Allosteric inhibition of the IRE1α RNase preserves cell viability and function during endoplasmic reticulum stress.

Depending on endoplasmic reticulum (ER) stress levels, the ER transmembrane multidomain protein IRE1α promotes either adaptation or apoptosis. Unfolded ER proteins cause IRE1α lumenal domain homo-oligomerization, inducing trans autophosphorylation that further drives homo-oligomerization of its cytosolic kinase/endoribonuclease (RNase) domains to activate mRNA splicing of adaptive XBP1 transcription factor. However, under high/chronic ER stress, IRE1α surpasses an oligomerization threshold that expands RNase substrate repertoire to many ER-localized mRNAs, leading to apoptosis. To modulate these effects, we developed ATP-competitive IRE1α Kinase-Inhibiting RNase Attenuators-KIRAs-that allosterically inhibit IRE1α's RNase by breaking oligomers. One optimized KIRA, KIRA6, inhibits IRE1α in vivo and promotes cell survival under ER stress. Intravitreally, KIRA6 preserves photoreceptor functional viability in rat models of ER stress-induced retinal degeneration. Systemically, KIRA6 preserves pancreatic ß cells, increases insulin, and reduces hyperglycemia in Akita diabetic mice. Thus, IRE1α powerfully controls cell fate but can itself be controlled with small molecules to reduce cell degeneration.

(Re)generating Human Beta Cells: Status, Pitfalls, and Perspectives.

Diabetes mellitus results from disturbed glucose homeostasis due to an absolute (type 1) or relative (type 2) deficiency of insulin, a peptide hormone almost exclusively produced by the beta cells of the endocrine pancreas in a tightly regulated manner. Current therapy only delays disease progression through insulin injection and/or oral medications that increase insulin secretion or sensitivity, decrease hepatic glucose production, or promote glucosuria. These drugs have turned diabetes into a chronic disease as they do not solve the underlying beta cell defects or entirely prevent the long-term complications of hyperglycemia. Beta cell replacement through islet transplantation is a more physiological therapeutic alternative but is severely hampered by donor shortage and immune rejection. A curative strategy should combine newer approaches to immunomodulation with beta cell replacement. Success of this approach depends on the development of practical methods for generating beta cells, either in vitro or in situ through beta cell replication or beta cell differentiation. This review provides an overview of human beta cell generation.

Fluoride removal from groundwater using Zirconium Impregnated Anion Exchange Resin.

Drinking water containing excess fluoride is a major health concern across the globe. The present study reports the feasibility of zirconium impregnated hybrid anion exchange resin (HAIX-Zr) for treating fluoride contaminated groundwater. The HAIX-Zr resin was prepared by impregnating ZrO2 nanoparticles on polymeric anion exchanger resin. Fluoride uptake by HAIX-Zr was quite rapid, 60% removal was obtained within 30 min. Kinetics of fluoride uptake by HAIX-Zr resin followed the pseudo-second-order kinetic model and adsorption data fitted best to Freundlich adsorption isotherm model. Maximum fluoride uptake capacity was observed as 12.0 mg/g. The defluoridation capacity of the resin decreases with increase in solution pH. The co-existing anions like chloride, phosphate, bicarbonate, nitrate, and sulphate at 100 mg/L concentration significantly affected fluoride removal and bicarbonate showed the highest interference. Continuous flow packed bed experiments were performed with real groundwater. To maintain a lower pH, weak acid cation exchange resin (INDION-236) was used before HAIX-Zr. It was observed that reducing the pH of the sample water to 4-4.5, increased the number of treated bed volumes fifteen times. Regeneration of fluoride-containing resin was done by passing 3% NaOH and 3% NaCl solution through an exhausted resin bed. The results revealed that HAIX-Zr can effectively remove fluoride from groundwater.

Evidence of Economically Sustainable Village-Scale Microenterprises for Arsenic Remediation in Developing Countries.

Although unknown 25 years ago, natural arsenic contamination of groundwater affects over 50 countries and up to 200 million people. The economic viability was analyzed and modeled of eighty-eight community-based arsenic mitigation systems existing for up to 20 years in India and Bangladesh. The performances of three community-based arsenic mitigation systems that are ethnically different and separated across two different countries were monitored closely for 24 months of self-sustainable, long-term operation at WHO standards through local, paid caretakers. Based on data from the use of hybrid ion exchange materials (HIX-Nano) and the broad set of field operations, Monte Carlo simulations were used to explore the conditions required for self-sustainable operation and job creation in low-income communities (<$2/day/capita). The results from field data and cost modeling provided clear evidence of economic growth and job creation for systems managed by villagers' committee through collection of monthly tariffs. Ethnicity and religion did not have perceptible impacts on day-to-day operations or cumulative long-term revenue. The cost of the treatment technology (i.e., HIX-Nano) had minimal impact on the operational profitability, while number of customers and water delivery significantly affected profitability. Local employment generation with income significantly higher than poverty level was the most enduring outcome and led to enhanced sustainability.

Human pancreatic neuro-insular network in health and fatty infiltration.

AIMS/HYPOTHESIS: Identification of a pancreatic neuro-insular network in mice suggests that a similar integration of islets and nerves may be present in the human pancreas. To characterise the neuro-insular network and the intra-pancreatic ganglia in a clinically related setting, we examined human pancreases in health and with fatty infiltration via 3-dimensional (3D) histology and compared the human pancreatic microenvironment with its counterpart in mice. METHODS: Human pancreatic specimens from individuals with normal BMI, high BMI (≥ 25) and type 2 diabetes were used to investigate the neuro-insular network. Transparent specimens were prepared by tissue clearing for transmitted light and deep-tissue fluorescence imaging to simultaneously visualise infiltrated adipocytes, islets and neurovascular networks. RESULTS: High-definition images of human islets reveal that both the sympathetic and parasympathetic nerves enter the islet core and reside in the immediate microenvironment of islet cells. Around the islets, the neuro-insular network is visualised with 3D histology to identify the intra-pancreatic ganglia (peri-lobular and intra-parenchymal ganglia) and the islet-ganglionic association. In humans, but not in mice, pancreatic fatty infiltration (BMI dependent) features adipocytes infiltrating into the parenchyma and accumulating in the peri-lobular space, in which the peri-lobular ganglia also reside. We identified the formation of adipose-ganglionic complexes in the peri-lobular space and enlargement of ganglia around adipocytes. In the specimen from the individual with type 2 diabetes, an increase in the number of nerve projections from the intra-parenchymal ganglia is associated with severe fatty infiltration. CONCLUSIONS/INTERPRETATION: We present new perspectives of human pancreas and islet innervation via 3D histology. Our results strongly suggest that fatty infiltration in the human pancreas creates a neurotrophic microenvironment and promotes remodelling of pancreatic innervation.

Ectopic serotonin production in ß-cell specific transgenic mice.

Genetically modified mice have been widely used in the field of ß-cell research. However, analysis of results gathered using genetically modified organisms should be interpreted carefully as the results may be confounded by several factors. Here, we showed the ectopic serotonin (5-HT) production in ß-cells of RIP-CreMgn, MIP-GFP, and MIP-Cre/ERT mice. These mice contained a human growth hormone (hGH) cassette to enhance transgene expression and hGH expression and Stat5 phosphorylation were detected in pancreatic islets of these mice. The expression level of tryptophan hydroxylase 1 (Tph1) was upregulated in pancreatic islets of transgenic mice with an hGH cassette but not in transgenic mice without an hGH cassette. Ectopic 5-HT production was not observed in ß-cell-specific prolactin receptor (Prlr) knockout mice or Stat5 knockout mice crossed with RIP-CreMgn. We further confirmed that 5-HT production in ß-cells of several transgenic mice was induced by hGH expression followed by the activation of the Prlr-Stat5-Tph1 pathway. These findings indicate that results obtained using transgenic mice containing the hGH cassette should be interpreted with care.

Gαi/o-coupled receptor signaling restricts pancreatic ß-cell expansion.

Gi-GPCRs, G protein-coupled receptors that signal via Gα proteins of the i/o class (Gαi/o), acutely regulate cellular behaviors widely in mammalian tissues, but their impact on the development and growth of these tissues is less clear. For example, Gi-GPCRs acutely regulate insulin release from pancreatic ß cells, and variants in genes encoding several Gi-GPCRs--including the α-2a adrenergic receptor, ADRA2A--increase the risk of type 2 diabetes mellitus. However, type 2 diabetes also is associated with reduced total ß-cell mass, and the role of Gi-GPCRs in establishing ß-cell mass is unknown. Therefore, we asked whether Gi-GPCR signaling regulates ß-cell mass. Here we show that Gi-GPCRs limit the proliferation of the insulin-producing pancreatic ß cells and especially their expansion during the critical perinatal period. Increased Gi-GPCR activity in perinatal ß cells decreased ß-cell proliferation, reduced adult ß-cell mass, and impaired glucose homeostasis. In contrast, Gi-GPCR inhibition enhanced perinatal ß-cell proliferation, increased adult ß-cell mass, and improved glucose homeostasis. Transcriptome analysis detected the expression of multiple Gi-GPCRs in developing and adult ß cells, and gene-deletion experiments identified ADRA2A as a key Gi-GPCR regulator of ß-cell replication. These studies link Gi-GPCR signaling to ß-cell mass and diabetes risk and identify it as a potential target for therapies to protect and increase ß-cell mass in patients with diabetes.

Follicle dynamics and global organization in the intact mouse ovary.

Quantitative analysis of tissues and organs can reveal large-scale patterning as well as the impact of perturbations and aging on biological architecture. Here we develop tools for imaging of single cells in intact organs and computational approaches to assess spatial relationships in 3D. In the mouse ovary, we use nuclear volume of the oocyte to read out quiescence or growth of oocyte-somatic cell units known as follicles. This in-ovary quantification of non-growing follicle dynamics from neonate to adult fits a mathematical function, which corroborates the model of fixed oocyte reserve. Mapping approaches show that radial organization of folliculogenesis established in the newborn ovary is preserved through adulthood. By contrast, inter-follicle clustering increases during aging with different dynamics depending on size. These broadly applicable tools can reveal high dimensional phenotypes and age-related architectural changes in other organs. In the adult mouse pancreas, we find stochastic radial organization of the islets of Langerhans but evidence for localized interactions among the smallest islets.

Aluminum-Cycle Ion Exchange Process for Hardness Removal: A New Approach for Sustainable Softening.

From a sustainability viewpoint, sodium exchange softening, although used widely, is under scrutiny due to its production of excess Na-laden spent regenerant and subsequent discharge to the environment. Many arid regions are introducing regulations disallowing dumping of concentrated sodium salts, the residuals from popular Na-exchange softening. The sodium content of the softened water is, also, always higher than in the feed, which poses a dietary health concern when used for drinking or cooking. An efficient, easy-to-operate hardness removal process with reduced sodium in both the treated water and in the spent regenerant is an unmet global need. Use of a cation exchange resin in Al3+-form for hardness removal, that is, exchange of divalent Ca2+ or Mg2+ with trivalent Al3+, is counterintuitive, and this is particularly so, because the aluminum ion to be exchanged has higher affinity than calcium. Nevertheless, ion exchange accompanied by precipitation of aluminum hydroxide allows progress of the cation exchange reaction leading to hardness removal. Experimental results demonstrated that calcium can be consistently removed for multiple cycles using a stoichiometric amount of AlCl3 as the regenerant. The process essentially operates at the maximum possible thermodynamic efficiency: removal of one equivalent of Ca2+ corresponds to use of one equivalent of Al3+ as a regenerant. During the Al-cycle process there is no increase in Na+ concentration and partial reduction in the total dissolved solids (TDS) of the treated water. It is noteworthy that the ion-exchange resin used, components of the fixed-bed column and operational protocol are nearly the same as traditional softening processes on Na-cycle. Thus, existing Na-cycle systems can be retrofitted into Al-cycle operation without major difficulty.

Rfx6 directs islet formation and insulin production in mice and humans.

Insulin from the beta-cells of the pancreatic islets of Langerhans controls energy homeostasis in vertebrates, and its deficiency causes diabetes mellitus. During embryonic development, the transcription factor neurogenin 3 (Neurog3) initiates the differentiation of the beta-cells and other islet cell types from pancreatic endoderm, but the genetic program that subsequently completes this differentiation remains incompletely understood. Here we show that the transcription factor Rfx6 directs islet cell differentiation downstream of Neurog3. Mice lacking Rfx6 failed to generate any of the normal islet cell types except for pancreatic-polypeptide-producing cells. In human infants with a similar autosomal recessive syndrome of neonatal diabetes, genetic mapping and subsequent sequencing identified mutations in the human RFX6 gene. These studies demonstrate a unique position for Rfx6 in the hierarchy of factors that coordinate pancreatic islet development in both mice and humans. Rfx6 could prove useful in efforts to generate beta-cells for patients with diabetes.

Serotonin regulates glucose-stimulated insulin secretion from pancreatic ß cells during pregnancy.

In preparation for the metabolic demands of pregnancy, ß cells in the maternal pancreatic islets increase both in number and in glucose-stimulated insulin secretion (GSIS) per cell. Mechanisms have been proposed for the increased ß cell mass, but not for the increased GSIS. Because serotonin production increases dramatically during pregnancy, we tested whether flux through the ionotropic 5-HT3 receptor (Htr3) affects GSIS during pregnancy. Pregnant Htr3a(-/-) mice exhibited impaired glucose tolerance despite normally increased ß cell mass, and their islets lacked the increase in GSIS seen in islets from pregnant wild-type mice. Electrophysiological studies showed that activation of Htr3 decreased the resting membrane potential in ß cells, which increased Ca(2+) uptake and insulin exocytosis in response to glucose. Thus, our data indicate that serotonin, acting in a paracrine/autocrine manner through Htr3, lowers the ß cell threshold for glucose and plays an essential role in the increased GSIS of pregnancy.

Activation of GLP-1 and gastrin signalling induces in vivo reprogramming of pancreatic exocrine cells into beta cells in mice.

AIMS/HYPOTHESIS: Lineage conversion of non-beta cells into insulin-producing cells has been proposed as a therapy for the cure of diabetes. Glucagon-like peptide-1 (GLP-1) and its derivatives can induce beta cell neogenesis in vitro and beta cell mass expansion in vivo, but GLP-1 signalling has not been shown to regulate cell fate decisions in vivo. We therefore tested the impact of GLP-1 receptor (GLP1R) expression on beta cell differentiation in vivo. METHODS: Mice overexpressing GLP1R in pancreatic exocrine cells were generated by Cre-mediated recombination in sex-determining region Y-box 9 (SOX9)-expressing cells and then treated with exendin-4 and/or gastrin. Histological analysis was performed to detect cellular reprogramming from the exocrine lineage into insulin-producing cells. RESULTS: Whereas no newly generated beta cells were detected in the mice treated with exendin-4 alone, treatment with gastrin only induced the conversion of exocrine cells into insulin-producing cells. Furthermore, the overexpression of GLP1R, together with gastrin and exendin-4, synergistically promoted beta cell neogenesis accompanied by the formation of islet-like clusters. These newly generated beta cells expressed beta cell specific transcription factors, such as pancreatic and duodenal homeobox 1 (PDX1), NK6 homeobox 1 (NKX6.1) and musculoaponeurotic fibrosarcoma oncogene family A (MafA). These mice showed no histological evidence of pancreatitis or pancreatic dysplasia in their acini and had normal plasma amylase levels. CONCLUSIONS/INTERPRETATION: Activation of GLP-1 and gastrin signalling induces beta cell neogenesis in the exocrine lineage without any deleterious pancreatic changes, which may lead to a potential therapy to cure diabetes by generating surrogate beta cells.

An siRNA screen in pancreatic beta cells reveals a role for Gpr27 in insulin production.

The prevalence of type 2 diabetes in the United States is projected to double or triple by 2050. We reasoned that the genes that modulate insulin production might be new targets for diabetes therapeutics. Therefore, we developed an siRNA screening system to identify genes important for the activity of the insulin promoter in beta cells. We created a subclone of the MIN6 mouse pancreatic beta cell line that expresses destabilized GFP under the control of a 362 base pair fragment of the human insulin promoter and the mCherry red fluorescent protein under the control of the constitutively active rous sarcoma virus promoter. The ratio of the GFP to mCherry fluorescence of a cell indicates its insulin promoter activity. As G protein coupled receptors (GPCRs) have emerged as novel targets for diabetes therapies, we used this cell line to screen an siRNA library targeting all known mouse GPCRs. We identified several known GPCR regulators of insulin secretion as regulators of the insulin promoter. One of the top positive regulators was Gpr27, an orphan GPCR with no known role in beta cell function. We show that knockdown of Gpr27 reduces endogenous mouse insulin promoter activity and glucose stimulated insulin secretion. Furthermore, we show that Pdx1 is important for Gpr27's effect on the insulin promoter and insulin secretion. Finally, the over-expression of Gpr27 in 293T cells increases inositol phosphate levels, while knockdown of Gpr27 in MIN6 cells reduces inositol phosphate levels, suggesting this orphan GPCR might couple to Gq/11. In summary, we demonstrate a MIN6-based siRNA screening system that allows rapid identification of novel positive and negative regulators of the insulin promoter. Using this system, we identify Gpr27 as a positive regulator of insulin production.

Neurogenin3 inhibits proliferation in endocrine progenitors by inducing Cdkn1a.

During organogenesis, the final size of mature cell populations depends on their rates of differentiation and expansion. Because transient expression of Neurogenin3 (Neurog3) in progenitor cells in the developing pancreas initiates their differentiation to mature islet cells, we examined the role of Neurog3 in cell cycle control during this process. We found that mitotically active pancreatic progenitor cells in mouse embryos exited the cell cycle after the initiation of Neurog3 expression. Transcriptome analysis demonstrated that the Neurog3-expressing cells dramatically up-regulated the mRNA encoding cyclin-dependent kinase inhibitor 1a (Cdkn1a). In Neurog3 null mice, the islet progenitor cells failed to activate Cdkn1a expression and continued to proliferate, showing that their exit from the cell cycle requires Neurog3. Furthermore, induced transgenic expression of Neurog3 in mouse ß-cells in vivo markedly decreased their proliferation, increased Cdkn1a levels, and eventually caused profound hyperglycemia. In contrast, in Cdkn1a null mice, proliferation was incompletely suppressed in the Neurog3-expressing cells. These studies reveal a crucial role for Neurog3 in regulating the cell cycle during the differentiation of islet cells and demonstrate that the subsequent down-regulation of Neurog3 allows the mature islet cell population to expand.

Use of copolymer polylactic and polyglycolic acid resorbable plates in repair of orbital floor fractures.

The fractures of the orbital floor are common after craniofacial trauma. Repair with resorbable plates is a viable reconstructive option; however, there are few reports in the literature. This study describes our experience using copolymer polylactic and polyglycolic acid (PLLA/PGA) orbital reconstruction plates (LactoSorb, Lorenz Surgical, Jacksonville, FL) in 29 cases of the orbital floor fracture repair. We conducted a retrospective review of 29 orbital floor fractures at a single institution repaired through transconjunctival, preseptal dissection using PLLA/PGA plates fashioned to repair the orbital floor defect. Associated fractures included zygomaticomaxillary, LeFort, and nasoethmoid fractures. There were six patients with complications. Four patients had transient diplopia with complete resolution of symptoms within 1 year. One patient had diplopia postoperatively, but was later lost to follow-up. Two patients have had persistent enophthalmos since 1 year. In each of these cases, the floor fracture was coincident with significant panfacial or neurotrauma. We did not encounter any adverse inflammatory reactions to the implant material itself. The study concluded that orbital floor fracture repair with resorbable plates is safe, relatively easy to perform, and in the majority of cases was effective without complications. In the presence of severe orbital trauma, more rigid implant materials may be appropriate.

The Role of Imatinib in Pediatric Type 1 Diabetes.

We report the first case of imatinib use in an adolescent with diabetes and suggest that it impacts the natural course of disease. A 14-year-old male patient presented in diabetic ketoacidosis (DKA) and was diagnosed with presumed autoantibody-negative type 1 diabetes (T1D) as well as myeloid neoplasm with platelet-derived growth factor receptor beta (PDGFRB) rearrangement. After starting exogenous insulin and imatinib, he experienced a 1.7-point reduction in glycated hemoglobin (HbA1c) and a 71% reduction in insulin requirement with sustained partial diabetes remission. Our case suggests imatinib as a potential therapeutic agent for pediatric T1D.

Hydrogen ion (H+) in waste acid as a driver for environmentally sustainable processes: opportunities and challenges.

Acid-base neutralization reaction in the aqueous phase is thermodynamically favorable and kinetically fast. Waste acid neutralization is also the most common waste management practice globally. However, waste acid neutralization is yet to be used for any work/energy generation because of the low concentrations of the waste acid and the high heat capacity of aqueous solutions. In this paper, we address potential processes that can effectively take advantage of the high energy inherent in neutralization reactions, in accordance with the goal of sustainable development.

ABCC8-Related Monogenic Diabetes Presenting Like Type 1 Diabetes in an Adolescent.

Background: Identifying cases of diabetes caused by single gene mutations between the more common type 1 diabetes (T1D) and type 2 diabetes (T2D) is a difficult but important task. We report the diagnosis of ATP-binding cassette transporter sub-family C member 8 (ABCC8)-related monogenic diabetes in a 35-year-old woman with a protective human leukocyte antigen (HLA) allele who was originally diagnosed with T1D at 18 years of age. Case Report: Patient A presented with polyuria, polydipsia, and hypertension at the age of 18 years and was found to have a blood glucose > 500 mg/dL (70-199 mg/dL) and an HbA1C (hemoglobin A1C) >14% (4%-5.6%). She had an unmeasurable C-peptide but no urine ketones. She was diagnosed with T1D and started on insulin therapy. Antibody testing was negative. She required low doses of insulin and later had persistence of low but detectable C-peptide. At the age of 35 years, she was found to have a protective HLA allele, and genetic testing revealed a pathogenic mutation in the ABCC8 gene. The patient was then successfully transitioned to sulfonylurea therapy. Discussion: Monogenic diabetes diagnosed in adolescence typically presents with mild to moderate hyperglycemia, positive family history and, in some cases, other organ findings or dysfunction. The patient in this report presented with very high blood glucose, prompting the diagnosis of T1D. When she was found to have a protective HLA allele, further investigation revealed the mutation in the sulfonylurea receptor gene, ABCC8. Conclusion: Patients suspected of having T1D but with atypical clinical characteristics such as negative autoantibodies, low insulin requirements, and persistence of C-peptide should undergo genetic testing for monogenic diabetes.

A genomic data archive from the Network for Pancreatic Organ donors with Diabetes.

The Network for Pancreatic Organ donors with Diabetes (nPOD) is the largest biorepository of human pancreata and associated immune organs from donors with type 1 diabetes (T1D), maturity-onset diabetes of the young (MODY), cystic fibrosis-related diabetes (CFRD), type 2 diabetes (T2D), gestational diabetes, islet autoantibody positivity (AAb+), and without diabetes. nPOD recovers, processes, analyzes, and distributes high-quality biospecimens, collected using optimized standard operating procedures, and associated de-identified data/metadata to researchers around the world. Herein describes the release of high-parameter genotyping data from this collection. 372 donors were genotyped using a custom precision medicine single nucleotide polymorphism (SNP) microarray. Data were technically validated using published algorithms to evaluate donor relatedness, ancestry, imputed HLA, and T1D genetic risk score. Additionally, 207 donors were assessed for rare known and novel coding region variants via whole exome sequencing (WES). These data are publicly-available to enable genotype-specific sample requests and the study of novel genotype:phenotype associations, aiding in the mission of nPOD to enhance understanding of diabetes pathogenesis to promote the development of novel therapies.