Albumin promoter-driven FlpO expression induces efficient genetic recombination in mouse liver.

Tissue-specific gene manipulations are widely used in genetically engineered mouse models. A single recombinase system, such as the one using Alb-Cre, has been commonly used for liver-specific genetic manipulations. However, most diseases are complex, involving multiple genetic changes and various cell types. A dual recombinase system is required for conditionally modifying different genes sequentially in the same cell or inducing genetic changes in different cell types within the same organism. A FlpO cDNA was inserted between the last exon and 3'-UTR of the mouse albumin gene in a bacterial artificial chromosome (BAC-Alb-FlpO). The founders were crossed with various reporter mice to examine the efficiency of recombination. Liver cancer tumorigenesis was investigated by crossing the FlpO mice with FSF-KrasG12D mice and p53frt mice (KPF mice). BAC-Alb-FlpO mice exhibited highly efficient recombination capability in both hepatocytes and intrahepatic cholangiocytes. No recombination was observed in the duodenum and pancreatic cells. BAC-Alb-FlpO-mediated liver-specific expression of mutant KrasG12D and conditional deletion of p53 gene caused the development of liver cancer. Remarkably, liver cancer in these KPF mice manifested a distinctive mixed hepatocellular carcinoma and cholangiocarcinoma phenotype. A highly efficient and liver-specific BAC-Alb-FlpO mouse model was developed. In combination with other Cre lines, different genes can be manipulated sequentially in the same cell, or distinct genetic changes can be induced in different cell types of the same organism.NEW & NOTEWORTHY A liver-specific Alb-FlpO mouse line was generated. By coupling it with other existing CreERT or Cre lines, the dual recombinase approach can enable sequential gene modifications within the same cell or across various cell types in an organism for liver research through temporal and spatial gene manipulations.

PTEN deficiency induces an extrahepatic cholangitis-cholangiocarcinoma continuum via aurora kinase A in mice.

BACKGROUND & AIMS: The PTEN-AKT pathway is frequently altered in extrahepatic cholangiocarcinoma (eCCA). We aimed to evaluate the role of PTEN in the pathogenesis of eCCA and identify novel therapeutic targets for this disease. METHODS: The Pten gene was genetically deleted using the Cre-loxp system in biliary epithelial cells. The pathologies were evaluated both macroscopically and histologically. The characteristics were further analyzed by immunohistochemistry, reverse-transcription PCR, cell culture, and RNA sequencing. Some features were compared to those in human eCCA samples. Further mechanistic studies utilized the conditional knockout of Trp53 and Aurora kinase A (Aurka) genes. We also tested the effectiveness of an Aurka inhibitor. RESULTS: We observed that genetic deletion of the Pten gene in the extrahepatic biliary epithelium and peri-ductal glands initiated sclerosing cholangitis-like lesions in mice, resulting in enlarged and distorted extrahepatic bile ducts in mice as early as 1 month after birth. Histologically, these lesions exhibited increased epithelial proliferation, inflammatory cell infiltration, and fibrosis. With aging, the lesions progressed from low-grade dysplasia to invasive carcinoma. Trp53 inactivation further accelerated disease progression, potentially by downregulating senescence. Further mechanistic studies showed that both human and mouse eCCA showed high expression of AURKA. Notably, the genetic deletion of Aurka completely eliminated Pten deficiency-induced extrahepatic bile duct lesions. Furthermore, pharmacological inhibition of Aurka alleviated disease progression. CONCLUSIONS: Pten deficiency in extrahepatic cholangiocytes and peribiliary glands led to a cholangitis-to-cholangiocarcinoma continuum that was dependent on Aurka. These findings offer new insights into preventive and therapeutic interventions for extrahepatic CCA. IMPACT AND IMPLICATIONS: The aberrant PTEN-PI3K-AKT signaling pathway is commonly observed in human extrahepatic cholangiocarcinoma (eCCA), a disease with a poor prognosis. In our study, we developed a mouse model mimicking cholangitis to eCCA progression by conditionally deleting the Pten gene via Pdx1-Cre in epithelial cells and peribiliary glands of the extrahepatic biliary duct. The conditional Pten deletion in these cells led to cholangitis, which gradually advanced to dysplasia, ultimately resulting in eCCA. The loss of Pten heightened Akt signaling, cell proliferation, inflammation, fibrosis, DNA damage, epigenetic signaling, epithelial-mesenchymal transition, cell dysplasia, and cellular senescence. Genetic deletion or pharmacological inhibition of Aurka successfully halted disease progression. This model will be valuable for testing novel therapies and unraveling the mechanisms of eCCA tumorigenesis.

A simple and effective genotyping workflow for rapid detection of CRISPR genome editing.

Genetically engineered mouse models play a pivotal role in the modeling of diseases, exploration of gene functions, and the development of novel therapies. In recent years, clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9)-mediated genome editing technology has revolutionized the process of developing such models by enabling precise genome modifications of the multiple interested genes simultaneously. Following genome editing, an efficient genotyping methodology is crucial for subsequent characterization. However, current genotyping methods are laborious, time-consuming, and costly. Here, using targeting the mouse trypsinogen genes as an example, we introduced common applications of CRISPR-Cas9 editing and a streamlined cost-effective genotyping workflow for CRISPR-edited mouse models, in which Sanger sequencing is required only at the initial steps. In the F0 mice, we focused on identifying the presence of positive editing by PCR followed by Sanger sequencing without the need to know the exact sequences, simplifying the initial screening. In the F1 mice, Sanger sequencing and algorithms decoding were used to identify the precise editing. Once the edited sequence was established, a simple and effective genotyping strategy was established to distinguish homozygous and heterozygous status by PCR from tail DNA. The genotyping workflow applies to deletions as small as one nucleotide, multiple-gene knockout, and knockin studies. This simplified, efficient, and cost-effective genotyping shall be instructive to new investigators who are unfamiliar with characterizing CRISPR-Cas9-edited mouse strains.NEW & NOTEWORTHY This study presents a streamlined, cost-effective genotyping workflow for clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) edited mouse models, focusing on trypsinogen genes. It simplifies initial F0 mouse screening using PCR and Sanger sequencing without needing exact sequences. For F1 mice, precise editing is identified through Sanger sequencing and algorithm decoding. The workflow includes a novel PCR strategy for distinguishing homozygous and heterozygous statuses in subsequent generations, effective for small deletions, multiple-gene knockouts, and knockins.

Development and validation of novel immune-inflammation-based clinical predictive nomograms in HER2-negative advanced gastric cancer.

Purpose: To explore the predictive value of multiple immune-inflammatory biomarkers including serum VEGFA and systemic immune-inflammation index (SII) in HER2-negative advanced gastric cancer (AGC) and establish nomograms for predicting the first-line chemotherapeutic efficacy, progression-free survival (PFS) and overall survival (OS) of patients with this fatal disease. Methods: From November 2017 to April 2022, 102 and 34 patients with a diagnosis of HER2-negative AGC at the First Affiliated Hospital of Bengbu Medical College were enrolled as development and validation cohorts, respectively. Univariate and multivariate analyses were performed to evaluate the clinical value of the candidate indicators. The variables were screened using LASSO regression analysis. Predictive models were developed using significant predictors and are displayed as nomograms. Results: Baseline VEGFA expression was significantly higher in HER2-negative AGC patients than in nonneoplastic patients and was associated with malignant serous effusion and therapeutic efficacy (all p<0.001). Multivariate analysis indicated that VEGFA was an independent predictor for first-line therapeutic efficacy and PFS (both p<0.01) and SII was an independent predictor for first-line PFS and OS (both p<0.05) in HER2-negative AGC patients. The therapeutic efficacy model had an R2 of 0.37, a Brier score of 0.15, and a Harrell's C-index of 0.82 in the development cohort and 0.90 in the validation cohort. The decision curve analysis indicated that the model added more net benefits than VEGFA assessment alone. The PFS/OS models had Harrell's C-indexes of 0.71/0.69 in the development cohort and 0.71/0.62 in the validation cohort. Conclusion: The established nomograms integrating serum VEGFA/SII and commonly available baseline characteristics provided satisfactory performance in predicting the therapeutic efficacy and prognosis of HER2-negative AGC patients.

A mouse model for high-efficient Flp-recombinase-mediated genetic manipulation in the pancreas.

BACKGROUND: Tissue and cell-specific gene targeting has been widely employed in biomedical research. In the pancreas, the commonly used Cre recombinase recognizes and recombines loxP sites. However, to selectively target different genes in distinct cells, a dual recombinase system is required. METHOD: We developed an alternative recombination system mediated by FLPo, which recognizes frt DNA sequences for pancreatic dual recombinase-mediated genetic manipulation. An IRES-FLPo cassette was targeted between the translation stop code and 3-UTR of the mouse pdx1 gene in a Bacterial Artificial Chromosome using recombineering technology. Transgenic BAC-Pdx1-FLPo mice were developed by pronuclear injection. RESULTS: Highly efficient recombination activity was observed in the pancreas by crossing the founder mice with Flp reporter mice. When the BAC-Pdx1-FLPo mice were bred with conditional FSF-KRasG12D and p53 F/F mice, pancreatic cancer developed in the compound mice. The characteristics of pancreatic cancer resembled those derived from conditional LSL-KRasG12D and p53 L/L mice controlled by pdx1-Cre. CONCLUSIONS: We have generated a new transgenic mouse line expressing FLPo, which enables highly efficient pancreatic-specific gene recombination. When combined with other available Cre lines, this system can be utilized to target different genes in distinct cells for pancreatic research.

Upregulation of the ERRγ-VDAC1 axis underlies the molecular pathogenesis of pancreatitis.

Emerging evidence suggest that transcription factors play multiple roles in the development of pancreatitis, a necroinflammatory condition lacking specific therapy. Estrogen-related receptor γ (ERRγ), a pleiotropic transcription factor, has been reported to play a vital role in pancreatic acinar cell (PAC) homeostasis. However, the role of ERRγ in PAC dysfunction remains hitherto unknown. Here, we demonstrated in both mice models and human cohorts that pancreatitis is associated with an increase in ERRγ gene expression via activation of STAT3. Acinar-specific ERRγ haploinsufficiency or pharmacological inhibition of ERRγ significantly impaired the progression of pancreatitis both in vitro and in vivo. Using systematic transcriptomic analysis, we identified that voltage-dependent anion channel 1 (VDAC1) acts as a molecular mediator of ERRγ. Mechanistically, we showed that induction of ERRγ in cultured acinar cells and mouse pancreata enhanced VDAC1 expression by directly binding to specific site of the Vdac1 gene promoter and resulted in VDAC1 oligomerization. Notably, VDAC1, whose expression and oligomerization were dependent on ERRγ, modulates mitochondrial Ca2+ and ROS levels. Inhibition of the ERRγ-VDAC1 axis could alleviate mitochondrial Ca2+ accumulation, ROS formation and inhibit progression of pancreatitis. Using two different mouse models of pancreatitis, we showed that pharmacological blockade of ERRγ-VDAC1 pathway has therapeutic benefits in mitigating progression of pancreatitis. Likewise, using PRSS1R122H-Tg mice to mimic human hereditary pancreatitis, we demonstrated that ERRγ inhibitor also alleviated pancreatitis. Our findings highlight the importance of ERRγ in pancreatitis progression and suggests its therapeutic intervention for prevention and treatment of pancreatitis.

Hematopoietic progenitor kinase 1 inhibits the development and progression of pancreatic intraepithelial neoplasia.

Ras plays an essential role in the development of acinar-to-ductal metaplasia (ADM) and pancreatic ductal adenocarcinoma (PDAC). However, mutant Kras is an inefficient driver for PDAC development. The mechanisms of the switching from low Ras activity to high Ras activity that are required for development and progression of pancreatic intraepithelial neoplasias (PanINs) are unclear. In this study, we found that hematopoietic progenitor kinase 1 (HPK1) was upregulated during pancreatic injury and ADM. HPK1 interacted with the SH3 domain and phosphorylated Ras GTPase-activating protein (RasGAP) and upregulated RasGAP activity. Using transgenic mouse models of HPK1 or M46, a kinase-dead mutant of HPK1, we showed that HPK1 inhibited Ras activity and its downstream signaling and regulated acinar cell plasticity. M46 promoted the development of ADM and PanINs. Expression of M46 in KrasG12D Bac mice promoted the infiltration of myeloid-derived suppressor cells and macrophages, inhibited the infiltration of T cells, and accelerated the progression of PanINs to invasive and metastatic PDAC, while HPK1 attenuated mutant Kras-driven PanIN progression. Our results showed that HPK1 plays an important role in ADM and the progression of PanINs by regulating Ras signaling. Loss of HPK1 kinase activity promotes an immunosuppressive tumor microenvironment and accelerates the progression of PanINs to PDAC.

Selecting an Appropriate Experimental Animal Model for Cholangiocarcinoma Research.

Cholangiocarcinoma (CCA) is a highly aggressive biliary tree malignancy with intrahepatic and extra-hepatic subtypes that differ in molecular pathogeneses, epidemiology, clinical manifestations, treatment, and prognosis. The overall prognosis and patient survival remains poor because of lack of early diagnosis and effective treatments. Preclinical in vivo studies have become increasingly paramount as they are helpful not only for the study of the fundamental molecular mechanisms of CCA but also for developing novel and effective therapeutic approaches of this fatal cancer. Recent advancements in cell and molecular biology have made it possible to mimic the pathogenicity of human CCA in chemical-mechanical, infection-induced inflammatory, implantation, and genetically engineered animal models. This review is intended to help investigators understand the particular strengths and weaknesses of the currently used in vivo animal models of human CCA and their related modeling techniques to aid in the selection of the one that is the best for their research needs.

Simple risk score to predict the likelihood of a positive EUS in idiopathic acute pancreatitis.

BACKGROUND AND AIMS: We sought to derive a risk score, DORM65, of known variables to predict the likelihood of a positive EUS in patients with idiopathic acute pancreatitis (IAP). METHODS: A retrospective cohort study of 180 patients with IAP was performed across 3 tertiary care centers between January 2018 and December 2021. Multivariate logistic regression modeling was performed to predict a positive EUS. Accuracy of the models was assessed by the area under the receiver-operating characteristic curve (AUROCC). RESULTS: The diagnostic yield of EUS was 58.9% (95% confidence interval [CI], 51.7-66.1). The DORM65 scores of 5 predictors present before EUS with the best discrimination were a delayed EUS (defined as ≥82 days from the last episode of AP), obesity, not having had a repeated transabdominal US, male sex, and age ≥65 years at the time of EUS. For those at the lowest risk score group, the positive EUS rate was 13.0% compared with 100% in those at the highest risk group (relative risk, 7.67; P < .001). A score of 3 or more had a positive predictive value of 86.0% with a sensitivity of 34.9% and specificity of 91.9%. The model had a high predictive accuracy (AUROCC, .774; 95% CI, .707-.841). Adding 3 additional predictors (no cholecystectomy, no MRCP, and a single episode of AP) did not increase the accuracy significantly (AUROCC, .805; 95% CI, .742-.867). CONCLUSIONS: DORM65 is easily calculated and accurately predicts a positive EUS in patients with IAP. Further validation is needed.

Rapid acceleration of KRAS-mutant pancreatic carcinogenesis via remodeling of tumor immune microenvironment by PPARδ.

Pancreatic intraepithelial neoplasia (PanIN) is a precursor of pancreatic ductal adenocarcinoma (PDAC), which commonly occurs in the general populations with aging. Although most PanIN lesions (PanINs) harbor oncogenic KRAS mutations that initiate pancreatic tumorigenesis; PanINs rarely progress to PDAC. Critical factors that promote this progression, especially targetable ones, remain poorly defined. We show that peroxisome proliferator-activated receptor-delta (PPARδ), a lipid nuclear receptor, is upregulated in PanINs in humans and mice. Furthermore, PPARδ ligand activation by a high-fat diet or GW501516 (a highly selective, synthetic PPARδ ligand) in mutant KRASG12D (KRASmu) pancreatic epithelial cells strongly accelerates PanIN progression to PDAC. This PPARδ activation induces KRASmu pancreatic epithelial cells to secrete CCL2, which recruits immunosuppressive macrophages and myeloid-derived suppressor cells into pancreas via the CCL2/CCR2 axis to orchestrate an immunosuppressive tumor microenvironment and subsequently drive PanIN progression to PDAC. Our data identify PPARδ signaling as a potential molecular target to prevent PDAC development in subjects harboring PanINs.

Aurora kinase a inhibitor MLN8237 suppresses pancreatic cancer growth.

Pancreatic ductal adenocarcinoma (PDAC) is notorious for high mortality due to limited options of appropriate chemotherapy drugs. Here we report that Aurora kinase-A expression is elevated in both human and mouse PDAC samples. MLN8237, an inhibitor of Aurora kinase-A, efficiently reduced the proliferation and motility of PDAC cells in vitro as well as tumor growth in orthotropic xenograft model and genetic pancreatic cancer animal models (p53/LSL/Pdx-Cre mice) in vivo. MLN8237 exhibited tumor inhibitory effect through inhibiting proliferation and migration, and inducing apoptosis and senescence. These results provide the molecular basis for a novel chemotherapy strategy for PDAC patients.

Ptf1a Promoter-Driven Cre Expression During Spermatogenesis Causes Germline Recombination.

OBJECTIVES: Two independently developed Ptf1a-Cre mouse lines, Ptf1atm1(cre)Hnak and Ptf1atm1(Cre)Cvw, are widely used in pancreatic research. Recently, Ptf1atm1(cre)Hnak line was reported to transmit unwanted paternal recombination. We aimed to investigate whether this exists in the Ptf1atm1(Cre)Cvw line. METHODS: Ptf1atm1(Cre)Cvw mice were crossed with R26-LSL-LacZ reporter mice. DNA recombination and gene expression were examined by recombination-specific polymerase chain reaction, reverse transcription-polymerase chain reaction, and X-Gal staining. RESULTS: R26 locus recombination was detected in the pancreas as well as the testes and sperm of the double transgenic mice. Positive ptf1a mRNA expression from testes revealed that there was endogenous Ptf1a promoter activity in this extrapancreatic tissue. Of the 15 progenies that inherited LacZ from the male double transgenic mice, 4 (26.7%) were positive for complete whole-body recombination. The presence of recombination in R26 only mice suggested that the recombination occurred before meiosis. CONCLUSIONS: Paternal germline recombination exists in the Ptf1atm1(Cre)Cvw mouse line. Ptf1a promoter-driven Cre expression during spermatogenesis before meiosis is the cause of germline recombination. Therefore, when male Ptf1a-Cre mice are used in compound mice breeding, it is necessary to genotype not only floxed alleles but also recombined alleles to examine unwanted recombinations.

Pancreatic Disorders in Patients with Inflammatory Bowel Disease.

Inflammatory bowel disease (IBD) can involve multiple organ systems, and pancreatic manifestations of IBD are not uncommon. The incidence of several pancreatic diseases is more frequent in patients with Crohn's disease and ulcerative colitis than in the general population. Pancreatic manifestations in IBD include a heterogeneous group of disorders and abnormalities ranging from mild, self-limited disorders to severe diseases. Asymptomatic elevation of amylase and/or lipase is common. The risk of acute pancreatitis in patients with IBD is increased due to the higher incidence of cholelithiasis and drug-induced pancreatitis in this population. Patients with IBD commonly have altered pancreatic histology and chronic pancreatic exocrine dysfunction. Diagnosing acute pancreatitis in patients with IBD is challenging. In this review, we discuss the manifestations and possible causes of pancreatic abnormalities in patients with IBD.

Exome Sequencing of Pancreatic Acinar Carcinoma Identified Distinctive Mutation Patterns.

OBJECTIVES: Pancreatic acinar cell carcinoma (ACC) is a rare pancreatic cancer. The advancement of treatment is hampered because of the limited knowledge of its molecular mechanism. METHODS: Whole-exome sequencing was performed on DNA extracted from 11 pure ACC surgical samples. Potential germline variants were removed on the basis of polymorphic databases, alternative allele frequency, coverage depth, and Catalogue of Somatic Mutations in Cancer (COSMIC) annotations after variant calling procedure. Mutation profiles and signatures were assessed through the Mutational Patterns package. RESULTS: A median of 34 somatic mutations were detected (range, 19-60). Three novel recurrent small deletions were identified. Common pancreatic ductal adenocarcinoma mutations or neuroendocrine tumor mutants were not found. FAT atypical cadherin 4, mucin 5B, titin, and zinc finger homeobox 3 were consistently mutated across 4 independent ACC studies. A high contribution of COSMIC mutational signature 1 was seen in ACC, indicating deamination of 5-methylcytosine. The majority of the patients had COSMIC signatures 6, 15, or 20, relating to defective DNA mismatch repair. Six patients showed COSMIC mutational signature 10 because of the altered activity of DNA polymerase epsilon. CONCLUSIONS: Distinct mutational signatures pathways were found in ACC and targeting them may improve clinical outcome.

Dissecting VEGF-induced acute versus chronic vascular hyperpermeability: Essential roles of dimethylarginine dimethylaminohydrolase-1.

Vascular endothelial cell growth factor (VEGF) is a key regulator of vascular permeability. Herein we aim to understand how acute and chronic exposures of VEGF induce different levels of vascular permeability. We demonstrate that chronic VEGF exposure leads to decreased phosphorylation of VEGFR2 and c-Src as well as steady increases of nitric oxide (NO) as compared to that of acute exposure. Utilizing heat-inducible VEGF transgenic zebrafish (Danio rerio) and establishing an algorithm incorporating segmentation techniques for quantification, we monitored acute and chronic VEGF-induced vascular hyperpermeability in real time. Importantly, dimethylarginine dimethylaminohydrolase-1 (DDAH1), an enzyme essential for NO generation, was shown to play essential roles in both acute and chronic vascular permeability in cultured human cells, zebrafish model, and Miles assay. Taken together, our data reveal acute and chronic VEGF exposures induce divergent signaling pathways and identify DDAH1 as a critical player and potentially a therapeutic target of vascular hyperpermeability-mediated pathogenesis.

Systemic anticoagulation is associated with decreased mortality and morbidity in acute pancreatitis.

BACKGROUND/OBJECTIVES: Acute pancreatitis (AP) is a procoagulant state, and markers of coagulopathy are associated with AP severity. We aimed to explore the association of systemic anticoagulation therapy before AP onset with the inpatient outcomes of patients with acute pancreatitis. METHODS: This case-control, retrospective study used data from the Nationwide Inpatient Sample (Jan 2014-Dec 2016). We used medical coding data to identify patients with a principal diagnosis of AP who were receiving systemic anticoagulation therapy. Patients with anticoagulation were matched to those without it on the propensity for having anticoagulation. The propensity for having anticoagulation was estimated using a logistic regression model, matching for age, gender, race, median household income for patients' zip code, Charlson comorbidity score, region of hospital, location of hospital (urban/rural), teaching status of hospital, if admission day was on a weekend, pancreatic cancer class, obesity, tobacco usage. Secondary outcomes were inpatient outcomes and hospital expenditures. RESULTS: A total of 190,474 patients admitted for acute pancreatitis were identified, out of which 7827 patients were on anticoagulation. After propensity matching, 5776 matched pairs were successfully identified. Patients with AP on anticoagulation tended to have lower risk for ICU admission, acute kidney injury, organ failure or inpatient mortality. However, the group with anticoagulation had longer hospital length of stay and higher hospital costs. CONCLUSIONS: Anticoagulation therapy may have a pivotal role in the pathogenesis and progression of AP. These data suggest a potential therapeutic role for anticoagulants in AP. Further studies are needed to better understand these observations.

Pancreas-specific CHRM3 activation causes pancreatitis in mice.

Hyperstimulation of the cholecystokinin 1 receptor (CCK1R), a G protein-coupled receptor (GPCR), in pancreatic acinar cells is commonly used to induce pancreatitis in rodents. Human pancreatic acinar cells lack CCK1R but express cholinergic receptor muscarinic 3 (M3R), another GPCR. To test whether M3R activation is involved in pancreatitis, a mutant M3R was conditionally expressed in pancreatic acinar cells in mice. This mutant receptor loses responsiveness to its native ligand, acetylcholine, but can be activated by an inert small molecule, clozapine-N-oxide (CNO). Intracellular calcium and amylase were elicited by CNO in pancreatic acinar cells isolated from mutant M3R mice but not WT mice. Similarly, acute pancreatitis (AP) could be induced by a single injection of CNO in the transgenic mice but not WT mice. Compared with the cerulein-induced AP, CNO caused more widespread acinar cell death and inflammation. Furthermore, chronic pancreatitis developed at 4 weeks after 3 episodes of CNO-induced AP. In contrast, in mice with 3 recurrent episodes of cerulein-included AP, pancreas histology was restored in 4 weeks. Furthermore, the M3R antagonist ameliorated the severity of cerulein-induced AP in WT mice. We conclude that M3R activation can cause the pathogenesis of pancreatitis. This model may provide an alternative approach for pancreatitis research.

Downregulation of GSTM2 enhances gemcitabine chemosensitivity of pancreatic cancer in vitro and in vivo.

Glutathione-S-transferases (GSTs) not only show cytoprotective role and their involvement in the development of anticancer drug resistance, but also transmit signals that control cell proliferation and apoptosis. However, the role of GST isoforms in chemotherapy resistance remains elusive in pancreatic cancer. Here, we demonstrated that gemcitabine treatment increased the GSTM2 expression in pancreatic cancer cell lines. Knockdown of GSTM2 by siRNA elevated apoptosis and decreased viability of pancreatic cancer cells treated with gemcitabine. Moreover, in vivo experiments further showed that shRNA induced GSTM2 downregulation enhanced drug sensitivity of gemcitabine in orthotopic pancreatic tumor mice. We also found that GSTM2 levels were lower in tumor tissues than in non-tumor tissues and higher GSTM2 expression was significantly associated with longer overall survival. In conclusion, our findings indicate that GSTM2 expression is essential for the survival of pancreatic cancer cells undergoing gemcitabine treatment and leads to chemo resistance. Downregulation of GSTM2 in pancreatic cancer may benefit gemcitabine treatment. GSTM2 expression in patients also shows significant correlation with overall survival. Thus, our study suggests that GSTM2 is a potential target for chemotherapy optimization and prognostic biomarker of pancreatic cancer.

Adipose-Derived Biogenic Nanoparticles for Suppression of Inflammation.

Extracellular vesicles secreted from adipose-derived mesenchymal stem cells (ADSCs) have therapeutic effects in inflammatory diseases. However, production of extracellular vesicles (EVs) from ADSCs is costly, inefficient, and time consuming. The anti-inflammatory properties of adipose tissue-derived EVs and other biogenic nanoparticles have not been explored. In this study, biogenic nanoparticles are obtained directly from lipoaspirate, an easily accessible and abundant source of biological material. Compared to ADSC-EVs, lipoaspirate nanoparticles (Lipo-NPs) take less time to process (hours compared to months) and cost less to produce (clinical-grade cell culture facilities are not required). The physicochemical characteristics and anti-inflammatory properties of Lipo-NPs are evaluated and compared to those of patient-matched ADSC-EVs. Moreover, guanabenz loading in Lipo-NPs is evaluated for enhanced anti-inflammatory effects. Apolipoprotein E and glycerolipids are enriched in Lipo-NPs compared to ADSC-EVs. Additionally, the uptake of Lipo-NPs in hepatocytes and macrophages is higher. Lipo-NPs and ADSC-EVs have comparable protective and anti-inflammatory effects. Specifically, Lipo-NPs reduce toll-like receptor 4-induced secretion of inflammatory cytokines in macrophages. Guanabenz-loaded Lipo-NPs further suppress inflammatory pathways, suggesting that this combination therapy can have promising applications for inflammatory diseases.