunocompromised nature, these immunocompromised animals require housing and maintenance under germfree conditions. In this protocol, we describe the use of these pigs in a subcutaneous tumor injection study with human PANC1 cells. The tumors demonstrate a steady, linear growth curve, reaching 1.0 cm within 30 days post injection. The model described here is focused on subcutaneous injections behind the ear. However, it is readily adaptable for other locations and additional human cell types.

Increased knowledge of reproduction and health of domesticated animals is integral to sustain and improve global competitiveness of U.S. animal agriculture, understand and resolve complex animal and human diseases, and advance fundamental research in sciences that are critical to understanding mechanisms of action and identifying future targets for interventions. Historically, federal and state budgets have dwindled and funding for the United States Department of Agriculture (USDA) National Institute of Food and Agriculture (NIFA) competitive grants programs remained relatively stagnant from 1985 through 2010. This shortage in critical financial support for basic and applied research, coupled with the underappreciated knowledge of the utility of non-rodent species for biomedical research, hindered funding opportunities for research involving livestock and limited improvements in both animal agriculture and animal and human health. In 2010, the National Institutes of Health and USDA NIFA established an interagency partnership to promote the use of agriculturally important animal species in basic and translational research relevant to both biomedicine and agriculture. This interagency program supported 61 grants totaling over $107 million with 23 awards to new or early-stage investigators. This article will review the success of the 9-year Dual Purpose effort and highlight opportunities for utilizing domesticated agricultural animals in research.

Precision livestock farming has become an important research focus with the rising demand of meat production in the swine industry. Currently, the farming practice is widely conducted by the technology of computer vision (CV), which automates monitoring pig activity solely based on video recordings. Automation is fulfilled by deriving imagery features that can guide CV systems to recognize animals' body contours, positions, and behavioral categories. Nevertheless, the performance of the CV systems is sensitive to the quality of imagery features. When the CV system is deployed in a variable environment, its performance may decrease as the features are not generalized enough under different illumination conditions. Moreover, most CV systems are established by supervised learning, in which intensive effort in labeling ground truths for the training process is required. Hence, a semi-supervised pipeline, VTag, is developed in this study. The pipeline focuses on long-term tracking of pig activity without requesting any pre-labeled video but a few human supervisions to build a CV system. The pipeline can be rapidly deployed as only one top-view RGB camera is needed for the tracking task. Additionally, the pipeline was released as a software tool with a friendly graphical interface available to general users. Among the presented datasets, the average tracking error was 17.99 cm. Besides, with the prediction results, the pig moving distance per unit time can be estimated for activity studies. Finally, as the motion is monitored, a heat map showing spatial hot spots visited by the pigs can be useful guidance for farming management. The presented pipeline saves massive laborious work in preparing training dataset. The rapid deployment of the tracking system paves the way for pig behavior monitoring. Lay Summary Collecting detailed measurements of animals through cameras has become an important focus with the rising demand for meat production in the swine industry. Currently, researchers use computational approaches to train models to recognize pig morphological features and monitor pig behaviors automatically. Though little human effort is needed after model training, current solutions require a large amount of pre-selected images for the training process, and the expensive preparation work is difficult for many farms to implement such practice. Hence, a pipeline, VTag, is presented to address these challenges in our study. With few supervisions, VTag can automatically track positions of multiple pigs from one single top-view RGB camera. No pre-labeled images are required to establish a robust pig tracking system. Additionally, the pipeline was released as a software tool with a friendly graphical user interface, that is easy to learn for general users. Among the presented datasets, the average tracking error is 17.99 cm, which is shorter than one-third of the pig body length in the study. The estimated pig activity from VTag can serve as useful farming guidance. The presented strategy saves massive laborious work in preparing training datasets and setting up monitoring environments. The rapid deployment of the tracking system paves the way for pig behavior monitoring. The presented pipeline, VTag, saves massive laborious work in preparing labeled training datasets and setting up environment for pig tracking tasks. VTag can be deployed rapidly and paves the way for pig behavior monitoring. USDA-NIFA [202067030-31339, 2020-67021-32460] Published version This study was supported by the USDA-NIFA grant 202067030-31339 and 2020-67021-32460

Recombinant proteins are essential for the treatment and diagnosis of clinical human ailments. The availability and biological activity of recombinant proteins is heavily influenced by production platforms. Conventional production platforms such as yeast, bacteria, and mammalian cells have biological and economical challenges. Transgenic livestock species have been explored as an alternative production platform for recombinant proteins, predominantly through milk secretion; the strategy has been demonstrated to produce large quantities of biologically active proteins. The major limitation of utilizing livestock species as bioreactors has been efforts required to alter the genome of livestock. Advancements in the genome editing field have drastically improved the ability to genetically engineer livestock species. Specifically, genome editing tools such as the CRISPR/Cas9 system have lowered efforts required to generate genetically engineered livestock, thus minimizing restrictions on the type of genetic modification in livestock. In this review, we discuss characteristics of transgenic animal bioreactors and how the use of genome editing systems enhances design and availability of the animal models.

New therapeutic strategies are direly needed in the fight against cancer. Over the last decade, several tumor ablation strategies have emerged as stand-alone or combination therapies. Histotripsy is the first completely noninvasive, nonthermal, and nonionizing tumor ablation method. Histotripsy can produce consistent and rapid ablations, even near critical structures. Additional benefits include real-time image guidance, high precision, and the ability to treat tumors of any predetermined size and shape. Unfortunately, the lack of clinically and physiologically relevant preclinical cancer models is often a significant limitation with all focal tumor ablation strategies. The majority of studies testing histotripsy for cancer treatment have focused on small animal models, which have been critical in moving this field forward and will continue to be essential for providing mechanistic insight. While these small animal models have notable translational value, there are significant limitations in terms of scale and anatomical relevance. To address these limitations, a diverse range of large animal models and spontaneous tumor studies in veterinary patients have emerged to complement existing rodent models. These models and veterinary patients are excellent at providing realistic avenues for developing and testing histotripsy devices and techniques designed for future use in human patients. Here, we provide a review of animal models used in preclinical histotripsy studies and compare histotripsy ablation in these models using a series of original case reports across a broad spectrum of preclinical animal models and spontaneous tumors in veterinary patients. Accepted version PMID: 34478363

Oocyte activation occurs at the time of fertilization and is a series of cellular events initiated by intracellular Ca2+ increases. Consequently, oocytes are alleviated from their arrested state in meiotic metaphase II (MII), allowing for the completion of meiosis. Oocyte activation is also an essential step for somatic cell nuclear transfer and an important tool to overcome clinical infertility. Traditional artificial activation methods aim to mimic the intracellular Ca2+ changes which occur during fertilization. Recent studies emphasize the importance of cytoplasmic Zn2+ on oocyte maturation and the completion of meiosis, thus suggesting artificial oocyte activation approaches that are centered around the concentration of available Zn2+in oocytes. Depletion of intracellular Zn2+ in oocytes with heavy metal chelators leads to successful oocyte activation in the absence of cellular Ca2+ changes, indicating that successful oocyte activation does not always depends on intracellular Ca2+ increases. Current findings lead to new approaches to artificially activate mammalian oocytes by reducing available Zn2+ contents, and the approaches improve the outcome of oocyte activation when combined with existing Ca2+-based oocyte activation methods. Here, we review the important role of Ca2+ and Zn2+ in mammalian oocyte activation and development of novel oocyte activation approaches based on Zn2+ availability.

Influenza viruses cause significant morbidity and mortality worldwide. Long-term or frequent use of approved anti-influenza agents has resulted in drug-resistant strains, thereby necessitating the discovery of new drugs. In this study, we found aprotinin, a serine protease inhibitor, as an anti-influenza candidate through screening of compound libraries. Aprotinin has been previously reported to show inhibitory effects on a few influenza A virus (IAV) subtypes (e.g., seasonal H1N1 and H3N2). However, because there were no reports of its inhibitory effects on the other types of influenza viruses, we investigated the inhibitory effects of aprotinin in vitro on a wide range of influenza viruses, including avian and oseltamivir-resistant influenza virus strains. Our cell-based assay showed that aprotinin had inhibitory effects on seasonal human IAVs (H1N1 and H3N2 subtypes), avian IAVs (H5N2, H6N5, and H9N2 subtypes), an oseltamivir-resistant IAV, and a currently circulating influenza B virus. We have also confirmed its activity in mice infected with a lethal dose of influenza virus, showing a significant increase in survival rate. Our findings suggest that aprotinin has the capacity to inhibit a wide range of influenza virus subtypes and should be considered for development as a therapeutic agent against influenza.

We have designed and synthesized twenty-six N-arylindazole-3-carboxamide (3a-p) and N-benzoylindazole (6a-j) derivatives to discover with excellent inhibition activities of α-MSH-stimulated melanogenesis. In the bio evaluation studies of these compounds, we discovered eighteen compounds, out of twenty-six exhibited more potent inhibition than the positive control arbutin. From the SAR studies, we identified 3k and 6g as lead compounds which displayed almost 5 and 9 times more potent inhibition of α-MSH-stimulated melanogenesis respectively than the reference arbutin. It is also evident the presence of electron withdrawing group at para position (R3) for the compounds (3a-p) and presence of +M group at ortho position (R5) for the compounds (6a-j) were crucial for their excellent inhibition activities of α-MSH-stimulated melanogenesis.

Background Saururus chinensis leaves have been used as traditional medicine in Korea for pain, intoxication, edema, and furuncle. According to previous reports, these leaves exert renoprotective, neuroprotective, and antioxidant effects by attenuating inflammatory responses. However, the beneficial effect of Saururus chinensis leaves on arthritis has not been elucidated. Thus, we evaluated the water extract of Saururus chinensis leaves (SHW) using type II collagen-induced arthritis (CIA) mice models. Methods Quantitative analysis of major components from SHW was performed by HPLC. Arthritis was induced by injection of type II collagen. Each group was orally administered SHW (100 mg/kg and 500 mg/kg). Methotrexate (MTX) was used as a positive control. Serum levels of interleukin-6, TNF-alpha, and type II collagen IgG in the animal models were measured using ELISA. Histological features were observed by H&E staining. Results Quantitative analysis of SHW showed the contents as 56.4 ± 0.52 mg/g of miquelianin, 7.75 ± 0.08 mg/g of quercetin 3-O-(2”-O-β -glucopyranosyl)-α-rhamnopyranoside, and 3.17 ± 0.02 mg/g of quercitrin. Treatment with 500 mg/kg SHW decreased the serum level of Interleukin-6 (IL-6), TNF-alpha, and collagen IgG in the CIA model. Moreover, SHW treatment diminished the swelling of hind limbs and monocyte infiltration in blood vessels in CIA animal models. The results indicate that SHW could decrease CIA-induced arthritis in vivo. Conclusions The results indicate that SHW could be used to improving arthritis by reducing inflammatory factors (IL-6 and TNF-alpha). However, further experiments are required to determine how SHW influences signal transduction in animal models. Electronic supplementary material The online version of this article (10.1186/s12906-018-2418-z) contains supplementary material, which is available to authorized users.

A novel HIF (hypoxia-inducible factor)-1α inhibitor, the (aryloxyacetylamino)benzoic acid derivative LW6, is an anticancer agent that inhibits the accumulation of HIF-1α. The aim of this study was to characterize and determine the structures of the metabolites of LW6 in ICR mice. Metabolite identification was performed using a predictive multiple reaction monitoring-information dependent acquisition-enhanced product ion (pMRM-IDA-EPI) method in negative ion mode on a hybrid triple quadrupole-linear ion trap mass spectrometer (QTRAP). A total of 12 metabolites were characterized based on their MS/MS spectra, and the retention times were compared with those of the parent compound. The metabolites were divided into five structural classes based on biotransformation reactions: amide hydrolysis, ester hydrolysis, mono-oxidation, glucuronidation, and a combination of these reactions. From this study, 2-(4-((3r,5r,7r)-adamantan-1-yl)phenoxy)acetic acid (APA, M7), the metabolite produced via amide hydrolysis, was found to be a major circulating metabolite of LW6 in mice. The results of this study can be used to improve the pharmacokinetic profile by lowering the clearance and increasing the exposure relative to LW6.

Colony-stimulating factor 2 (CSF2) functions in the reproductive tract to modulate the function of the preimplantation embryo. The β subunit of the CSF2 receptor (CSF2RB) is not expressed in the embryo, and signal transduction is therefore different than for myeloid cells where the receptor is composed of α (CSF2RA) and β subunits. Here, we produced embryos in which exons 5 and 6 of CSF2RA were disrupted using the CRISPR/Cas 9 system to test whether CSF2RA signaling was essential for actions of CSF2 in the bovine embryo. Wild-type and CSF2RA knockout embryos were treated with 10 ng/mL CSF2 or vehicle at day 5 of development. Blastocysts were harvested at day 8 to determine transcript abundance of 90 genes by real-time polymerase chain reaction (PCR). Responses in female blastocysts were examined separately from male blastocysts because actions of CSF2 are sex-dependent. For wild-type embryos, CSF2 altered expression of 10 genes in females and 20 in males. Only three genes were affected by CSF2 in a similar manner for both sexes. Disruption of CSF2RA prevented the effect of CSF2 on expression for 9 of 10 CSF2-regulated genes in females and 19 of 20 genes in males. The results confirm the importance of CSF2RA for regulation of gene expression by CSF2 in the blastocyst.Summary sentenceDisruption of CSF2RA blocked most CSF2-induced changes in gene expression in the blastocyst, indicating that, despite the absence of the β subunit of the receptor, the preimplantation embryo uses CSF2RA for signal transduction.

Development of viviparity in mammals requires that the placenta evolves as an intermediate interface between the fetus and maternal uterus. In addition to the retention of the fetus and secretion of nutrients to support growth and development to term, it is essential that viviparous species modify or inhibit the maternal immune system from recognizing the semi-allogeneic fetus. Following blastocyst hatching from its zona pellucida, trophoblast differentiation provides the initial communication to the maternal endometrium to regulate maintenance of progesterone production from the corpus luteum and biological pathways in uterine and conceptus development necessary in the establishment and maintenance of pregnancy. Many conceptus factors have been proposed to serve in the establishment and maintenance of pregnancy. CRISPR-Cas9 gene-editing technology provides a specific and efficient method to generate animal models to perform loss-of-function studies to investigate the role of specific conceptus factors. The utilization of CRISPR-Cas9 gene editing has provided a direct approach to investigate the specific role of conceptus factors in the development and establishment of pregnancy in the pig. This technology has helped address a number of questions concerning peri-implantation development and has altered our understanding of maternal recognition and maintenance of pregnancy in the pig.

Membrane-damaging antimicrobial agents have great potential to treat multidrug-resistant or multidrug-tolerant bacteria against which conventional antibiotics are not effective. However, their therapeutic applications are often hampered due to their low selectivity to bacterial over mammalian membranes or their potential for cross-resistance to a broad spectrum of cationic membrane-active antimicrobial agents. We discovered that the diarylurea derivative compound PQ401 has antimicrobial potency against multidrug-resistant and multidrug-tolerant Staphylococcus aureus. PQ401 selectively disrupts bacterial membrane lipid bilayers in comparison to mammalian membranes. Unlike cationic membrane-active antimicrobials, the neutral form of PQ401 rather than its cationic form exhibits maximum membrane activity. Overall, our results demonstrate that PQ401 could be a promising lead compound that overcomes the current limitations of membrane selectivity and cross-resistance. Also, this work provides deeper insight into the design and development of new noncharged membrane-targeting therapeutics to combat hard-to-cure bacterial infections., Resistance or tolerance to traditional antibiotics is a challenging issue in antimicrobial chemotherapy. Moreover, traditional bactericidal antibiotics kill only actively growing bacterial cells, whereas nongrowing metabolically inactive cells are tolerant to and therefore “persist” in the presence of legacy antibiotics. Here, we report that the diarylurea derivative PQ401, previously characterized as an inhibitor of the insulin-like growth factor I receptor, kills both antibiotic-resistant and nongrowing antibiotic-tolerant methicillin-resistant Staphylococcus aureus (MRSA) by lipid bilayer disruption. PQ401 showed several beneficial properties as an antimicrobial lead compound, including rapid killing kinetics, low probability for resistance development, high selectivity to bacterial membranes compared to mammalian membranes, and synergism with gentamicin. In contrast to well-studied membrane-disrupting cationic antimicrobial low-molecular-weight compounds and peptides, molecular dynamic simulations supported by efficacy data demonstrate that the neutral form of PQ401 penetrates and subsequently embeds into bacterial lipid bilayers more effectively than the cationic form. Lastly, PQ401 showed efficacy in both the Caenorhabditis elegans and Galleria mellonella models of MRSA infection. These data suggest that PQ401 may be a lead candidate for repurposing as a membrane-active antimicrobial and has potential for further development as a human antibacterial therapeutic for difficult-to-treat infections caused by both drug-resistant and -tolerant S. aureus.

Cutibacterium acnes is capable of inducing inflammation in acne and can lead to a chronic prostatic infection. The diverse pathogenicity among different strains of C. acnes has been presented, but simple appropriate animal models for the evaluation of this bacterium are lacking. In this study, the nematode Caenorhabditis elegans was used as an invertebrate infection model. We revealed that C. acnes type strain ATCC 6919 caused lethal infections to C. elegans in solid and liquid culture media (p

(1S,5R)-4-((E)-3,4-dihydroxy-5-methoxystryryl)-6,6-dimethylbicylco[3.1.1]hept-3-en-2-one (SP-8356) is a novel (1S)-(−)-verbenone derivative that is currently in preclinical development for the treatment of ischemic stroke and atherosclerosis. This report aimed at characterization of the metabolism and pharmacokinetic properties of SP-8356. Following intravenous dose in rats and dogs, plasma concentrations of SP-8356 declined rapidly with high clearance (CL) and short half-life; after oral administration in both species, its plasma levels were below the quantitation limit. Fourteen circulating metabolites, formed by mono-oxygenation, demethylation, glucuronidation, catechol O-methylation, sulfation and oxidation (bioactivation) followed by glutathione (GSH) conjugation, were tentatively identified in both species. Urinary excretion of SP-8356 appeared to be minimal in rats, compared to its metabolites. GSH conjugate of SP-8356 was also formed during incubation with rat liver S9 fraction consistent with oxidative bioactivation; this bioactivation was almost completely inhibited by the cofactors for glucuronidation, sulfation and methylation, indicating that it may be abolished by competing metabolic reactions in the body. The human pharmacokinetics of SP-8356 was predicted to be similar to that of the animals based on the current in vitro metabolic stability results. In summary, rapid phase II metabolism appears to be mainly responsible for its suboptimal pharmacokinetics, such as high CL and low oral absorption. Because of competing metabolic reactions, potential safety risks related to SP-8356 bioactivation may be low.

Historically, genetic engineering in livestock proved to be challenging. Without stable embryonic stem cell lines to utilize, somatic cell nuclear transfer (SCNT) had to be employed to produce many of the genetically engineered (GE) livestock models. Through the genetic engineering of somatic cells followed by SCNT, GE livestock models could be generated carrying site-specific modifications. Although successful, only a few GE livestock models were generated because of low efficiency and associated birth defects. Recently, there have been major strides in the development of genome editing tools: Zinc-Finger Nucleases (ZFNs), Transcription activator-like effector nucleases (TALENS), and Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated 9 (Cas9) system. These tools rely on the generation of a double strand DNA break, followed by one of two repair pathways: non-homologous end joining (NHEJ) or homology directed repair (HDR). Compared to the traditional approaches, these tools dramatically reduce time and effort needed to establish a GE animal. Another benefit of utilizing genome editing tools is the application of direct injection into developing embryos to induce targeted mutations, therefore, eliminating side effects associated with SCNT. Emerging technological advancements of genome editing systems have dramatically improved efficiency to generate GE livestock models for both biomedical and agricultural purposes. Although the efficiency of genome editing tools has revolutionized GE livestock production, improvements for safe and consistent application are desired. This review will provide an overview of genome editing techniques, as well as examples of GE livestock models for agricultural and biomedical purposes.

The ten-eleven translocation (TET) family (TET1/2/3) initiates conversion of 5-methylcytosine to 5-hydroxymethylcytosine, thereby orchestrating the DNA demethylation process and changes in epigenetic marks during early embryogenesis. In this study, CRISPR/Cas9 technology and a TET-specific inhibitor were applied to elucidate the role of TET family in regulating pluripotency in preimplantation embryos using porcine embryos as a model. Disruption of TET1 unexpectedly resulted in the upregulation of NANOG and ESRRB transcripts, although there was no change to the level of DNA methylation in the promoter of NANOG. Surprisingly, a threefold increase in the transcript level of TET3 was observed in blastocysts carrying modified TET1, which may explain the upregulation of NANOG and ESRRB. When the activity of TET enzymes was inhibited by dimethyloxalylglycine (DMOG) treatment, a dioxygenase inhibitor, to investigate the role of TET1 while eliminating the potential compensatory activation of TET3, reduced level of pluripotency genes including NANOG and ESRRB, and increased level of DNA methylation in the NANOG promoter was detected. Blastocysts treated with DMOG also presented a lower inner cell mass/TE ratio, implying the involvement of TET family in lineage specification in blastocysts. Our results indicate that the TET family modulates proper expression of NANOG, a key pluripotency marker, by controlling its DNA methylation profile in the promoter during embryogenesis. This study suggests that TET family is a critical component in pluripotency network of porcine embryos by regulating gene expression involved in pluripotency and early lineage specification.

The purpose of this study was to investigate the pharmacokinetics and metabolism of streptochlorin and its derivative 5-hydroxy-2'-isobutyl streptochlorin (HIS) in mice. Plasma concentration of streptochlorin declined rapidly resulting in a high sustemic plasma clearance (CLp) (5.8±1.7 L/h/kg), a large volume of distribution (Vss) (1.4±0.9 L/kg) and a short half-life (t1/2) (0.4±0.1 h) after a single intravenous administration (5 mg/kg). Oral bioavailability (F) was 10.3±3.4% after a single oral administration (10 mg/kg). HIS also showed a rapid plasma decline with a high CLp (11.3±8.8 L/h/kg), a high Vss (0.8±1.0 L/kg) and a short t1/2 (0.070±0.004 h) following intravenous administration. It was not detected in plasma after oral administration. Metabolic stability studies using mouse liver microsomes and S9 fractions predicted a high hepatic clearance for both compounds, consistent with the in vivo data. Metabolite identification studies revealed three metabolic pathways for streptochlorin: monooxygenation, glucuronidation of the indole moiety and oxidative opening of the 4-chlorooxazole ring. HIS was metabolized via monooxygenation of the isobutyl chain and glucuronidation of the indole ring. These results may aid in structural optimization to mitigate the metabolic liability of streptochlorin.

A series of catechol and dioxolane analogs containing thiazole CGA derivatives have been synthesized and evaluated for their inhibitory activity against α-MSH. The inhibitory activity was improved by replacing an α,β-unsaturated carbonyl of previously reported caffeamides with thiazole motif. Surprisingly, compound 7d , one of the derivatives of dioxolane analogs, displayed the most potent inhibitory activity with an IC 50 of 0.90 μM. Further studies on metabolic stability and bioactivation potential were also accomplished.

Summary Adjusting to a continuously changing environment is a key feature of life. For metazoans, environmental changes include alterations in the gut microbiota, which can affect both memory and behavior. The bacteriovorous nematode Caenorhabditis elegans discriminates between pathogenic and non-pathogenic food sources, avoiding the consumption of pathogens. Here, we demonstrate the role of the intestine in regulating C. elegans avoidance to Pseudomonas aeruginosa by an insulin-like neuropeptide encoded by ins-11 . The transcriptional expression of ins-11 is controlled through transcription factor hlh-30 and the p38 mitogen-activated protein kinase (MAPK) pathway. ins-11 negatively controls signal pathways in neurons that regulate aversive learning behavior. Attenuation of ins-11 increased avoidance behavior and survival on pathogenic bacteria but decreased opportunities to find a food source as well as lowered energy storage and the number of viable progeny. Our findings support a role for the intestine in avoidance and identify an advantageous role for negative feedback that allows C. elegans to actively balance noxious and favorable environments.

Treatment of Staphylococcus aureus infections is complicated by the development of antibiotic tolerance, a consequence of the ability of S. aureus to enter into a nongrowing, dormant state in which the organisms are referred to as persisters. We report that the clinically approved anthelmintic agent bithionol kills methicillin-resistant S. aureus (MRSA) persister cells, which correlates with its ability to disrupt the integrity of Gram-positive bacterial membranes. Critically, bithionol exhibits significant selectivity for bacterial compared with mammalian cell membranes. All-atom molecular dynamics (MD) simulations demonstrate that the selectivity of bithionol for bacterial membranes correlates with its ability to penetrate and embed in bacterial-mimic lipid bilayers, but not in cholesterol-rich mammalian-mimic lipid bilayers. In addition to causing rapid membrane permeabilization, the insertion of bithionol increases membrane fluidity. By using bithionol and nTZDpa (another membrane-active antimicrobial agent), as well as analogs of these compounds, we show that the activity of membrane-active compounds against MRSA persisters positively correlates with their ability to increase membrane fluidity, thereby establishing an accurate biophysical indicator for estimating antipersister potency. Finally, we demonstrate that, in combination with gentamicin, bithionol effectively reduces bacterial burdens in a mouse model of chronic deep-seated MRSA infection. This work highlights the potential repurposing of bithionol as an antipersister therapeutic agent.

Cassia tora Linn. is an annual or perennial plant of the Fabaceae/Leguminosae family. It is used in traditional medicine for various biological activities including anti-constipation, anti-inflammatory, visual acuity, and hepato-protective activities. The present study was carried out to investigate the potential toxicity of C. tora L. seed ethanol extract (CTSEE) following a 13-week repeated oral administration to Sprague-Dawley rats. CTSEE was administered orally to male and female rats for 13 weeks at 0 (control), 500, 1000, and 2000 mg/kg/day (n = 10, for male and female rats for each dose). Additional recovery groups from the control group and high dose group were observed for a 4-week recovery period. At the end of the treatment and recovery periods, animals were sacrificed, and their organs were weighed and blood samples collected. There were no treatment-related adverse effects in clinical signs, body weight, food consumption, estrous cycle, sperm parameters, urinalysis, hematology, serum biochemistry, necropsy findings, organ weight, and histopathology at any doses tested. Under the present experimental conditions, the no-observed-adverse-effect level of the CTSEE was >2000 mg/kg/day in both genders, and no target organs were identified.

Thymic stromal lymphopoietin (TSLP) plays an important role in the differentiation and proliferation of Th2 cells, resulting in eosinophilic inflammation and numerous allergic diseases. Baicalein (1), a major component of Scutellaria baicalensis, was found to be the first small molecule to block TSLP signaling pathways. It inhibited effectively eosinophil infiltration in house dust mite-induced and ovalbumin-challenged mouse models. Structure-activity relationship studies identified compound 11a, a biphenyl flavanone analog, as a novel human TSLP inhibitor for the discovery and development of new anti-allergic drugs.

Background The CRISPR/Cas9 system can effectively introduce site-specific modifications to the genome. The efficiency is high enough to induce targeted genome modifications during embryogenesis, thus increasing the efficiency of producing genetically modified animal models and having potential clinical applications as an assisted reproductive technology. Because most of the CRISPR/Cas9 systems introduce site-specific double-stranded breaks (DSBs) to induce site-specific modifications, a major concern is its potential off-targeting activity, which may hinder the application of the technology in clinics. In this study, we investigated off-targeting events in genome edited pigs/fetuses that were generated through direct injection of the CRISPR/Cas9 system into developing embryos; off-targeting activity of four different sgRNAs targeting RAG2, IL2RG, SCD5, and Ig Heavy chain were examined. Results First, bioinformatics analysis was applied to identify 27 potential off-targeting genes from the sgRNAs. Then, PCR amplification followed by sequencing analysis was used to verify the presence of off-targeting events. Off-targeting events were only identified from the sgRNA used to disrupt Ig Heavy chain in pigs; frequency of off-targeting was 80 and 70% on AR and RBFOX1 locus respectively. A potential PAM sequence was present in both of the off-targeting genes adjacent to probable sgRNA binding sites. Mismatches against sgRNA were present only on the 5′ side of AR, suggesting that off-targeting activities are systematic events. However, the mismatches on RBFOX1 were not limited to the 5′ side, indicating unpredictability of the events. Conclusions The prevalence of off-targeting is low via direct injection of CRISPR/Cas9 system into developing embryos, but the events cannot be accurately predicted. Off-targeting frequency of each CRISPR/Cas9 system should be deliberately assessed prior to its application in clinics. Electronic supplementary material The online version of this article (10.1186/s12896-019-0517-7) contains supplementary material, which is available to authorized users.

Breast cancer exhibits high lethality in women because it is frequently detected at an advanced stage and aggressive forms such as triple-negative breast cancer (TNBC), which are often characterized by metastasis through colonization of secondary tumors. Thus, developing therapeutic agents that target the metastatic process is crucial to successfully treat aggressive breast cancer. We evaluated SP-8356, an anti-inflammatory synthetic verbenone derivative, with respect to its regulation of breast cancer cell behavior and cancer progression. Treatment of SP-8356 arrested cell cycle and reduced growth in various types of breast cancer cells with mild cytotoxicity. Particularly, SP-8356 significantly reduced the motility and invasiveness of TNBC cells. Assays using an in vivo xenograft mouse model confirmed the cell-specific anti-proliferative and anti-metastatic activity of SP-8356. Functional studies revealed that SP-8356 suppressed serum response element-dependent reporter gene expression and NF-κB-related signaling, resulting in downregulation of many genes related to cancer invasion. We conclude that SP-8356 suppresses breast cancer progression through multimodal functions, including inhibition of NF-κB signaling and growth-related signaling pathways.

The objective of this study was to characterize pharmacokinetics and metabolism of (±)-cremastranone (CMT) in mouse. Plasma concentrations of CMT following a single oral dose (10 mg/kg) were all below quantitation limit throughout 24-h time course, indicating poor oral bioavailability. Its plasma levels declined rapidly, with a half-life (t

Traditionally, genetic engineering in the pig was a challenging task. Genetic engineering of somatic cells followed by somatic cell nuclear transfer (SCNT) could produce genetically engineered (GE) pigs carrying site-specific modifications. However, due to difficulties in engineering the genome of somatic cells and developmental defects associated with SCNT, a limited number of GE pig models were reported. Recent developments in genome-editing tools, such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) 9 system, have markedly changed the effort and time required to produce GE pig models. The frequency of genetic engineering in somatic cells is now practical. In addition, SCNT is no longer essential in producing GE pigs carrying site-specific modifications, because direct injection of genome-editing systems into developing embryos introduces targeted modifications. To date, the CRISPR/Cas9 system is the most convenient, cost-effective, timely and commonly used genome-editing technology. Several applicable biomedical and agricultural pig models have been generated using the CRISPR/Cas9 system. Although the efficiency of genetic engineering has been markedly enhanced with the use of genome-editing systems, improvements are still needed to optimally use the emerging technology. Current and future advances in genome-editing strategies will have a monumental effect on pig models used in agriculture and biomedicine.

LW6, an (aryloxyacetylamino)benzoic acid derivative, was recently identified to be an inhibitor of hypoxia-inducible factor-1α (HIF-1α), which is an attractive target for cancer therapeutics. Although LW6 is known to act by inhibiting the accumulation of HIF-1α, pharmacokinetics needs to be evaluated to assess its potential as an anti-tumor agent. Here, we investigated the plasma pharmacokinetics and metabolism of LW6 in mice. LW6 exhibited a small volume of distribution (0.5 ± 0.1 L/kg), and a short terminal half-life (0.6 ± 0.1 hr). Following intravenous or oral administration, LW6 was rapidly converted to its active metabolite, (4-adamantan-1-yl-phenoxy)acetic acid (APA). Although LW6 was rapidly absorbed, its oral bioavailability, estimated using AUClastvalues, was low (1.7 ± 1.8 %). It was slowly degraded in mouse liver microsomes (t1/2 &gt, 1 hr) and serum (t1/2 &gt, 6 hr). About 54% or 44.8% of LW6 was available systemically as APA in the mouse after a single intravenous or oral administration, respectively. Thus, our results indicated the need to simultaneously consider the active metabolite as well as the parent compound for successful evaluation during lead optimization.

A series of acylated xanthone C-glucosides were identified from the methanolic extract of whole Iris rossii Baker. The major constituent was characterized as 6'-O-acetyl mangiferin (OAM), and complete structure elucidation was carried out using 2D NMR (COSY, HSQC, and HMBC) and LC-IT-TOF-MS analyses. The present study is the first to report the anti-inflammatory effects of 6'-O-acetyl mangiferin from Iris rossii Baker on the lipopolysaccharide (LPS)-induced inflammatory response in RAW264.7 macrophage cells. OAM strongly suppressed protein expression of inducible nitric oxide (iNOS) and cyclooxygenase-2 (COX-2), thereby inhibiting the production of nitric oxide (NO), prostaglandin E2 (PGE2), and cytokines such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6). Furthermore, OAM inhibited the LPS-induced phosphorylation of ERK, JNK, and p38, which led to the blockade of nuclear factor-kappa B (NF-κB) and inhibitor kappa B (IκB)-α activation. These results suggest that the anti-inflammatory effects of OAM may be attributed to the downregulation of COX-2 and iNOS via the suppression of NF-κB and the MAPK signaling pathway in RAW264.7 macrophages.

A variety of external and internal factors modulate the activity of GnRH neurons to control fertility in mammals. A direct, vasoactive intestinal peptide (VIP)-mediated input to GnRH neurons originating from the suprachiasmatic nucleus is thought to relay circadian information within this network. In the present study, we examined the effects of VIP on GnRH neuron activity in male and female mice at different stages of the estrous cycle. We carried out cell-attached recordings in slices from GnRH-green fluorescent protein mice and calcium imaging in slices from a mouse line expressing the genetically encoded calcium indicator GCaMP3 selectively in GnRH neurons. We show that 50%–80% of GnRH neurons increase their firing rate in response to bath-applied VIP (1nM–1000nM) in both male and female mice and that this is accompanied by a robust increase in intracellular calcium concentrations. This effect is mediated directly at the GnRH neuron likely through activation of high-affinity VIP receptors. Because suprachiasmatic nucleus-derived timing cues trigger the preovulatory surge only on the afternoon of proestrus in female mice, we examined the effects of VIP during the estrous cycle at different times of day. VIP responsiveness in GnRH neurons did not vary significantly in diestrous and proestrous mice before or around the time of the expected preovulatory surge. These results indicate that the majority of GnRH neurons in male and female mice express functional VIP receptors and that the effects of VIP on GnRH neurons do not alter across the estrous cycle.

// Yun-Jung Choi 1,* , Kiho Lee 2,3,* , Woo-Jin Park 1 , Deug-Nam Kwon 1 , Chankyu Park 1 , Jeong Tae Do 1 , Hyuk Song 1 , Seong-Keun Cho 5 , Kwang-Wook Park 6 , Alana N. Brown 3,4 , Melissa S. Samuel 3,4 , Clifton N. Murphy 3,4 , Randall S. Prather 3,4 and Jin-Hoi Kim 1 1 Animal Biotechnology to Stem Cell and Regenerative Biotechnology, Humanized Pig Research Center (SRC), Konkuk University, Seoul, Republic of Korea 2 Department of Animal and Poultry Sciences, Virginia Tech, Blacksburg, VA, USA 3 Division of Animal Science, National Swine Resource and Research Center, University of Missouri, Columbia, MO, USA 4 National Swine Resource and Research Center, University of Missouri, Columbia, MO, USA 5 Department of Animal Science, Pusan National University, Miryang, Gyeongnam, Republic of Korea 6 Department of Animal Science and Technology, Sunchon National University, Suncheon, Jeonnam, Republic of Korea * These authors have contributed equally to this work Correspondence to: Randall S. Prather, email: // Jin-Hoi Kim, email: // Keywords : IL2RG, X-SCID, immunodeficiency, somatic cell nuclear transfer, TALEN, Pathology Section Received : May 08, 2016 Accepted : July 13, 2016 Published : July 24, 2016 Abstract In this study, we described the phenotype of monoallelic interleukin 2 receptor gamma knockout ( mIL2RG +/Δ69-368 KO) pigs. Approximately 80% of mIL2RG +/Δ69-368 KO pigs (8/10) were athymic, whereas 20% (2/10) presented a rudimentary thymus. The body weight of IL2RG +/Δ69-368 KO pigs developed normally. Immunological analysis showed that mIL2RG +/Δ69-368 KO pigs possessed CD25 + CD44- or CD25-CD44 + cells, whereas single (CD4 or CD8) or double (CD4/8) positive cells were lacking in mIL2RG +/Δ69-368 KO pigs. CD3 + cells in the thymus of mIL2RG +/Δ69-368 KO pigs contained mainly CD44 + cells and/or CD25 + cells, which included FOXP3 + cells. These observations demonstrated that T cells from mIL2RG +/Δ69-368 KO pigs were able to develop to the DN3 stage, but failed to transition toward the DN4 stage. Whole-transcriptome analysis of thymus and spleen, and subsequent pathway analysis revealed that a subset of genes differentially expressed following the loss of IL2RG might be responsible for both impaired T-cell receptor and cytokine-mediated signalling. However, comparative analysis of two mIL2RG +/Δ69-368 KO pigs revealed little variability in the down- and up-regulated gene sets. In conclusion, mIL2RG +/Δ69-368 KO pigs presented a T-B+NK- SCID phenotype, suggesting that pigs can be used as a valuable and suitable biomedical model for human SCID research.

SUMMARY Most in vitro culture conditions are less‐than‐optimal for embryo development. Here, we used a transcriptional‐profiling database to identify culture‐induced differences in gene expression in porcine blastocysts compared to in vivo‐produced counterparts. Genes involved in glycine transport (SLC6A9), glycine metabolism (GLDC, GCSH, DLD, and AMT), and serine metabolism (PSAT1, PSPH, and PHGDH) were differentially expressed. Addition of 10 mM glycine to the culture medium (currently containing 0.1 mM) reduced the abundance of SLC6A9 transcript and increased total cell number, primarily in the trophectoderm lineage (P = 0.003); this was likely by decreasing the percentage of apoptotic nuclei. As serine and glycine can be reversibly metabolized by serine hydroxymethyltransferase 2 (SHMT2), we assessed the abundance of SHMT2 transcript as well as its functional role by inhibiting it with aminomethylphosphonic acid (AMPA), a glycine analog, during in vitro culture. Both AMPA supplementation and elevated glycine decreased the mRNA abundance of SHMT2 and tumor protein p53 (TP53), which is activated in response to cellular stress, compared to controls (P ≤ 0.02). On the other hand, mitochondrial activity of blastocysts, mtDNA copy number, and abundance of mitochondria‐related transcripts did not differ between control and 10 mM glycine culture conditions. Despite improvements to these metrics of blastocyst quality, transfer of embryos cultured in 10 mM glycine did not result in pregnancy whereas the transfer of in vitro‐produced embryos cultured in control medium yielded live births. Mol. Reprod. Dev. 83: 246–258, 2016. © 2016 The Authors.

Hepatitis E virus (HEV), the causative agent of hepatitis E, is an important but incompletely understood pathogen causing high mortality during pregnancy and leading to chronic hepatitis in immunocompromised individuals. The underlying mechanisms leading to hepatic damage remain unknown; however, the humoral immune response is implicated. In this study, immunoglobulin (Ig) heavy chain J(H)(−/−) knockout gnotobiotic pigs were generated using CRISPR/Cas9 technology to deplete the B-lymphocyte population, resulting in an inability to generate a humoral immune response to genotype 3 HEV infection. Compared to wild-type gnotobiotic piglets, the frequencies of B lymphocytes in the Ig heavy chain J(H)(−/−) knockouts were significantly lower, despite similar levels of other innate and adaptive T-lymphocyte cell populations. The dynamic of acute HEV infection was subsequently determined in heavy chain J(H)(−/−) knockout and wild-type gnotobiotic pigs. The data showed that wild-type piglets had higher viral RNA loads in feces and sera compared to the J(H)(−/−) knockout pigs, suggesting that the Ig heavy chain J(H)(−/−) knockout in pigs actually decreased the level of HEV replication. Both HEV-infected wild-type and J(H)(−/−) knockout gnotobiotic piglets developed more pronounced lymphoplasmacytic hepatitis and hepatocellular necrosis lesions than other studies with conventional pigs. The HEV-infected J(H)(−/−) knockout pigs also had significantly enlarged livers both grossly and as a ratio of liver/body weight compared to phosphate-buffered saline-inoculated groups. This novel gnotobiotic pig model will aid in future studies into HEV pathogenicity, an aspect which has thus far been difficult to reproduce in the available animal model systems. IMPORTANCE According to the World Health Organization, approximately 20 million HEV infections occur annually, resulting in 3.3 million cases of hepatitis E and >44,000 deaths. The lack of an efficient animal model that can mimic the full-spectrum of infection outcomes hinders our ability to delineate the mechanism of HEV pathogenesis. Here, we successfully generated immunoglobulin heavy chain J(H)(−/−) knockout gnotobiotic pigs using CRISPR/Cas9 technology, established a novel J(H)(−/−) knockout and wild-type gnotobiotic pig model for HEV, and systematically determined the dynamic of acute HEV infection in gnotobiotic pigs. It was demonstrated that knockout of the Ig heavy chain in pigs decreased the level of HEV replication. Infected wild-type and J(H)(−/−) knockout gnotobiotic piglets developed more pronounced HEV-specific lesions than other studies using conventional pigs, and the infected J(H)(−/−) knockout pigs had significantly enlarged livers. The availability of this novel model will facilitate future studies of HEV pathogenicity.

Artificial oocyte activation is an essential step in somatic cell nuclear transfer (SCNT) and can enhance viability of embryos as a form of assisted reproductive technology (ART) in clinics. Most artificial activation methods have been developed to increase cytosolic calcium (Ca

Harlequin ichthyosis (HI) is a severe genetic skin disorder and caused by mutation in the ATP-binding cassette A12 (ABCA12) gene. The retinoid administration has dramatically improved long-term survival of HI, but improvements are still needed. However, the ABCA12 null mice failed to respond to retinoid treatment, which impedes the development of novel cure strategies for HI. Here we generated an ethylnitrosourea mutagenic HI pig model (named Z9), which carries a novel deep intronic mutation IVS49-727 AG in the ABCA12 gene, resulting in abnormal mRNA splicing and truncated protein production. Z9 pigs exhibit significant clinical symptom as human patients with HI. Most importantly, systemic retinoid treatment significantly prolonged the life span of the mutant pigs via improving epidermal maturation, decreasing epidermal apoptosis, and triggering the expression of ABCA6. Taken together, this pig model perfectly resembles the clinical symptom and molecular pathology of patients with HI and will be useful for understanding mechanistic insight and developing therapeutic strategies.

Eye diseases characterized by excessive angiogenesis such as wet age-related macular degeneration, proliferative diabetic retinopathy, and retinopathy of prematurity are major causes of blindness. Cremastranone is an antiangiogenic, naturally occurring homoisoflavanone with efficacy in retinal and choroidal neovascularization models and antiproliferative selectivity for endothelial cells over other cell types. We undertook a cell-based structure-activity relationship study to develop more potent cremastranone analogues, with improved antiproliferative selectivity for retinal endothelial cells. Phenylalanyl-incorporated homoisoflavonoids showed improved activity and remarkable selectivity for retinal microvascular endothelial cells. A lead compound inhibited angiogenesis in vitro without inducing apoptosis and had efficacy in the oxygen-induced retinopathy model in vivo.

With the aim of developing novel scaffolds as anticancer agents and inhibitors of NF-κB activity, 60 novel benzofuran- and 2,3-dihydrobenzofuran-2-carboxylic acid N-(substituted)phenylamide derivatives (1a-s, 2a-k, 3a-s, and 4a-k) were designed and synthesized from the reference lead compound KL-1156, which is an inhibitor of NF-κB translocation to the nucleus in LPS-stimulated RAW 264.7 macrophage cells. The novel benzofuran- and 2,3-dihydrobenzofuran-2-carboxamide derivatives exhibited potent cytotoxic activities (measured by the sulforhodamine B assay) at low micromolar concentrations against six human cancer cell lines: ACHN (renal), HCT15 (colon), MM231 (breast), NUGC-3 (gastric), NCI-H23 (lung), and PC-3 (prostate). In addition, these compounds also inhibited LPS-induced NF-κB transcriptional activity. The +M effect and hydrophobic groups on the N-phenyl ring potentiated the anticancer activity and NF-κB inhibitory activity, respectively. However, according to the results of structure-activity relationship studies, only benzofuran-2-carboxylic acid N-(4'-hydroxy)phenylamide (3m) was the lead scaffold with both an outstanding anticancer activity and NF-κB inhibitory activity. This novel lead scaffold may be helpful for investigation of new anticancer agents that act through inactivation of NF-κB.

We investigated to compare species differences in amitriptyline (AMI) metabolism among mouse, rat, dog, and human liver microsomes. We developed a method for simultaneous determination of metabolic stability and metabolite profiling using predictive multiple reaction monitoring information-dependent acquisition-enhanced product ion (MRM-IDA-EPI) scanning. In the cofactor-dependent microsomal metabolism study, AMI was metabolized more rapidly in rat and human liver microsomes incubated with NADPH than UDPGA. AMI incubated with NADPH+UDPGA in rat, dog, or mouse liver microsomes disappeared rapidly with a half-life of 3.5, 8.4, or 9.2 min, respectively, but slowly in human liver microsomes with a half-life of 96 min. In total, 9, 10, 11, and 6 putative metabolites of AMI were detected in mouse, rat, dog, and human liver microsomes, respectively, based on mass spectrometric analyses. Kinetic analysis of metabolites in liver microsomes from each species over 120 min showed common metabolic routes of AMI, such as N-demethylation, hydroxylation, and glucuronidation, and subtle interspecies differences in AMI metabolism. The main metabolic routes in mouse, rat, dog, and human liver microsomes were hydroxylation followed by glucuronide conjugation, methyl hydroxylation, and N-demethylation, respectively. The MRM-IDA-EPI method can provide quantitative and qualitative information about metabolic stability and metabolite profiling simultaneously. Moreover, time course analysis of metabolites can not only eliminate false identification of metabolites, but also provide a rationale for proposed metabolic pathways. The MRM-IDA-EPI method combined with time course analysis of metabolites is useful for investigating drug metabolism at the early drug discovery stage.

Faulty epigenetic reprogramming of somatic nuclei is thought to be the main reason for low cloning efficiency by somatic cell nuclear transfer (SCNT). Histone deacetylase inhibitors (HDACi), such as Scriptaid, improve developmental competence of SCNT embryos in several species. Another HDACi, Oxamflatin, is about 100 times more potent than Scriptaid in the ability to inhibit nuclear-specific HDACs. The present study determined the effects of Oxamflatin treatment on embryo development, DNA methylation, and gene expression. Oxamflatin treatment enhanced blastocyst formation of SCNT embryos in vitro. Embryo transfer produced more pigs born and fewer mummies from the Oxamflatin-treated group compared to the Scriptaid-treated positive control. Oxamflatin also decreased DNA methylation of POU5F1 regulatory elements and centromeric repeat elements in day-7 blastocysts. When compared to in vitro-fertilized (IVF) embryos, the methylation status of POU5F1, NANOG, and centromeric repeat was similar in the cloned embryos, indicating these genes were successfully reprogrammed. However, compared to the lack of methylation of XIST in day-7 IVF embryos, a higher methylation level in day-7 cloned embryos was observed, implying that X chromosomes were activated in day-7 IVF blastocysts, but were not fully activated in cloned embryos, i.e., reprogramming of XIST was delayed. A time-course analysis of XIST DNA methylation on day-13, -15, -17, and -19 in vivo embryos revealed that XIST methylation initiated at about day 13 and was not completed by day 19. The methylation of the XIST gene in day-19 control cloned embryos was delayed again when compared to in vivo embryos. However, methylation of XIST in Oxamflatin-treated embryos was comparable with in vivo embryos, which further demonstrated that Oxamflatin could accelerate the delayed reprogramming of XIST gene and thus might improve cloning efficiency.

The objective of the present work was to investigate the potential for pharmacokinetic drug-drug interactions between glimepiride (GMP) and piperazine dithioctate (PDT) in rats to support the development of an orally combined product of the two drugs. An LC-MS/MS bioanalytical method was developed for simultaneous quantification of GMP and thioctic acid (TA) in rat plasma. The accuracy, precision, linearity, selectivity, and recovery were all within an acceptable range. The oral plasma exposure of the GMP solution was more than 14-times greater than that of the GMP suspension at a dose of 0.5 mg/kg, suggesting a dissolution-limited absorption of the GMP suspension. Oral co-administration of PDT (72 mg/kg) with GMP suspension (0.5 mg/kg) reduced the plasma GMP exposure by approximately 80% without a significant change in t1/2 and tmax. Oral co-administration of PDT with GMP solution had no significant effect on the plasma pharmacokinetics of GMP. PDT lowered the pH (from ca. 7 to 5.6) and the dissolved GMP concentration in the GMP suspension. It was also shown that GMP was more soluble at pH 7 than at 5.7 in an aqueous solution, and the oral plasma exposure of a GMP suspension at pH 7.0 was substantially higher than that of a suspension at pH 5.7. These results suggest that the pH-dependent solubility of GMP was likely responsible for PDT's effect on the oral absorption of GMP. In conclusion, the current work suggests a possibility of drug-drug interaction between GMP and PDT upon oral co-administration.

Diabetes mellitus is a chronic disease with accompanying severe complications. Various animal models, mostly rodents due to availability of genetically modified lines, have been used to investigate the pathophysiology of diabetes. Using pigs for diabetic research can be beneficial because of their similarity in size, pathogenesis pathway, physiology, and metabolism with human. However, the use of pigs for diabetes research has been hampered due to only few pig models presenting diabetes symptoms. In this study, we have successfully generated insulin-deficient pigs by generating the indels of the porcine INS gene in somatic cells using CRISPR/Cas9 system followed by somatic cell nuclear transfer. First, somatic cells carrying a modified INS gene were generated using CRISPR/Cas9 system and their genotypes were confirmed by T7E1 assay; targeting efficiency was 40.4% (21/52). After embryo transfer, three live and five stillborn piglets were born. As expected, INS knockout piglets presented high blood glucose levels and glucose was detected in the urine. The level of insulin and c-peptide in the blood serum of INS knockout piglets were constant after feeding and the expression of insulin in the pancreas was absent in those piglets. This study demonstrates effectiveness of CRISPR/Cas9 system in generating novel pig models. We expect that these insulin-deficient pigs can be used in diabetes research to test the efficacy and safety of new drugs and the recipient of islet transplantation to investigate optimal transplantation strategies.

At the time of fertilization, the sperm activates the egg and induces embryonic development by triggering an elevation in the egg's intracellular free Ca

Francisella tularensis is a highly infectious Gram-negative intracellular pathogen that causes tularemia. Because of its potential as a bioterrorism agent, there is a need for new therapeutic agents. We therefore developed a whole-animal Caenorhabditis elegans - F. tularensis pathosystem for high-throughput screening to identify and characterize potential therapeutic compounds. We found that the C. elegans p38 mitogen-activate protein (MAP) kinase cascade is involved in the immune response to F. tularensis , and we developed a robust F. tularensis -mediated C. elegans killing assay with a Z′ factor consistently of >0.5, which was then utilized to screen a library of FDA-approved compounds that included 1,760 small molecules. In addition to clinically used antibiotics, five FDA-approved drugs were also identified as potential hits, including the anti-inflammatory drug diflunisal that showed anti- F. tularensis activity in vitro . Moreover, the nonsteroidal anti-inflammatory drug (NSAID) diflunisal, at 4× MIC, blocked the replication of an F. tularensis live vaccine strain (LVS) in primary human macrophages and nonphagocytic cells. Diflunisal was nontoxic to human erythrocytes and HepG2 human liver cells at concentrations of ≥32 μg/ml. Finally, diflunisal exhibited synergetic activity with the antibiotic ciprofloxacin in both a checkerboard assay and a macrophage infection assay. In conclusion, the liquid C. elegans - F. tularensis LVS assay described here allows screening for anti- F. tularensis compounds and suggests that diflunisal could potentially be repurposed for the management of tularemia.

Maternal-to-zygotic transition is an event that developmental control of early embryos is switched from oocyte-derived factors to the zygotic genome. Ability to inhibit DNA replication, transcription, and translation is an important tool in studying events, such as zygotic genome activation, during embyogenesis. Here, we describe approaches to block DNA replication, transcription, and translation using chemical inhibitors. Then we also demonstrate how the transcript level of a maternally inherited gene, ten-eleven translocation methylcytosine dioxygenase 3, responses to the chemical treatments.

The emergence of antibiotic-resistant and -tolerant bacteria is a major threat to human health. Although efforts for drug discovery are ongoing, conventional bacteria-centered screening strategies have thus far failed to yield new classes of effective antibiotics. Therefore, new paradigms for discovering novel antibiotics are of critical importance. Caenorhabditis elegans, a model organism used for in vivo, offers a promising solution for identification of anti-infective compounds. Areas covered: This review examines the advantages of C. elegans-based high-throughput screening over conventional, bacteria-centered in vitro screens. It discusses major anti-infective compounds identified from large-scale C. elegans-based screens and presents the first clinically-approved drugs, then known bioactive compounds, and finally novel small molecules. Expert opinion: There are clear advantages of using a C. elegans-infection based screening method. A C. elegans-based screen produces an enriched pool of non-toxic, efficacious, potential anti-infectives, covering: conventional antimicrobial agents, immunomodulators, and anti-virulence agents. Although C. elegans-based screens do not denote the mode of action of hit compounds, this can be elucidated in secondary studies by comparing the results to target-based screens, or conducting subsequent target-based screens, including the genetic knock-down of host or bacterial genes.