Mouse Model Research Articles

Mouse models for pancreatic ductal adenocarcinoma are affected by the cre-driver used to promote KRASG12D activation.

The fundamental biology of pancreatic ductal adenocarcinoma has been greatly impacted by the characterization of genetically engineered mouse models that allow temporal and spatial activation of oncogenic KRAS (KRASG12D). One of the most commonly used models involves targeted insertion of a cre-recombinase into the Ptf1a gene. However, this approach disrupts the Ptf1a gene, resulting in haploinsufficiency that likely affects sensitivity to oncogenic KRAS (KRASG12D). This study aims to determine if Ptf1a haploinsufficiency affected the acinar cell response to KRASG12D before and after induction of pancreatic injury. We performed morphological and molecular analysis of three genetically engineered mouse models that express a tamoxifen-inducible cre-recombinase to activate KrasG12D in acinar cells of the pancreas. The cre-recombinase was targeted to the acinar-specific transcription factor genes, Ptf1a or Mist1/Bhlha15, or expressed within a BAC-derived Elastase transgene. Histological and RNA-seq analyses were used to delineate differences between the models. Up to two months after tamoxifen induction of KRASG12D, morphological changes were negligible. However, induction of pancreatic injury by cerulein resulted in widespread PanIN lesions in Ptf1acreERT pancreata within seven days and maintained for at least five weeks post-injury, which was not seen in the models with two functional Ptf1a alleles. RNA-seq analysis prior to injury induction suggested Ptf1acreERT and Mist1creERT mice have unique profiles of gene expression that predict a differential response to injury. Multiplex analysis of pancreatic tissue confirmed different inflammatory responses between the models. These findings suggest Ptf1a haploinsufficiency in Ptf1acreERT mouse models promotes KRASG12D priming of genes for promotion of PDAC.

Eradication of Pseudomonas aeruginosa Persister Cells by Eravacycline.

Pseudomonas aeruginosa is a leading bacterial pathogen that causes persistent infections. One major reason that antibiotics fail to clear such infections is the presence of a dormant subpopulation called persister cells. To eradicate persister cells, it is important to change drug development from traditional strategies that focus on growth inhibition to the search for new leads that can kill dormant cells. In this study, we demonstrate that eravacycline can effectively accumulate in P. aeruginosa persister cells, leading to strong killing during wakeup, including persister cells in both planktonic cultures and biofilms of the wild-type strain and its mucoid mutant. The effects of eravacycline on persister control were further validated in vivo using a lung infection model in mice. Collectively, these results demonstrate the possibility to control persister cells of bacterial pathogens by targeting dormancy.

Design, Synthesis, and Biological Evaluation of New Selective PDE4 Inhibitors for Topical Treatment of Psoriasis.

Psoriasis is a complex and chronic inflammatory disease. Current drugs help control the symptoms of psoriasis but make no cure, urging discovery of novel drugs. We report in this paper the discovery of new phosphodiesterase 4 (PDE4) inhibitors for treatment of psoriasis. We designed and synthesized 45 new compounds, among which 14h exhibited IC50 of 0.57 nM for PDE4D and >4100-fold selectivity over other PDE families. Compound 14h inhibited release of inflammatory cytokines of TNF-α (IC50 = 34.2 μM) and IL-6 (IC50 = 40.9 μM) in Raw264.7 cells and reduced the expression of IL-1β and IL-17A in the skin of psoriasis mice. In addition, 14h alleviated IMQ-induced psoriasis in the mouse model and reduced the erythema level, scales, and thickness of the back skin of the mice. In short, our results suggested that PDE4 inhibitor 14h is a strong candidate for the topical treatment of psoriasis.

Bacteriocin Microcin J25's antibacterial infection effects and novel non-microbial regulatory mechanisms: differential regulation of dopaminergic receptors.

The antibacterial and immunomodulatory activities of bacteriocins make them attractive targets for development as anti-infective drugs. Although the importance of the enteric nervous system (ENS) in the struggle against infections of the intestine has been demonstrated, whether it is involved in bacteriocins anti-infective mechanisms is poorly defined. Here, we demonstrated that the bacteriocin Microcin J25 (J25) significantly alleviated diarrhea and intestinal inflammation in piglets caused by enterotoxigenic Escherichia coli (ETEC) infection. Mechanistically, macrophage levels were significantly downregulated after J25 treatment, and this was replicated in a mouse model. Omics analysis and validation screening revealed that J25 treatment induced significant changes in the dopaminergic neuron pathway, but little change in microbial structure. The alleviation of inflammation may occur by down-regulating dopamine receptor (DR) D1 and the downstream DAG-PKC pathway, thus inhibiting arachidonic acid decomposition, and the inhibition of macrophages may occur through the up-regulation of DRD5 and the downstream cAMP-PKA pathway, thus inhibiting NF-κB. Our studies' findings provide insight into the changes and possible roles of the ENS in J25 treatment of ETEC infection, providing a more sophisticated foundational understanding for developing the application potential of J25.

Enhancing the Hypolipidemic and Functional Properties of Flammulina velutipes Root Dietary Fiber via Steam Explosion

Flammulina velutipes is an edible mushroom widely cultivated in China. As a by-product of Flammulina velutipes, the roots are rich in high-quality dietary fiber (DF). In order to obtain high-quality soluble dietary fiber (SDF), steam explosion (SE) is used as an effective modification method to improve the extraction rate and avoid the loss of active substances. Mounting evidence shows that SDF alleviates lipid metabolism disorders. However, it is not well understood how the influence of SDF with SE pretreatment could benefit lipid metabolism. In this study, we extracted a soluble dietary fiber from Flammulina velutipes root with an SE treatment, named SE-SDF, using enzymatic assisted extraction. The physicochemical and structural properties of the SE-SDF were investigated, and its hypolipidemic effects were also analyzed using oleic-acid-induced HepG2 cells. In addition, the anti-obesity and hypolipidemic effects of SE-SDF were investigated using a high-fat diet (HFD) mouse model. The results indicate that SE treatment (1.0 MPa, 105 s) increased the SDF content to 8.73 ± 0.23%. The SE-SDF was primarily composed of glucose, galactose, and mannose. In HFD-fed mice, SE-SDF significantly reduced weight gain and improved lipid profiles, while restoring liver function and reducing injury. This work provides an effective method for the processing of fungi waste and adds to its economic value. In future studies, the structural characteristics and the anti-obesity and gut microbiota regulation mechanisms of SE-SDF will be explored in depth, supporting its high-value utilization in healthcare products.

The novel estrogen receptor beta agonist EGX358 and APOE genotype influence memory, vasomotor, and anxiety outcomes in an Alzheimer’s mouse model

IntroductionAlzheimer’s disease (AD) prevalence and severity are associated with increased age, female sex, and apolipoprotein E4 (APOE4) genotype. Although estrogen therapy (ET) effectively reduces symptoms of menopause including hot flashes and anxiety, and can reduce dementia risk, it is associated with increased risks of breast and uterine cancer due to estrogen receptor alpha (ERβ)-mediated increases in cancer cell proliferation. Because ERβ activation reduces this cell proliferation, selective targeting of ERβ may provide a safer method of improving memory and reducing hot flashes in menopausal women, including those with AD. APOE genotype influences the response to ET, although it is unknown whether effects of ERβ activation vary by genotype.MethodsHere, we tested the ability of long-term oral treatment with a novel highly selective ERβ agonist, EGX358, to enhance object recognition and spatial recognition memory, reduce drug-induced hot flashes, and influence anxiety-like behaviors in female mice expressing 5 familial AD mutations (5xFAD-Tg) and human APOE3 (E3FAD) or APOE3 and APOE4 (E3/4FAD). Mice were ovariectomized at 5 months of age and were then treated orally with vehicle (DMSO) or EGX358 (10 mg/kg/day) via hydrogel for 8 weeks. Spatial and object recognition memory were tested in object placement (OP) and object recognition (OR) tasks, respectively, and anxiety-like behaviors were tested in the open field (OF) and elevated plus maze (EPM). Hot flash-like symptoms (change in tail skin temperature) were measured following injection of the neurokinin receptor agonist senktide (0.5 mg/kg).ResultsEGX358 enhanced object recognition memory in E3FAD and E3/4FAD mice but did not affect spatial recognition memory. EGX358 also reduced senktide-induced tail temperature elevations in E3FAD, but not E3/4FAD, females. EGX358 did not influence anxiety-like behaviors or body weight.DiscussionThese data indicate that highly selective ERβ agonism can facilitate object recognition memory in both APOE3 homozygotes and APOE3/4 heterozygotes, but only reduce the magnitude of a drug-induced hot flash in APOE3 homozygotes, suggesting that APOE4 genotype may blunt the beneficial effects of ET on hot flashes. Collectively, these data suggest a potentially beneficial effect of selective ERβ agonism for memory and hot flashes in females with AD-like pathology, but that APOE genotype plays an important role in responsiveness.

Pharmacogenomic screening identifies and repurposes leucovorin and dyclonine as pro-oligodendrogenic compounds in brain repair.

Oligodendrocytes are critical for CNS myelin formation and are involved in preterm-birth brain injury (PBI) and multiple sclerosis (MS), both of which lack effective treatments. We present a pharmacogenomic approach that identifies compounds with potent pro-oligodendrogenic activity, selected through a scoring strategy (OligoScore) based on their modulation of oligodendrogenic and (re)myelination-related transcriptional programs. Through in vitro neural and oligodendrocyte progenitor cell (OPC) cultures, ex vivo cerebellar explants, and in vivo mouse models of PBI and MS, we identify FDA-approved leucovorin and dyclonine as promising candidates. In a neonatal chronic hypoxia mouse model mimicking PBI, both compounds promote neural progenitor cell proliferation and oligodendroglial fate acquisition, with leucovorin further enhancing differentiation. In an adult MS model of focal de/remyelination, they improve lesion repair by promoting OPC differentiation while preserving the OPC pool. Additionally, they shift microglia from a pro-inflammatory to a pro-regenerative profile and enhance myelin debris clearance. These findings support the repurposing of leucovorin and dyclonine for clinical trials targeting myelin disorders, offering potential therapeutic avenues for PBI and MS.

N-CADHERIN+/CD168- subpopulation determines therapeutic variations of UC-MSCs for cardiac repair after myocardial infarction.

The efficiency of mesenchymal stem cells (MSCs) in treating myocardial infarction (MI) remains inconsistent, which limits their therapeutic applications. Therefore, exploring the mechanism for the inconsistent efficacy of MSCs and identification the criteria for screening MSCs are important for improving the efficiency of MSCs. Mouse model after MI was utilized to test the role of MSCs from different donors and the functional subpopulation in improving cardiac function. Heterogeneity of MSCs was identified using single-cell RNA sequencing (scRNA-seq) of MSC-GY. GSEA and Scissor analyses were used to find the functional subpopulations of MSCs that promote angiogenesis. The role of functional subpopulations in promoting angiogenesis was verified by detecting the secretory proteins, the ratio of N-CADHERIN+/CD168- subpopulations in MSCs, and the tube formation, migration, and proliferation of HUVECs after treatment with conditional medium (CM) derived from different MSCs. We found that umbilical cord-derived MSCs(UC-MSCs) from different donors have varied therapeutic efficacy in MI mice and UC-MSCs with higher therapeutic effectiveness exhibited the most potent pro-angiogenic effects by secreting elevated levels of angiogenesis-related proteins, such as MYDGF, VEGFA, and FGF2. ScRNA-seq of 10,463 UC-MSCs revealed that the N-CADHERIN+/CD168- subpopulation was closely associated with pro-angiogenic effects, and the ratio of this cell subpopulation was positively correlated with the angiogenic potential of MSCs. We also found that the N-CADHERIN+/CD168- subpopulation was the functional subpopulation of MSCs in improving cardiac function of MI mice. Our study identified that the N-CADHERIN+/CD168- subpopulation was the functional subpopulation of MSCs in treating MI, which was essential for the development and utilization of MSCs in MI treatment.

Abstract IA005: Tumor cell-based liquid biopsy to characterize prostate cancer

Abstract Circulating Tumor Cells (CTCs) hold the promise of comprehensive yet noninvasive molecular characterization of cancer cells during the course of tumor evolution and progression. To dissect the epigenetic profile of prostate CTCs, we undertook paired DNA methylation and RNA sequencing of single CTCs isolated from blood specimens of patients with prostate cancer. Across the genome, our study identified 40 core Partial Methylation Domains (PMDs) that arise early in tumorigenesis and are shared by all individual CTCs across multiple patients. Key among these is a single genomic locus harboring multiple interferon-inducible genes along with all the CD1 gene family members, implicated in innate immunity. Based on mouse models, we propose that early biallelic silencing of multiple genes residing within a single PMD may contribute to the escape from immune surveillance in prostate and potentially other cancers. Studies of CTC heterogeneity within individual patients are limited by the very small number of cells that can be isolated from standard 10ml blood volumes. To enable such analyses, we applied a new high throughput microfluidic platform, capable of processing an entire leukapheresis product, interrogating 2-3 L of blood volume equivalent. Applying this technology to patients with metastatic prostate cancer, we successfully enriched thousands of CTCs from individual cases, enabling the characterization of intra-patient heterogeneity at the level of cell morphology and marker expression, chromosome copy number variation and exome-wide genotyping, and transcriptionally-defined tumor cell subpopulations. Such high-volume microfluidic enrichment of CTCs constitutes a new dimension in liquid biopsies. Citation Format: Daniel A Haber, Shyamala Maheswaran, Hongshan Guo, Joanna Vuille, Shih-Bo Huang, Ben S Wittner, Lecia V Sequist, Richard J Lee, Patricia AR Brunker, David T Miyamoto, Avanish Mishra, Mehmet Toner. Tumor cell-based liquid biopsy to characterize prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr IA005.

Identification of 2,4-Di-tert-Butylphenol as an Antimicrobial Agent Against Cutibacterium acnes Bacteria from Rwandan Propolis

Background/Objectives: Acne is the most prevalent dermatological condition among humans, affecting approximately 80% of adolescents during puberty. To date, numerous compounds have been used for acne treatment, including erythromycin ointments and antiseptics, with varying degrees of success. The emergence of erythromycin-resistant C. acnes strains has spurred the search for new antimicrobial agents, particularly from natural sources. Methods: Propolis collected in Rwanda was extracted and fractionated by flash chromatography and tested against C. acnes growth by using NCLSI recommendations. Results: In our research, we identified a molecule, 2,4-Di-tert-butylphenol (2,4-DTBP) which inhivbited the C. acnes growth at a concentration of 16 µg/mL. Based on these results, we formulated an ointment (1%) using OFAP18 and petroleum jelly for the potential treatment of acne using a mouse model. Conclusions: In vitro and in vivo evidence suggests that 2,4-DTBP has anti-inflammatory properties and could effectively manage the overgrowth of C. acnes as well as serve as a potent alternative for the formulation of an active propolis ointment for acne treatment.

Intratumoral administration of poly-ICLC enhances the antitumor effects of anti-PD-1.

Immune checkpoint inhibitors are effective to treat hepatocellular carcinoma (HCC) yet only successful in a small part of patients. This study aimed to investigate whether poly-ICLC, an immune stimulant, can enhance the antitumor effects of anti-PD-1 on mouse HCC. We established two syngeneic HCC mouse models with BNL cells in BALB/c mice and Hep-55.1 C cells in C57BL/6 J mice. Mice with subcutaneous HCC tumors received one of five treatments: control, anti-PD-1, intratumoral (IT) poly-ICLC, anti-PD-1 plus intramuscular (IM) poly-ICLC, or anti-PD-1 plus IT poly-ICLC. Tumor volumes were measured, CD8+ T lymphocytes in tumors and spleen were analyzed, and interferon-γ activity was assessed by ELISpot. Immune cell types and abundance were evaluated with NanoString nCounter IO360 panels. Cotreatment with poly-ICLC significantly enhanced the antitumor effects of anti-PD-1, with IT administration being more effective than IM. IT poly-ICLC also induced more significant CD8+ T cell infiltration and interferon-γ activity in the tumor and spleen, and more upregulation of both interferon-γ and M1 macrophage signals in the tumor microenvironment while downregulating several cancer-promoting pathways. Combination therapy with poly-ICLC, especially through IT route, and anti-PD-1 provides significantly greater antitumor effects than anti-PD-1 monotherapy in syngeneic mouse models of HCC.

Triterpenoids from Potentilla chinensis inhibit RANKL-induced osteoclastogenesis in vitro and lipopolysaccharide-induced osteolytic bone loss in vivo.

In this study, a phytochemical investigation on the methanol extract of Potentilla chinensis led to the isolation of eleven triterpenoids including ursolic acid (1), pomolic acid (2), tormentic acid (3), 2-epi-corosolic acid (4), 3-epi-corosolic acid (ECA, 5), 3β-hydroxyurs-11-en-13β(28)-olide (6), euscaphic acid (7), 2-epi-tormentic acid (8), corosolic acid (9), uvaol (10), and 3-O-acetylpomolic acid (11). Among them, ECA (5) showed potential anti-osteoclastogenic activity. To the best of our knowledge, this represents the first isolation of ECA (5) from P. chinensis as well as the first investigation of its effects on osteoclast formation. Further study revealed that ECA inhibited RANKL-induced mature osteoclast formation in vitro without compromising cell viability. Mechanistically, ECA attenuated RANKL-induced mitogen-activated protein kinases (MAPKs) and nuclear factor-κB (NF-κB) activation, leading to the inhibition of c-Fos and nuclear factor of activated T cells cytoplasmic 1 (NFATc1) activation. Moreover, ECA protected against LPS-induced inflammatory bone loss and osteoclast formation in a mouse model. However, ECA did not inhibit LPS-induced inflammatory responses in macrophages. Our findings suggest that ECA mitigates LPS-induced inflammatory bone loss in mice by inhibiting RANKL-induced activation of key osteoclastogenic transcription factors, including c-Fos and NFATc1, and may be a potential natural triterpenoid for preventing or treating osteolytic diseases.

Opa1 and MT-Nd6 mutations induce early mitochondrial changes in the retina and prelaminar optic nerve of hereditary optic neuropathy mouse models

Abstract Hereditary optic neuropathies (HON), including dominant optic atrophy and Leber's hereditary optic neuropathy, are genetic disorders characterized by retinal ganglion cell (RGC) degeneration leading to vision loss, mainly associated with mitochondrial dysfunction. In this study, we analyzed mitochondrial distribution and ultrastructure in the retina and longitudinal optic nerve sections of presymptomatic HON mouse models with Opa1 and Nd6 deficiency to identify early mitochondrial changes. Our results show significant mitochondrial fragmentation and increased mitophagy in Opa1+/- mice, indicating early mitochondrial changes prior to neuronal loss. Conversely Nd6P25L mice exhibited mitochondrial hypertrophy, suggesting an adaptive response to compensate for altered energy metabolism. These pre-symptomatic mitochondrial changes were mainly observed in the unmyelinated portion of the RGC axons, where the transmission of the visual information requires high energy expenditure, constituting the specific point of vulnerability in HON. These findings highlight early focal mitochondrial changes prior to neuronal loss in HON and provide insight into presymptomatic therapeutic approaches.

Myoferlin alleviates pressure overload-induced cardiac hypertrophy and dysfunction by inhibiting NLRP3-mediated pyroptosis

Myoferlin (MYOF) is a muscle-derived secretory protein. Recent studies have found that MYOF protects against cell damage. However, the role of MYOF in cardiac hypertrophy remains unclear. Increasing evidence suggests that NLRP3 (NOD-like receptor protein 3) and the pyroptosis cascade play critical roles in the development of cardiac hypertrophy and inflammation. To investigate the role of MYOF in cardiac hypertrophy, we conducted a transverse aortic constriction (TAC) experiment in a mouse model. We found that MYOF can improve cardiac hypertrophy and cardiac function. Furthermore, our study confirmed a connection between cardiac hypertrophy and myocardial pyroptosis. Cardiac hypertrophy significantly increased the proportion of apoptotic cells and upregulated apoptosis-associated speck-like protein containing a CARD (ASC), caspase-1, and gasdermin D (GSDMD). This suggests that pharmacological or genetic inhibition of NLRP3 can effectively reduce cardiac hypertrophy. An abnormal increase in NLRP3 can reverse the cardioprotective effects of MYOF. Our findings indicate that MYOF is a potential therapeutic agent for cardiac hypertrophy.

Abstract B073: Leveraging micronuclei DNA from erythrocytes for early detection of hepatocellular carcinoma

Abstract Background: Hepatocellular carcinoma (HCC) is the most common form of liver cancer and accounts for ∼90% of cases globally. Detection of early-stage HCC is vital as it allows for potentially curative treatment, yet remains a significant challenge. Micronuclei (MN) are a hallmark of genomic instability. An increased frequency of MN is commonly observed in tumor cells as well as other somatic cells, including erythrocytes, in cancer patients. Here, we have established a technique (WO2021/228246 A1), for the isolation and analysis of micronuclei DNA (MN-DNA) in erythrocytes from peripheral blood. Significant changes in read densities at specific genomic locations within MN-DNA were identified in patients, termed as tumor- associated MN-DNA (taMN-DNA) features. This study evaluates the potential of these taMN- DNA features for early-stage HCC detection across human and murine model. Methods: To explore the potential of MN-DNA in cancer detection, we first collected 1-2 mL of whole blood from HDs (N = 53) and HCC patients (N = 53). MN-DNA was isolated and purified from erythrocytes, followed by whole-genome sequencing. Participants were randomly divided into training, test cohorts in an 8:2 ratio. We applied machine learning algorithms to leverage these features for cancer detection in the human model. Additionally, to investigate the formation of taMN-DNA features, we employed a well-established mouse model that mimics HCC development. Mice were injected with the genotoxic agent diethylnitrosamine (DEN) 2 weeks after birth, and developed malignant macroscopic liver nodules at approximately 40 weeks of age. Results: In the human cohort, we enrolled 106 participants, with more than half of the HCC cases were diagnosed at early-stage (stage 0-II). The cancer detection model utilizing taMN- DNA features achieved an overall accuracy of 86.3%, with a sensitivity of 81.8% and specificity of 90.9%. For early-stage, the model demonstrated sensitivities of 54.5% at a specificity of 90.9%. In the DEN-induced HCC mouse model, unsupervised hierarchical clustering based on these selected taMN-DNA features clearly separated WT and tumor mice into two distinct groups. Notably, mice that were not tumorigenic at 36 weeks post-treatment in the DEN-treated group fell into the WT group when classified by the same taMN-DNA features, indicating that the formation of taMN-DNA signatures is associated with the presence of tumors specifically. Conclusions: This pilot study highlights the potential of MN-DNA as a promising tool for early HCC detection. Leveraging these taMN-DNA features can accurately distinguish early-stage HCC patients from HDs with high sensitivity. In both human and murine models, there is a significant relationship between these signatures and tumor presence, suggesting that taMN- DNA features might reflect the diseased state across mammalian species. Research sponsor: Timing Biotech. Citation Format: Hui Lin, Haobo Sun, Xingyun Yao, Xiaoxiao Fan, Fei Meng, Honghao Liang, Xiaofei Gao. Leveraging micronuclei DNA from erythrocytes for early detection of hepatocellular carcinoma [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr B073.

Optimization of in vivo antimalarial efficacy of combinations of aqueous leaf extracts of Artemisia annua L., Vernonia amygdalina Del, and Microglossa pyrifolia (Lam.) Kuntze using factorial design.

Malaria continues to be among the leading causes of mortality in Africa including Uganda, with the emergence of parasite resistance to the first-line therapeutics (Artemisinin- based Combination Therapy). To find new therapeutics, this study has reported an in vivo antimalarial efficacy of combinations of Artemisia annua (Aa), Vernonia amygdalina (Va), and Microglossa pyrifolia (Mp) in mice model using factorial design. The Aa and Va were extracted by hot infusion, and Mp by cold maceration using distilled water. The dry extracts were screened for different phytochemicals, and later subjected to in vivo antimalarial activity using Peter's 4-day suppressive test. The 23 factorial design used Aa, Va, and Mp aqueous extracts as independent variables at two levels (-1 and 1), and the percentage chemo suppression and survival time as response variables. The data was analyzed using Design Expert 13 and GraphPad Prism employing ANOVA linear regression modelling and t-test respectively. All the extracts had alkaloids, phenols, saponins, terpenoids, cardiac glycosides, tannins, steroids, and carbohydrates. The various combinations showed chemo suppression from 41.5 to 91.0% and survival time of 19 to 23days. The first three combinations having lower levels of Aa (200mg/kg) exhibited higher chemo suppression (> 90%) compared to Artemisinin-Lumefantrine positive control at 4mg/kg with 87.5%. Lower levels of Aa in the combinations contributed to high chemo suppression while higher levels of Va prolonged survival times. Interactions between Aa and Mp showed higher chemo suppression, and that between Aa and Va increased survival time. An optimized prediction of 94.4% chemo suppression was made by the ANOVA model at lower levels of Aa and Va, and a higher level of Mp, which is similar to an experimental run which gave a response of 90. 6%. An optimum combination of the three plants as a natural herbal antimalarial therapy was obtained using factorial design, and it offers an alternative to first line Artemisinin based Combination Therapy (ACTs) as parasite resistance looms. This combination could be further developed into a standard phytopharmaceutical and subjected to Randomized Controlled Trials (RCTs).

Encapsulation of soybean lunasin and amaranth unsaponifiable matter in liposomes induces cell cycle arrest in an allograft melanoma mouse model.

Melanoma is the most aggressive type of skin cancer and can metastasize during primary tumor formation. This research aimed to determine the relationship between the prevention of melanoma development in a mouse model treated with liposomes loaded with soybean lunasin and amaranth unsaponifiable matter (UM + LunLip) and cell cycle arrest. Tumors excised from C57BL/6 mice treated topically or subcutaneously with UM + LunLip were subjected to immunohistochemistry. Markers related to cell cycle inhibition (p16, p21, p27, and p53) and markers involved in cell cycle progression (cyclin-dependent kinase, CDK6, and cyclin D1) were assessed. The results showed that UM + LunLip had antitumor activity in C57BL/6 mice treated either topically or subcutaneously by p16, p21, p27, and p53 overexpression (up to 572-, 134-, 30-, and 57-fold change, FC, respectively) in the tumors of mice treated with 30mg UM + LunLip/kg body weight compared with the tumor-bearing untreated control. However, CDK6 and cyclin D1 expression was not inhibited (up to 1.37 FC and 2.09 FC, respectively), which is a typical behavior of cyclin D in melanoma. Therefore, melanoma tumor development was prevented by the overexpression of cell cycle inhibitors p16, p21, p27, and p53 due to UM + LunLip treatments. Since the topical application was effective, less invasive, and more practical for the user, this application will be recommended for future steps in in vivo studies.

Whole genome sequencing of CRISPR/Cas9-engineered NF-κB reporter mice for validation and variant discovery.

Targeted knockout, mutations, or knock-in of genomic DNA fragments in model organisms have been used widely for functional and cell-tracking studies. The desired genetic perturbation is often accomplished by recombination-based or CRISPR/Cas9-based genome engineering. For validating the intended genetic modification, a local region surrounding the targeted locus is typically examined based on enzymatic cleavage and consequent length patterns, e.g. in a Southern analysis. Despite its wide use, this approach is open to incomplete and ambiguous readouts. With decreasing costs of high-throughput sequencing, it is becoming feasible to consider a large-scale validation of a new strain after a targeted genetic perturbation. Here we describe a dataset of whole-genome sequences and the variant analysis results from four novel reporter mouse strains. This served to validate the strains and identified all the off-target effects on the genome, thereby increasing the genetic diversity of genomic sequences over those represented in the public databases for inbred mice.

The CLEC3B inhibits cellular proliferation and metastasis of cholangiocarcinoma through Wnt/β-catenin pathway

Purpose Cholangiocarcinoma (CCA) is a cancer of the biliary system, including intrahepatic and extrahepatic cholangiocarcinoma, and is highly aggressive. C-type lectins family member 3b (CLEC3B) is a Ca2+ binding transmembrane protein with different biological functions in a variety of cancers. The objective of this study was to explore the biological function of CLEC3B in CCA. Methods The CLEC3B gene was identified using the TCGA database and survival analysis of the cholangiocarcinoma clinical cohort. The expression CLEC3B cholangiocarcinoma and correlation with prognosis was investigated in our patient cohort. The effects of CLEC3B on proliferation, apoptosis, migration and invasion were verified in QBC939 and HUCCT1 cells. The effect of CLEC3B on the tumor formation was proved by xenograft tumor model in nude mice. The signaling pathway of CLEC3B in regulating CCA progression was further analyzed RNA sequencing and western blot. Results CLEC3B was decreased in the cholangiocarcinoma in the database. The mRNA and protein expression level of CLEC3B were significantly lower and correlated with poor overall survival in cholangiocarcinoma of our patient cohort. In vitro experiments proved that overexpression of CLEC3B can inhibit proliferation, migration and invasion in bile duct cancer cells. The CLEC3B was correlated with epithelial–mesenchymal transition and apoptosis. The calcium could promote the biological function of CLEC3B. The vivo study indicated that CLEC3B inhibited tumor formation. RNA sequencing indicating CLEC3B may transduce signal through e Wnt/β-catenin signaling pathway. Conclusions The CLEC3B inhibits cellular proliferation and migration of cholangiocarcinoma through the Wnt/β-catenin pathway.

Acute intermittent hypoxia elicits sympathetic neuroplasticity independent of peripheral chemoreflex activation and spinal cord tissue hypoxia in a rodent model of high-thoracic spinal cord injury.

The loss of medullary control of spinal circuits controlling the heart and blood vessels is a unifying mechanism linking both hemodynamic instability and the risk for cardiovascular diseases (CVD) following spinal cord injury (SCI). As such, new avenues to regulate sympathetic activity are essential to mitigate CVD in this population. Acute intermittent hypoxia (AIH) induces a type of neuroplasticity known as long-term facilitation (LTF), a persistent increase in nerve activity post-AIH in spinal motor circuits. Whether LTF occurs within the sympathetic circuit following SCI is largely unknown. We aimed to test whether AIH elicits sympathetic LTF (i.e., sLTF) and attenuates hypoactivity in sub-lesional splanchnic sympathetic circuits in a male rat model of SCI. In 3 experimental series, we tested whether 1) high-thoracic contusion SCI induces hypoactivity in splanchnic sympathetic nerve activity, 2) AIH elicits sLTF following SCI, and 3) sLTF requires carotid chemoreflex or spinal cord tissue hypoxia. Our results indicate that a single-session of AIH therapy (10 × 1 min of FiO2 = 0.1, interspersed with 2 min of FiO2 = 1.0) delivered at 2 weeks following SCI attenuates SCI-induced sympathetic hypoactivity by eliciting sLTF 90 min post-treatment that is independent of peripheral chemoreflex activation and/or spinal cord hypoxia. These findings advance our mechanistic understanding of AIH in the field and yield new insights into factors underpinning AIH-induced sLTF following SCI in a rat model. Our findings also set the stage for the chronic application of AIH to alleviate secondary complications resulting from sympathetic hypoactivity following SCI.