Generation of INS-jGCaMP7f knock-in Ca reporter human embryonic stem cell line, GZLe001-C, using CRISPR/Cas9-based gene targeting.

As a member of the single-fluorophore genetically encoded calcium indicators (GECIs), jGCaMP7f is widely applied to investigate intracellular Ca concentrations. Here, we established an INS-jGCaMP7f knock-in H1 human embryonic stem cell (hESC) line by integrating jGCaMP7f gene into insulin locus via CRISPR/Cas9 system. The reporter cell line not only effectively labelled the insulin-producing cells induced from hESC, but also reflected the cytosolic change of Ca level in response to different stimuli.

A differentiation protocol for generating pancreatic delta cells from human pluripotent stem cells.

In this protocol, we detail a seven-stage differentiation methodology for generating pancreatic delta cells (SC-delta cells) from human pluripotent stem cells (hPSCs). In the first step, definitive endoderm is generated by activin A and CHIR99021, followed by induction of primitive gut tube and posterior foregut by treatment with FGF7, SANT1, LDN193189, PdBU, and retinoic acid (RA). The subsequent endocrine generation and directed SC-delta cell induction is achieved by a combined treatment of the FGF7 with FGF2 during stage 4 and 5, together with RA, XXI, ALK5 inhibitor II, SANT1, Betacellulin and LDN193189.

Advancing High-Throughput MS-Based Protein Quantification: A Case Study on Quantifying 10 Major Food Allergens by LC-MS/MS Using a One-Sample Multipoint External Calibration Curve.

The LC-MS-based method has emerged as the preferred approach for quantifying food allergens. However, the preparation of a traditional calibration curve (MSCC) is labor-intensive and error-prone. Here, a sensitive and robust LC-MS/MS method for quantifying 10 major food allergens was developed and validated, where the one-sample multipoint external calibration curve (OSCC) was employed instead of MSCC.

Integrating a Multiple Isotopologue Reaction-Monitoring Technique and LC-MS/MS for Quantitation of Small Molecules: Ten Mycotoxins in Cereals as an Example.

Accurate quantification of mycotoxin in cereals is crucial for ensuring food safety and human health. However, the preparation of traditional multisample external calibration curves (MSCCs) is labor-intensive and error-prone. Here, a multiple isotopologue reaction-monitoring (MIRM)-LC-MS/MS method for accurate quantitation of ten major mycotoxins in cereals was successfully developed and validated, where a novel one-sample multipoint calibration curve (OSCC) strategy is used instead of MSCCs.

A role and mechanism for redox sensing by SENP1 in β-cell responses to high fat feeding.

Pancreatic β-cells respond to metabolic stress by upregulating insulin secretion, however the underlying mechanisms remain unclear. Here we show, in β-cells from overweight humans without diabetes and mice fed a high-fat diet for 2 days, insulin exocytosis and secretion are enhanced without increased Ca influx. RNA-seq of sorted β-cells suggests altered metabolic pathways early following high fat diet, where we find increased basal oxygen consumption and proton leak, but a more reduced cytosolic redox state.

An integrated calibration strategy for the development and validation of an LC-MS/MS method for accurate quantification of egg allergens (Gal d 1-6) in foods.

Accurate determination of egg allergens in food is vital for allergen management and labeling. However, quantifying egg allergens with mass spectrometry poses challenges and lacks validation methods. Here, we developed and validated an LC-MS/MS method for quantifying egg allergens (Gal d 1-6) in foods.

Ca2+ Oscillations, Waves, and Networks in Islets From Human Donors With and Without Type 2 Diabetes.

Pancreatic islets are highly interconnected structures that produce pulses of insulin and other hormones, maintaining normal homeostasis of glucose and other nutrients. Normal stimulus-secretion and intercellular coupling are essential to regulated secretory responses, and these hallmarks are known to be altered in diabetes. In the current study, we used calcium imaging of isolated human islets to assess their collective behavior.

P2Y1 purinergic receptor identified as a diabetes target in a small-molecule screen to reverse circadian β-cell failure.

The mammalian circadian clock drives daily oscillations in physiology and behavior through an autoregulatory transcription feedback loop present in central and peripheral cells. Ablation of the core clock within the endocrine pancreas of adult animals impairs the transcription and splicing of genes involved in hormone exocytosis and causes hypoinsulinemic diabetes. Here, we developed a genetically sensitized small-molecule screen to identify druggable proteins and mechanistic pathways involved in circadian β-cell failure.

Arginine-vasopressin mediates counter-regulatory glucagon release and is diminished in type 1 diabetes.

Insulin-induced hypoglycemia is a major treatment barrier in type-1 diabetes (T1D). Accordingly, it is important that we understand the mechanisms regulating the circulating levels of glucagon. Varying glucose over the range of concentrations that occur physiologically between the fed and fuel-deprived states (8 to 4 mM) has no significant effect on glucagon secretion in the perfused mouse pancreas or in isolated mouse islets (in vitro), and yet associates with dramatic increases in plasma glucagon.

β-Cell Knockout of SENP1 Reduces Responses to Incretins and Worsens Oral Glucose Tolerance in High-Fat Diet-Fed Mice.

SUMOylation reduces oxidative stress and preserves islet mass at the expense of robust insulin secretion. To investigate a role for the deSUMOylating enzyme sentrin-specific protease 1 (SENP1) following metabolic stress, we put pancreas/gut-specific SENP1 knockout (pSENP1-KO) mice on a high-fat diet (HFD). Male pSENP1-KO mice were more glucose intolerant following HFD than littermate controls but only in response to oral glucose.

Improvement of islet transplantation by the fusion of islet cells with functional blood vessels.

Pancreatic islet transplantation still represents a promising therapeutic strategy for curative treatment of type 1 diabetes mellitus. However, a limited number of organ donors and insufficient vascularization with islet engraftment failure restrict the successful transfer of this approach into clinical practice. To overcome these problems, we herein introduce a novel strategy for the generation of prevascularized islet organoids by the fusion of pancreatic islet cells with functional native microvessels.

A role for alternative splicing in circadian control of exocytosis and glucose homeostasis.

The circadian clock is encoded by a negative transcriptional feedback loop that coordinates physiology and behavior through molecular programs that remain incompletely understood. Here, we reveal rhythmic genome-wide alternative splicing (AS) of pre-mRNAs encoding regulators of peptidergic secretion within pancreatic β cells that are perturbed in and β-cell lines. We show that the RNA-binding protein THRAP3 (thyroid hormone receptor-associated protein 3) regulates circadian clock-dependent AS by binding to exons at coding sequences flanking exons that are more frequently skipped in clock mutant β cells, including transcripts encoding () and ().

Improved glucose tolerance with DPPIV inhibition requires β-cell SENP1 amplification of glucose-stimulated insulin secretion.

Pancreatic islet insulin secretion is amplified by both metabolic and receptor-mediated signaling pathways. The incretin-mimetic and DPPIV inhibitor anti-diabetic drugs increase insulin secretion, but in humans this can be variable both in vitro and in vivo. We examined the correlation of GLP-1 induced insulin secretion from human islets with key donor characteristics, glucose-responsiveness, and the ability of glucose to augment exocytosis in β-cells.

Iodine-Catalyzed Regioselective Oxidative Cyclization of Aldehyde Hydrazones with Electron-Deficient Olefins for the Synthesis of Mefenpyr-Diethyl.

A regioselective synthesis of polysubstituted dihydropyrazoles and pyrazoles through an iodine-catalyzed oxidative cyclization strategy of aldehyde hydrazones with electron-deficient olefins is described. The protocol adopts very mild reaction conditions and provides desirable yields. The reaction is supposed to proceed via a cascade C-H functionalization, C-N bond formation, and oxidation sequential processes.

β Cell tone is defined by proglucagon peptides through cAMP signaling.

Paracrine interactions between pancreatic islet cells have been proposed as a mechanism to regulate hormone secretion and glucose homeostasis. Here, we demonstrate the importance of proglucagon-derived peptides (PGDPs) for α to β cell communication and control of insulin secretion. Signaling through this system occurs through both the glucagon-like peptide receptor (Glp1r) and glucagon receptor (Gcgr).

The proliferation and invasion of osteosarcoma are inhibited by miR-101 via targetting ZEB2.

Having a better grasp of the molecular mechanisms underlying carcinogenesis and progression in osteosarcoma would be helpful to find novel therapeutic targets. Different types of cancers have presented abnormal expression of miRNA-101 (miR-101). Nevertheless, we still could not figure out what expression of miR-101 in human osteosarcoma is and its biological function.

Incorporation of Viral Glycoprotein VSV-G Improves the Delivery of DNA by Erythrocyte Ghost into Cells Refractory to Conventional Transfection.

The objective of this study was to formulate a novel gene delivery system based on the erythrocyte ghost (EG) integrated with fusogenic viral glycoprotein vesicular stomatitis virus glycoprotein G (VSV-G). VSV-G proteins were harvested as condition medium of Ad293 cells carrying a VSV-G transgene and then incorporated into EG. Plasmid DNA was condensed by various transfection reagents.

Niacin receptor GPR109A inhibits insulin secretion and is down-regulated in type 2 diabetic islet beta-cells.

Objectives: We previously found niacin receptor GPR109A was expressed in murine islet beta-cells, and signaling through GPR109A inhibited glucose stimulated insulin secretion (GSIS). However, the expression of GPR109A in human islets and its functional relevance is still not known.

Methods: The expression of GPR109A was examined by antibody staining and in situ hybridization on pancreatic paraffin sections.

Incorporation of VSV-G produces fusogenic plasma membrane vesicles capable of efficient transfer of bioactive macromolecules and mitochondria.

The objective of this study was to determine if plasma membrane vesicles (PMVs) could be exploited for efficient transfer of macro-biomolecules and mitochondria. PMVs were derived from mechanical extrusion, and made fusogenic (fPMVs) by incorporating the glycoprotein G of vesicular stomatitis virus (VSV-G). Confocal microscopy examination revealed that cytoplasmic proteins and mitochondria were enclosed in PMVs as evidenced by tracing with cytoplasmically localized and mitochondria-targeted EGFP, respectively.