Pancreatic Cell Lines Research Articles

Fabrication of phospholipid polymer-modified alginate hydrogels for bioartificial pancreas.

The bioartificial pancreas, composed of a semi-permeable hydrogel encapsulating insulin-secreting cells, has attracted attention as a treatment for type 1 diabetes. In this study, we developed phospholipid polymer-modified alginate hydrogel beads that encapsulated spheroids of the pancreatic beta cell line MIN6. The hydrogel beads were composed of methacrylated alginic acid, which enabled both ionic and covalent cross-linking, resulting in a hydrogel that was more stable than conventional alginate hydrogels. Furthermore, modification of biocompatible 2-methacryloyloxyethyl phosphorylcholine (MPC) polymers suppressed protein adsorption to the hydrogel beads. Hydrogel beads encapsulating MIN6 spheroids maintained the cell viability and insulin secretion ability in response to glucose levels invitro. Allogenic transplantation of gel beads lowered blood glucose levels in diabetic mice for 30 days, whereas gel beads without MPC polymer modification failed to regulate blood glucose levels. These results indicate that MPC polymer modification of hydrogels provides a new strategy for the fabrication of functional bioartificial pancreas and transplantable biomaterials for cell therapies.

The In Vitro Cytotoxic Potential of Biosynthesized Silver Nanoparticles in MIA PaCa-2 Cells Supported with an In Silico Study

Pancreatic cancer affects many people and is quite aggressive. Metallic nanoparticles may be an alternative treatment for this disease. In this work, the antiproliferative activity of biosynthesized silver nanoparticles was evaluated. Stenocereus queretaroensis peel extract was used as a reducing agent for nanoparticle synthesis; the characterization was carried out using spectroscopic techniques, X-ray diffraction, and microscopy. The antiproliferative effect was evaluated in the MIA PaCa-2 pancreatic tumoral cell line, and a molecular docking test was run with the STAT3 protein. The results obtained show that it was possible to synthesize silver nanoparticles from the plant extract, which was confirmed through characterization studies. The average size of the nanoparticles was measured to be 48.8 nm, and they predominantly exhibited a spherical shape. The antiproliferative effect was demonstrated in the pancreatic cell line, with an IC50 value of 15.66 µg/mL. The molecular docking analysis predicted a strong interaction with the STAT3 protein, with a binding energy value of −6.47 obtained. With these results, it is concluded that biosynthesized silver nanoparticles inhibit pancreatic tumor cell growth and may represent an innovative cancer treatment.

The Microenvironment in an Experimental Model of Acute Pancreatitis Can Modify the Formation of the Protein Corona of sEVs, with Implications on Their Biological Function.

A considerable number of the physiological functions of extracellular vesicles are conditioned by the protein corona attached to their surface. The composition of this corona is initially defined during their intracellular synthesis, but it can be subsequently modified by interactions with the microenvironment. Here, we evaluated how the corona of small extracellular vesicles exposed to the inflammatory environment generated in acute pancreatitis is modified and what functional changes occur as a result of these modifications. Small extracellular vesicles obtained from a pancreatic cell line were incubated with the ascitic fluid generated in experimental acute pancreatitis in rats. Using proteomic techniques, we detected the appearance of new proteins and an increase the uptake of extracellular vesicles by certain cell types and the response induced in inflammatory cells. The inhibition of different pattern recognition receptors reversed this activation, indicating that some of these effects could be due to binding of damage-associated molecular patterns to the corona. All of this indicates that in pathologies such as acute pancreatitis, characterized by an inflammatory response and intense tissue damage, the microenvironment substantially influences the corona of extracellular vesicles, thus altering their behavior and enhancing their inflammatory activity.

Long-term in vitro expansion of a human fetal pancreas stem cell that generates all three pancreatic cell lineages

The mammalian pancreas consists of three epithelial compartments: the acini and ducts of the exocrine pancreas and the endocrine islets of Langerhans. Murine studies indicate that these three compartments derive from a transient, common pancreatic progenitor. Here, we report derivation of 18 human fetal pancreas organoid (hfPO) lines from gestational weeks 8-17 (8-17 GWs) fetal pancreas samples. Four of these lines, derived from 15 to 16 GWs samples, generate acinar-, ductal-, and endocrine-lineage cells while expanding exponentially for >2 years under optimized culture conditions. Single-cell RNA sequencing identifies rare LGR5+ cells in fetal pancreas and in hfPOs as the root of the developmental hierarchy. These LGR5+ cells share multiple markers with adult gastrointestinal tract stem cells. Organoids derived from single LGR5+ organoid-derived cells recapitulate this tripotency invitro. We describe a human fetal tripotent stem/progenitor cell capable of long-term expansion invitro and of generating all three pancreatic cell lineages.

Rapid Screening to Identify Antivirals against Persistent and Acute Coxsackievirus B3 Infection.

Enteroviruses cause significant morbidity and mortality worldwide, and Coxsackievirus B3 (CVB3) is one of the most commonly reported. Coxsackieviruses establish persistent infection, characterized as infection that is not cleared from host cells generating a continuous infection. No antivirals targeting persistent or acute infection are available, and CVB3 may respond differently depending on the type of infection. Therefore, there is an urgent need for new antiviral drugs to combat acute and persistent CVB3 infection. We developed a system to study persistent CVB3 infection with pancreatic ductal cell line PANC-1, and we used an epithelial cell line, Vero-E6 cells, to study acute CVB3 infection. We maintained persistently infected cells for over a year. Now, in an effort to identify antivirals, using the National Institutes of Health's Developmental Therapeutics Program (DTP), we screened thousands of compounds for activity against acute and persistent CVB3 infection, and among the hits was Ro 5-3335, a 1,4-benzodiazepine nordazepam that acts as a RUNX1-CBFβ leukemia inhibitor. Ro 5-3335 has previously been reported to inhibit HIV-1 gene expression through interference with Tat-mediated transactivation. We confirmed Ro 5-3335's antiviral activity against CVB3 in both acute and persistent infection, in several cell types and at pharmacologically favorable conditions. We show that Ro 5-3335 has minimal cytotoxicity and is antiviral over several rounds of replication. We identified viral egress as a putative target. We also show efficacy against other RNA viruses, but it is ineffective against a model DNA virus. Overall, Ro 5-3335 is a promising antiviral that may target CVB3 infection.

Antigen-independent single-cell circulating tumor cell detection using deep-learning-assisted biolasers

Circulating tumor cells (CTCs) in the bloodstream are important biomarkers for clinical prognosis of cancers. Current CTC identification methods are based on immuno-labeling, which depends on the differential expression of specific antigens between the cancer cells and white blood cells. Here we present an antigen-independent CTC detection method utilizing a deep-learning-assisted single-cell biolaser. Single-cell lasers were measured from nucleic-acid-stained cells inside optical cavities. A Deep Cell-Laser Classifier (DCLC) was developed to detect tumor cells from a patient CTC-derived pancreatic cell line using their unique single-cell lasing mode patterns. We further showed that the knowledge learned from one type of pancreatic cancer cell line can be transferred to detect other pancreatic cancer cell lines by the DCLC in zero-shot. A sensitivity of 94.3% and a specificity of 99.9% were achieved. Finally, enumeration was performed on CTCs obtained from pancreatic cancer patients. We further demonstrated the DCLC’s ability in zero-shot generalization of enumeration on lung cancer patients’ CTCs. The counting trends were consistent with those observed using conventional immunofluorescence imaging techniques. Employing our DCLC model, single-cell lasers open new avenues for both future biological studies and clinical applications, including classification of cell types and identification of rare cells.

Circ_0124346 facilitates cell proliferation of pancreatic adenocarcinoma cells by regulating lipid metabolism via miR-223-3p/ACSL3 axis

BackgroundBoth lipid metabolism and cyclic RNAs (circRNAs) have been found to be involved in pancreatic adenocarcinoma (PAAD) progression, but the relationship between lipid metabolism and circRNAs remains unclear.MethodsThe expression levels of miR-223-3p, circ_0124346, and acyl-CoA synthetase long chain family member 3 (ACSL3) were determined through qRT-PCR and Western blot analysis. Cell proliferation was evaluated using the CCK-8 and EdU incorporation assays. Cholesterol (CH) and triglyceride (TG) levels were quantified using relevant kits. The relationships between miR-223-3p and circ_0124346 or ACSL3 mRNA were examined by bioinformatics analysis, luciferase reporter, RNA-RNA pull-down, and RIP assays.ResultsWe observed a significant elevation in circ_0124346 expression in both pancreatic adenocarcinoma (PAAD) tissues and cell lines, and its expression level was shown to be correlated with tumor size. Circ_0124346 stimulated cell proliferation and facilitated lipid synthesis in PAAD cells. Additionally, we found that circ_0124346 functioned as a sponge for miR-223-3p, preventing miR-223-3p’s binding to the 3'-UTR of ACSL3 mRNA, which subsequently led to an elevation in ACSL3 expression and promoted lipid synthesis. Accordingly, circ_0124346 knockdown resulted in a significant decrease in lipid synthesis and cell proliferation in PAAD cells, with partial reversal of these effects achieved via inhibiting miR-223-3p or overexpressing ACSL3.ConclusionOur study demonstrated that circ_0124346 regulates lipid metabolism in PAAD cells via the miR-223-3p/ACSL3 axis, suggesting that targeting circ_0124346 may serve as a potential strategy for treating PAAD and assisting in its diagnosis.

Investigation of the efficacy of siRNA-mediated KRAS gene silencing in pancreatic cancer therapy.

Pancreatic carcinoma is an aggressive cancer that progresses without many symptoms. The difficulty of early diagnosis and an inadequate response to traditional treatments also cause the survival rate of pancreatic cancer to be low. Current research is focusing on methods of diagnosis and treatment, such as gene therapy, to increase survival rates. Small interfering ribonucleic acid (siRNA) has emerged as a promising advanced therapeutic strategy for cancer treatment. This study sought to silence the KRAS gene in the human pancreatic carcinoma cell line using a complex of small interfering ribonucleic acid (siRNA) and gold nanoparticles (AuNP). In this study, 25 nM siRNA and gold nanoparticles at 0.5 mg/ml, 0.25 mg/ml, and 0.125 mg/ml concentrations were used to silence the KRAS gene in the CAPAN-1 cell line. Real-time PCR analysis, agarose gel electrophoresis, and double staining were carried out, and xCelligence real-time cell analysis (RTCA) was used to measure proliferation. The PCR analysis revealed crossing point (CP) values of actin beta (ACTB) ranging from 33.04 to 35.98, which was in the expected range for all samples. The interaction between the gold nanoparticle/siRNA complex in the double staining analysis revealed that the most effective concentration of gold nanoparticle was 0.125 mg/ml. The WST-1 technique showed that siRNA/AuPEI cells in application groups had a viability rate of over 90%, indicating no toxicity or side effects. The xCELLigence RTCA®showed that at hour 72, there was a significant difference in proliferation in the 0.5 mg/mL PEI/AuNP-siRNA, 0.25 mg/mL PEI/AuNP-siRNA, and 0.125 mg/mL PEI/AuNP-siRNA application groups compared to the control and siRNA groups (p<0.05). By hour 96, all three groups were statistically different from the control and siRNA groups in terms of proliferation (p<0.05). The results of this analysis suggest that the AuPEI/siRNA complex can be effectively used to silence the target gene, but more studies are needed to verify these results.

Mirror-Image Random Nonstandard Peptides Integrated Discovery (MI-RaPID) Technology Yields Highly Stable and Selective Macrocyclic Peptide Inhibitors for Matrix Metallopeptidase 7.

Matrix metallopeptidase 7 (MMP7) plays a crucial role in cancer metastasis and progression, making it an attractive target for therapeutic development. However, the development of selective MMP7 inhibitors is challenging due to the conservation of active sites across various matrix metalloproteinases (MMPs). Here, we have developed mirror-image random nonstandard peptides integrated discovery (MI-RaPID) technology to discover innate protease-resistant macrocyclic peptides that specifically bind to and inhibit human MMP7. One identified macrocyclic peptide against D-MMP7, termed D20, was synthesized in its mirror-image form, D'20, consisting of 12 D-amino acids, one cyclic β-amino acid, and a thioether bond. Notably, it potently inhibited MMP7 with an IC50 value of 90 nM, and showed excellent selectivity over other MMPs with similar substrate specificity. Moreover, D'20 inhibited the migration of pancreatic cell line CFPAC-1, but had no effect on the cell proliferation and viability. D'20 exhibited excellent stability in human serum, as well as in simulated gastric and intestinal fluids. This study highlights that MI-RaPID technology can serve as a powerful tool to develop in vivo stable macrocyclic peptides for therapeutic applications.

Mirror‐Image Random Nonstandard Peptides Integrated Discovery (MI‐RaPID) Technology Yields Highly Stable and Selective Macrocyclic Peptide Inhibitors for Matrix Metallopeptidase 7

AbstractMatrix metallopeptidase 7 (MMP7) plays a crucial role in cancer metastasis and progression, making it an attractive target for therapeutic development. However, the development of selective MMP7 inhibitors is challenging due to the conservation of active sites across various matrix metalloproteinases (MMPs). Here, we have developed mirror‐image random nonstandard peptides integrated discovery (MI‐RaPID) technology to discover innate protease‐resistant macrocyclic peptides that specifically bind to and inhibit human MMP7. One identified macrocyclic peptide against D‐MMP7, termed D20, was synthesized in its mirror‐image form, D’20, consisting of 12 D‐amino acids, one cyclic β‐amino acid, and a thioether bond. Notably, it potently inhibited MMP7 with an IC50 value of 90 nM, and showed excellent selectivity over other MMPs with similar substrate specificity. Moreover, D’20 inhibited the migration of pancreatic cell line CFPAC‐1, but had no effect on the cell proliferation and viability. D’20 exhibited excellent stability in human serum, as well as in simulated gastric and intestinal fluids. This study highlights that MI‐RaPID technology can serve as a powerful tool to develop in vivo stable macrocyclic peptides for therapeutic applications.

Activating p53Y220C with a Mutant-Specific Small Molecule.

TP53 is the most commonly mutated gene in cancer, but it remains recalcitrant to clinically meaningful therapeutic reactivation. We present here the discovery and characterization of a small molecule chemical inducer of proximity that activates mutant p53. We named this compound TRanscriptional Activator of p53 (TRAP-1) due to its ability to engage mutant p53 and BRD4 in a ternary complex, which potently activates mutant p53 and triggers robust p53 target gene transcription. Treatment of p53Y220C expressing pancreatic cell lines with TRAP-1 results in rapid upregulation of p21 and other p53 target genes and inhibits the growth of p53Y220C-expressing cell lines. Negative control compounds that are unable to form a ternary complex do not have these effects, demonstrating the necessity of chemically induced proximity for the observed pharmacology. This approach to activating mutant p53 highlights how chemically induced proximity can be used to restore the functions of tumor suppressor proteins that have been inactivated by mutation in cancer.

TGF-β-Induced PAUF Plays a Pivotal Role in the Migration and Invasion of Human Pancreatic Ductal Adenocarcinoma Cell Line Panc-1.

Pancreatic adenocarcinoma upregulated factor (PAUF) was initially identified as a secreted protein that is substantially expressed in pancreatic ductal adenocarcinoma (PDAC). PAUF also affects invasiveness, motility, and the proliferation of cells in several types of cancer. Recently, PAUF was reported to play a pivotal role in the TLR4-mediated migration and invasion of PDAC cells. However, the mechanism inducing PAUF expression and its functional role in TGF-β-stimulated PDAC cells have not yet been studied. Thus, we first assessed whether TGF-β regulates PAUF expression in several PDAC cell lines and found a significant increase in PAUF expression in Smad signaling-positive Panc-1 cells treated with TGF-β. We also found that the PAUF promoter region contains a Smad-binding element. TGF-β-treated Panc-1 cells showed an increase in PAUF promoter activity, but this effect was not observed in TGF-β-stimulated Smad4-null BxPC-3 cells. Restoring Smad4 expression increased the PAUF promoter activity and expression in Smad4-overexpressing BxPC-3 cells treated with TGF-β. We further found that PAUF aggravated the TGF-β-induced epithelial-mesenchymal transition (EMT) in Panc-1 and BxPC-3 cells via the activation of MEK-ERK signaling. These results indicate that TGF-β/Smad signaling-mediated upregulation of PAUF plays a crucial role in EMT progression by activating the TGF-β-mediated MEK-ERK signaling pathway.

Oncolytic Coxsackievirus B3 Strain PD-H Is Effective Against a Broad Spectrum of Pancreatic Cancer Cell Lines and Induces a Growth Delay in Pancreatic KPC Cell Tumors In Vivo

Pancreatic cancer is one of the deadliest cancers globally, with limited success from existing therapies, including chemotherapies and immunotherapies like checkpoint inhibitors for patients with advanced pancreatic ductal adenocarcinoma (PDAC). A promising new approach is the use of oncolytic viruses (OV), a form of immunotherapy that has been demonstrated clinical effectiveness in various cancers. Here we investigated the potential of the oncolytic coxsackievirus B3 strain (CVB3) PD-H as a new treatment for pancreatic cancer. In vitro, PD-H exhibited robust replication, as measured by plaque assays, and potent lytic activity, as assessed by XTT assays, in most pancreatic tumor cell lines, outperforming two other coxsackievirus strains tested, H3N-375/1TS and CVA21. Thus, H3N-375/1TS showed efficient replication and lytic efficiency in distinctly fewer tumor cell lines, while most tumor cells were resistant to CVA21. The oncolytic efficiency of the three OV largely correlated with mRNA expression levels of viral receptors and their ability to induce apoptosis, as measured by cleaved caspase 3/7 activity in the tumor cells. In a syngeneic mouse model with subcutaneous pancreatic tumors, intratumoral administration of PD-H significantly inhibited tumor growth but did not completely stop tumor progression. Importantly, no virus-related side effects were observed. Although pancreatic tumors respond to PD-H treatment, its therapeutic efficacy is limited. Combining PD-H with other treatments, such as those aiming at reducing the desmoplastic stroma which impedes viral infection and spread within the tumor, may enhance its efficacy.

Targeting Human Pancreatic Cancer with a Fluorophore-Conjugated Mucin 4 (MUC4) Antibody: Initial Characterization in Orthotopic Cell Line Mouse Models.

Background/Objectives: Pancreatic cancer is the third leading cause of death related to cancer. The only possible cure presently is complete surgical resection; however, this is limited by difficulty in clearly defining tumor margins. Enhancement of the visualization of pancreatic ductal adenocarcinoma (PDAC) tumor margins using near-infrared dye-conjugated tumor-specific antibodies was pioneered by using anti-CEA, anti-CA19.9, and anti-MUC5AC in orthotopic mouse models of pancreatic cancer. Recently, an antibody to Mucin 4 (MUC4) conjugated to a fluorescent probe has shown promise in targeting colon tumors in orthotopic mouse models. Methods: In the present study, we targeted pancreatic cancer using an anti-MUC4 antibody conjugated to IRDye800 (anti-MUC4-IR800) in orthotopic mouse models. Two pancreatic cancer human cell lines were used, SW1990 and CD18/HPAF. Results: Anti-MUC4-IR800 targeted the two pancreatic cancer cell line tumors in orthotopic mouse models with high tumor-to-pancreas ratios and high tumor-to-liver ratios, with greater targeting seen in SW1990. Conclusions: The present results suggest anti-MUC4-IR800's potential to be used in fluorescence-guided surgical resection of pancreatic cancer.

The L-type amino acid transporter 1 enhances drug delivery to the mouse pancreatic beta cell line (MIN6)

l-type amino acid transporter 1 (LAT1) is a membrane transporter responsible for carrying large, neutral l-configured amino acids as well as appropriate (pro)drugs into a cell. It has shown a great potential to improve drug delivery across the blood-brain barrier and to increase cell uptake into several brain and cancer cell types. However, besides the brain, the LAT1-utilizing compounds are also delivered more efficiently into the pancreas in vivo. In this study, we quantified the expression of LAT1 along several other membrane transporters in mouse pancreatic β-cell line (MIN6). Furthermore, we studied the function of LAT1 in MIN6 cells, and its ability to deliver non-steroidal anti-inflammatory drug (NSAID)-derived prodrugs there. The results showed that LAT1 was highly abundant in MIN6 cells, with an even expression on cell pseudoislets. The l-leucine uptake as a probe substrate was efficient, with comparable affinity and capacity to previously studied immortalized mouse microglia (BV2). The NSAID-derived prodrugs utilized LAT1 for their delivery and were uptaken into MIN6 cells 2–300 times more efficiently when compared to their parent drugs. A similar increase in pancreatic delivery was observed also in vivo, where the pancreatic exposure was 2–10 times higher with selected prodrugs, indicating an excellent correlation between in vitro uptake and in vivo pancreatic delivery. Finally, the LAT1-utilizing prodrugs were able to reverse the effects of cytokines on insulin secretion in MIN6 cells, showing that improved delivery via LAT1 can enhance drug effects in the mouse pancreatic β-cell line.

Nano-synthesis, characterization, theoretical, biological and catalytic studies of Mn(II), Cu(II), Cd(II), and Th(IV) complexes based on antipyrine azo dye ligand

The design and detailed characterization of the azo dye ligand obtained from the reaction of 4-aminoantipyrine diazonium salt with 3-hydroxybenzaldehyde (HL) and its novel metal chelates with Mn2+, Cu2+, Cd2+ and Th4+ ions have been presented. Utilizing modern spectroscopic and analytical techniques, these complexes were found to adopt an octahedral geometry, demonstrate inner sphere d2sp3 hybridization for Th(IV) complex and outer sphere sp3d2 hybridization for other complexes. Particle sizes calculated using the Debye-Scherrer equation on XRD patterns were 32.05 nm for Mn(II), 34.07 nm for Cu(II), 30.90 nm for Cd(II), and 22.05 nm for Th(IV). These substances' anticancer activity was assessed on human lung cancer cells (A-549) and pancreatic carcinoma cell lines (PANC-1). Cd(II) complex exhibited higher cytotoxicity against the A-549 cell line than the reference drug vinblastine sulfate (VinBLAStine Sulfate) which revealing its potential application as a new anticancer agent. Quantum chemical computations using the DMOL3 module's density functional theory (DFT) and molecular docking studies with the Schrödinger suite demonstrated significant inhibitory effects of the free organic compound and Cu(II) chelate on the FGFR1 protein, crucial for cell development and differentiation. Additionally, the study examined the catalytic activity of HL and its metal chelates in the oxidative degradation of methyl violet 2B dye in presence of H2O2. The order was followed by the pseudo-first-order rate constants: Th(IV) chelate (0.9556 min⁻¹) > HL (0.4911 min⁻¹) > Cu(II) chelate (0.5979 min⁻¹) > Mn(II) chelate (0.2645 min⁻¹) > Cd(II) chelate (0.2280 min⁻¹).

Antimicrobial, Anti-biofilm, Anti-swarming, Anti-quorum Sensing Activities, and Cytotoxicity of Propolis Samples from Northeast of Türkiye

Aim of study: Studies on propolis have increased as it has been revealed that it contains biologically active molecules. In the current study, it was aimed to analyze biological activity, and cytotoxicity of ethanolic extract of three different propolis samples from Türkiye. Material and methods: The antibacterial activity of the extracts against 14 microorganisms was assessed using the agar well diffusion method and the microdilution method. Chromobacter violeceum was used in quorum-sensing assay, and Pseudomonas aeruginosa PAO1 strain was used in swarming and biofilm assays. Using the MTT test, the cytotoxic effect of the extracts was examined on the lung adenocarcinoma cell line (A549), pancreatic tumoral cell line (AR42J), breast cancer cell line (MDA-MB-231), and normal epithelial cell line (Vero). Main results: All propolis extracts were effective against 8/14 microorganisms included in the study. While all propolis extracts have shown anti-quorum sensing activity, there was not any anti-swarming and anti-biofilm activity in each sample. It was demonstrated that every propolis sample had a dose-dependent cytotoxic effect on the examined cell lines. Research highlights: Due to the biological activity shown by the propolis samples included in the study, it is considered that it has the potential to influence the creation of novel medications in the future.

Pancreatic Differentiation of Oral Minor Salivary Gland Stem Cells.

Stem cells from various sources including major salivary glands have been used to establish pancreatic differentiation in an attempt to provide new treatment options for patients with diabetes mellitus. In contrast, the potential of using the more easily accessible intraoral minor salivary glands has not been evaluated so far. Salivary stem cells were isolated from normal labial minor salivary glands that were removed during the excision of a mucocele and were attempted to differentiate into pancreatic cell lines using a culture medium enriched with activin A, retinoic acid and GLP-1.Real time RT-PCR was used to evaluate the expression of the genes of pancreatic transcription factors MafA, Ptf1a, Hb9 and Arx. Complementary, 22 labial minor salivary gland paraffin-embedded specimens were examined using immunohistochemistry for the presence of the relevant gene products of the pancreatic transcription factors Arx, MafA, Ptf1a and Pdx1. The differentiated salivary stem cells(cells of passage 3) expressed the genes of the pancreatic transcription factors MafA, Ptf1a, Hb9 and Arx even on the first day of the experiment while immunohistochemistry also confirmed the presence of the protein products of Arx, MafA, Ptf1a as well as Pdx1[> 50% of the specimens for Arx(5/8) and MafA(7/8), < 50% for Ptf1a(5/11) and Pdx1(5/11)] in ducts, mesenchymal connective tissue and acinar cells. Labial minor salivary glands may share gene and protein characteristics with pancreas suggesting a possible usefulness for pancreatic regeneration or substitution in cases of deficiency.

Structural and quantitative characterization of membrane N-glycans from MIN6 mouse pancreatic beta cells using liquid chromatography-quadrupole-Orbitrap tandem mass spectrometry

MIN6, a mouse pancreatic beta cell line, is used in diabetes research, and the cellular N-glycoproteins in membrane are important in regulating the metabolism of insulin secretion. However, the identities of N-glycans in MIN6 cells are yet to be fully elucidated. In this study, the structures of N-glycans were analyzed using liquid chromatography-electrospray ionization-higher energy collisional dissociation-tandem mass spectrometry. The abundances (%) of each N-glycan relative to the total N-glycans (100 %) were also obtained. Fifty N-glycans (with relative abundance of each > 0.5 %) were obtained, revealing 22 bisecting N-acetylglucosamine (GlcNAc; associated with cell adhesion and growth; sum of relative abundance of each: 27.1 %), 21 core-fucosylated (associated with glucose sensing and insulin secretion regulation; 28.3 %), and 16 sialylated (N-acetylneuraminic acid; related to the expression of glucose transporters and diabetes;15.5 %) N-glycans. Membranes contained higher bisecting GlcNAc and core-fucosylation, similar sialylation, but less high-mannosylation than the lysate (the cellular contents). Notably, all bisecting GlcNAc N-glycans were categorized into structures with (16.6 %) or without (10.5 %) core-fucosylation and with (6.9 %) or without (20.2 %) sialylation. The bisecting GlcNAc structures were not found in human islets; moreover, sialylation levels were 6.9 times higher than for human islets. These structural characteristics of N-glycans affect their cell adhesion and distribution through homologous interactions between beta cells, leading to increased insulin secretion efficiency. This study is the first to identify the structures and quantities of 50 N-glycans in MIN6 cell membranes that may play an important role in regulating the functions of pancreatic beta cells.

Large-scale multi-omic analysis identifies noncoding somatic driver mutations and nominates ZFP36L2 as a driver gene for pancreatic ductal adenocarcinoma.

Identification of somatic driver mutations in the noncoding genome remains challenging. To comprehensively characterize noncoding driver mutations for pancreatic ductal adenocarcinoma (PDAC), we first created genome-scale maps of accessible chromatin regions (ACRs) and histone modification marks (HMMs) in pancreatic cell lines and purified pancreatic acinar and duct cells. Integration with whole-genome mutation calls from 506 PDACs revealed 314 ACRs/HMMs significantly enriched with 3,614 noncoding somatic mutations (NCSMs). Functional assessment using massively parallel reporter assays (MPRA) identified 178 NCSMs impacting reporter activity (19.45% of those tested). Focused luciferase validation confirmed negative effects on gene regulatory activity for NCSMs near CDKN2A and ZFP36L2 . For the latter, CRISPR interference (CRISPRi) further identified ZFP36L2 as a target gene (16.0 - 24.0% reduced expression, P = 0.023-0.0047) with disrupted KLF9 binding likely mediating the effect. Our integrative approach provides a catalog of potentially functional noncoding driver mutations and nominates ZFP36L2 as a PDAC driver gene.