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Alternative splicing (AS) within the β-cell has been proposed as one potential pathway that may exacerbate autoimmunity and unveil novel immunogenic epitopes in type 1 diabetes (T1D). We used a computational strategy to prioritize pathogenic splicing events in human islets treated with interleukin-1β plus interferon-γ as an ex vivo model of T1D and coupled this analysis with a k-mer–based approach to predict RNA-binding proteins involved in AS. In total, 969 AS events were identified in cytokine-treated islets, with a majority (44.8%) involving a skipped exon. ExonImpact identified 129 events predicted to affect protein structure. AS occurred with high frequency in MHC class II–related mRNAs, and targeted quantitative PCR validated reduced inclusion of exon 5 in the MHC class II gene HLA-DMB. Single-molecule RNA fluorescence in situ hybridization confirmed increased HLA-DMB splicing in β-cells from human donors with established T1D and autoantibody positivity. Serine/arginine-rich splicing factor 2 was implicated in 37.2% of potentially pathogenic events, including exon 5 exclusion in HLA-DMB. Together, these data suggest that dynamic control of AS plays a role in the β-cell response to inflammatory signals during T1D evolution.

SummaryLoss of pancreatic β cell mass, identity, and function contribute to the development of diabetes. Here, we show that the endoplasmic reticulum (ER) calcium sensor, stromal interaction molecule 1 (STIM1), is critical for the maintenance of β cell function in female mice. When mice with β cell-specific deletion of STIM1 (STIM1Δβ) were challenged with high-fat diet, β cell dysfunction was observed in female, but not male, mice. Impaired glucose tolerance was accompanied by reductions in β cell mass, a concomitant increase in α cell mass, and significant reductions in the expression of markers of β cell maturity, including MafA and UCN3. Mechanistic assays demonstrated that the sexually dimorphic phenotype observed in STIM1Δβ mice was due in part to loss of signaling through the noncanonical 17-β estradiol receptor, GPER1. Together, these data suggest that STIM1 orchestrates pancreatic β cell function and identity through GPER1-mediated estradiol signaling.

Kimura’s disease (KD) is a rare lymphoproliferative fibroinflammatory disorder that commonly affects the subcutaneous tissue and lymph nodes of the head and neck. The condition is a reactive process involving T helper type 2 cytokines. Concurrent malignancies have not been described. Differential diagnosis with lymphoma can be challenging without tissue biopsy. Here, we present the first reported case of coexisting KD and eosinophilic nodular sclerosis Hodgkin lymphoma of the right cervical lymphatics in a 72-year-old Taiwanese man.

Utilization of proper preclinical models accelerates development of immunotherapeutics and the study of the interplay between human malignant cells and immune cells. Lysosomal acid lipase (LAL) is a critical lipid hydrolase that generates free fatty acids and cholesterol. Ablation of LAL suppresses immune rejection and allows growth of human lung cancer cells in lal(−/−) mice. In the lal(−/−) lymph nodes, the percentages of both T- and B-regulatory cells (Tregs and Bregs, respectively) are increased, with elevated expression of programmed death-ligand 1 and IL-10, and decreased expression of interferon-γ. Levels of enzymes in the glucose and glutamine metabolic pathways are elevated in Tregs and Bregs of the lal(−/−) lymph nodes. Pharmacologic inhibitor of pyruvate dehydrogenase, which controls the transition from glycolysis to the citric acid cycle, effectively reduces Treg and Breg elevation in the lal(−/−) lymph nodes. Blocking the mammalian target of rapamycin or reactivating peroxisome proliferator-activated receptor γ, an LAL downstream effector, reduces lal(−/−) Treg and Breg elevation and PD-L1 expression in lal(−/−) Tregs and Bregs, and improves human cancer cell rejection. Treatment with PD-L1 antibody also reduces Treg and Breg elevation in the lal(−/−) lymph nodes and improves human cancer cell rejection. These observations conclude that LAL-regulated lipid metabolism is essential to maintain antitumor immunity.

Increased circulating levels of incompletely processed insulin (i.e. proinsulin) are observed clinically in both type 1 and type 2 diabetes; however, the mechanisms underlying impaired proinsulin processing remain incompletely understood. Here, we identify the sarcoendoplasmic reticulum Ca2+ ATPase-2 (SERCA2) pump and β cell ER Ca2+ as key regulators of systemic glucose tolerance and proinsulin processing. We generated mice with a β cell-specific SERCA2 deletion (βS2KO) and SERCA2 deficient INS-1 cells to show that SERCA2 loss increases systemic and pancreatic levels of proinsulin protein and leads to aberrant localization of proinsulin within the proximal β cell secretory pathway. These defects in proinsulin processing were linked to reduced maturation of the proinsulin processing enzymes PC1/3 and PC2, suggesting a model whereby chronic ER Ca2+ depletion in the β cell, which is observed in many pathological conditions, impairs the spatial regulation of prohormone trafficking, processing, and maturation within the β cell secretory pathway.

Kimura disease (KD) is a rare, chronic proliferative condition presenting as a subcutaneous mass predominantly located in the head and neck region; it is characterized by eosinophilia and elevated serum IgE levels. IgG4-related disease (IgG4RD) is a fibroinflammatory condition characterized by swelling in single or multiple organs and the infiltration of IgG4 plasma cells. Herein, we presented two cases. Case 1 is a 38-year-old man with a painless mass in his right postauricular region, and Case 2 is a 36-year-old man with painless lymphadenopathy in his bilateral postauricular region. After surgical excision, they showed good recovery with no relapse. Although Cases 1 and 2 shared several overlapping pathological manifestations, there were a few differences that allowed the differentiation of KD and IgG4RD.

Neuropsychiatric systemic lupus erythematous (NPSLE) encompasses various psychiatric and neurological manifestations that develop in patients with systemic lupus erythematous (SLE), secondary to the involvement of the central nervous system (CNS). Although neuropsychiatric manifestations are commonly described in NPSLE, catatonia has been less frequently reported in patients with SLE. The roles of benzodiazepines (BZDs), immunosuppression, therapeutic plasma exchange (TPE), and electroconvulsive therapy (ECT) have all been reported in the management of catatonia. Furthermore, another research reported that catatonic symptoms associated with NPSLE were considerably improved by TPE. We, herein, report a case of catatonia in a patient with newly diagnosed NPSLE who exhibited a favorable prognosis through the early initiation of systemic immunosuppressants and TPE. Furthermore, we have reviewed the literature on the role of medication and plasmapheresis (PP), or TPE, in the treatment of catatonia that is associated with SLE.

Avian species have immune system to fight invading pathogens. The immune system comprises innate and adaptive immunity. Innate immunity relies on pattern recognition receptors to sense particular molecules present in pathogens, i.e. pathogen-associated molecular patterns (PAMPs), or danger signals in the environment, i.e. danger-associated molecular patterns (DAMPs). Cytoplasmic retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) and nucleotide-binding oligomerization domain-like receptors (NLRs) are the sensors recognizing cytoplasmic PAMP and/or DAMP. Among common avian species, chickens do not have RIG-I whereas ducks and finches do. Therefore, the other RLR member, melanoma differentiation-associated gene 5 (MDA5), is believed to play an important role to recognize intracellular pathogens in chickens. Chicken MDA5 has been identified and its function determined. Chicken MDA5 maintains the same domain architecture compared with MDA5 analogs in other animal species. The expression of chicken MDA5 was upregulated when a synthetic double-stranded RNA (dsRNA), polyriboinosinic:polyribocytidylic acids (poly(I:C)), was transfected into chicken cells, whereas that did not change when cells were incubated with poly(I:C). The enhanced expression of chicken MDA5 in chicken cells upregulated the expression of chicken interferon-[Formula: see text] (IFN-[Formula: see text]). The infection of dsRNA infectious bursal disease virus (IBDV) in non-immune cells triggered the activation of chicken MDA5 signaling pathway, leading to the production of IFN-[Formula: see text] and subsequent response of IFN-stimulated genes. Furthermore, in immune cells like macrophages, chicken MDA5 participated in sensing the infection of IBDV by activating downstream antiviral genes and molecules and modulating adaptive immunity.On the contrary, one of cytoplasmic NLR member, NLR family pyrin domain containing 3 (NLRP3), was cloned and functionally characterized in chicken cells. Chicken NLRP3 conserved the same domain architecture compared with NLRP3 analogs in other animal species. Chicken NLRP3 was highly expressed in kidney, bursa of Fabricius and spleen. The production of mature chicken interleukin 1 [Formula: see text] (IL-1[Formula: see text] in chicken macrophages was stimulated by lipopolysaccharide (LPS) treatment followed by short ATP exposure.In summary, chicken MDA5 was a cytoplasmic dsRNA sensor that mediated the production of type I IFN upon ligand engagement, whereas NLRP3 sensed danger signals, such as ATP, in the cytoplasm and cleaved pro-IL-1[Formula: see text] to produce mature IL-1[Formula: see text]. Chicken MDA5 was not only involved in the activation of innate immune responses in non-immune and immune cells, but it also participated in modulating adaptive immunity in immune cells. Chicken NLRP3 participated in the production of mature chicken IL-1[Formula: see text] upon ligand engagement.

STIM1 is an endoplasmic reticulum (ER) calcium (Ca2+) sensor that replenishes ER Ca2+ stores in a process known as store operated calcium entry (SOCE). Previously, we have shown that SOCE is impaired in the diabetic pancreatic β cell; however, the consequences of reduced β cell SOCE have not been well studied. To this end, mice with pancreatic β cell specific deletion of STIM1 (STIM1Δβ) were generated by crossing STIM1flox/flox with Ins-Cre mice and fed a high fat diet (HFD) beginning at 8 wk of age. Male STIM1Δβ mice did not show changes in glucose tolerance or body composition. In contrast, female HFD-fed STIM1Δβ mice were heavier (25.1 vs. 28.3 g, p Disclosure P. Sohn: None. P. Krishnan: None. C. Lee: None. T. Kono: None. C. Evans-molina: Advisory Panel; Self; Provention Bio, Inc., Consultant; Self; Dompe, Other Relationship; Self; Bristol-Myers Squibb Company, Nimbus Pharmaceuticals, Pfizer Inc. Funding National Institutes of Health (5T32DK064466-17, 5F30DK123996); U.S. Department of Veterans Affairs (2-I01BX001733); National Institute of Diabetes and Digestive and Kidney Diseases (1R01DK093954-06A1); Indiana University School of Medicine; Gates Millennium Scholars Program

Store-operated calcium entry (SOCE) functions to refill endoplasmic reticulum (ER) Ca2+ stores in response to ER Ca2+ depletion through gating of plasmalemmal Orai channels by the ER Ca2+ sensor STIM1. Dysfunctional β cell SOCE has been observed in response to cytokine-mediated stress, whereas select histone deacetylase (HDAC) inhibitors are able to protect against cytokine-mediated β cell death and have been shown to improve glucose homeostasis in diabetic mice. Here, we tested the relationship between HDAC inhibition and SOCE in the pancreatic β cell. To this end, we investigated the effects of the HDAC inhibitor sodium butyrate (NaB) on Ca2+ signaling, insulin secretion, and gene expression in cytokine-treated mouse islets, human islets, and STIM1 knockout (KO) 832/13 INS-1 β cells. In mouse islets treated with IL-1β, IFN-γ and TNF-⍺, NaB increased glucose-induced phase Ca2+ responses and restored Ca2+ oscillations. In IL-1β-treated INS-1 β cells, NaB rescued SOCE and ER Ca2+ store depletion, and increased glucose-stimulated insulin secretion (GSIS), while additionally improving cell viability and reducing apoptosis. Chronic (24hrs) but not acute (8hrs) NaB treatment prevented IL-1β-mediated reduction in the SOCE response, suggesting changes in gene expression may underlie these effects. To test this, RT-qPCR for SOCE molecular components was performed and revealed increased STIM1 expression in cytokine-treated mouse islets and INS-1 cells, while no change was observed in Orai1/2 levels. Consistent with these results, NaB was unable to restore SOCE in IL-1β-treated STIM1 KO INS-1 cells. Lastly, NaB treatment in human islets from donors with type 2 diabetes significantly increased levels of GSIS. Taken together, these data suggest that HDAC inhibition restores insulin secretion under pro-inflammatory conditions through improved SOCE and STIM1 upregulation. Disclosure C. Lee: None. T. Kono: None. S. A. Weaver: None. P. Sohn: None. C. Evans-molina: Advisory Panel; Self; Provention Bio, Inc., Consultant; Self; Dompe, Other Relationship; Self; Bristol-Myers Squibb Company, Nimbus Pharmaceuticals, Pfizer Inc.

MicroRNAs (miRNAs) are small non-coding RNAs that function as modulators of gene expression. We previously identified upregulation of miR-146a-5p in pancreatic islets treated with proinflammatory cytokines and in human pancreatic sections from individuals with type 1 diabetes. While other studies have associated overexpression of miR-146a-5p with β cell apoptosis and defective insulin secretion, the molecular mechanisms through which these effects are mediated remain elusive. To this end, we transduced MIN6 cells with lentiviral vectors (i) overexpressing and (ii) inhibiting the expression of miR-146a-5p and developed stable cell lines resistant to puromycin. Monoclonal cell populations were developed by single clone selection and then treated with/without proinflammatory cytokines (IL-1β, IFN-γ and TNF-α) for 24 hours. Further characterization of the cells was performed using mRNA sequencing, western blot, RT-PCR of cognate targets and Seahorse mitochondrial functional assays. Overexpression of miR-146a-5p led to increased expression of cleaved caspase-3 and reduced insulin secretion under basal and cytokine-treated conditions, whereas inhibition of miR-146a-5p reversed these effects. Similarly, miR-146a-5p inhibition increased mitochondrial copy number, mitochondrial membrane potential, basal respiration rate, maximal respiration rate, spare respiratory capacity, and ATP production, suggesting a protective effect on the mitochondria, even upon exposure to proinflammatory cytokines. Consistent with these findings, bioinformatic analysis of sequencing data and validation of targets such as MAPT, MYRIP, NRG1, GPX1 also showed enrichment of pathways related to insulin secretion, apoptosis, mitochondrial function. Overall, findings from our study show for the first time that miR-146a-5p upregulation may alter β cell health through reduced mitochondrial function. Disclosure P. Krishnan: None. F. Syed: None. S. A. Weaver: None. C. Lee: None. C. Evans-molina: Advisory Panel; Self; Provention Bio, Inc., Consultant; Self; Dompe, Other Relationship; Self; Bristol-Myers Squibb Company, Nimbus Pharmaceuticals, Pfizer Inc.

Impaired proinsulin processing is observed in both type 1 and type 2 diabetes. We have previously shown that reductions in endoplasmic reticulum (ER) calcium (Ca2+) in the pancreatic β cell arising from impaired activity of the Sarcoendoplasmic Reticulum Ca2+ ATPase (SERCA) pump are associated with increased proinsulin secretion. However, the mechanisms responsible for reduced proinsulin processing in the context of SERCA2 deficiency remain incompletely understood. To test this, we developed mice with β cell specific SERCA2 deletion (βS2KO mice) and S2KO INS1 cells. βS2KO mice exhibited age-dependent glucose intolerance and reduced glucose-stimulated insulin secretion without evidence of impaired insulin sensitivity. ER Ca2+ levels in islets from βS2KO mice were significantly reduced, while serum proinsulin/insulin (PI/I) ratios and whole pancreas PI/I content were elevated. Immunoblot analysis of βS2KO islets and S2KO INS-1 cells revealed reduced active forms of the proinsulin processing enzymes, PC1/3, PC2 and CPE. Restoration of SERCA2b via adenoviral transduction in S2KO INS1 cells was sufficient to restore PC1/3 and PC2 maturation and enzyme activity. Brefeldin A treatment in INS1 cells recapitulated the impairments in PC1/3 and PC2 maturation observed in S2KO cells, suggesting a disturbance in protein trafficking between the ER and Golgi. Consistent with this, trafficking assays were performed using a vesicular stomatitis virus G (VSVG) protein construct and revealed a significantly slower rate of VSVG movement from the ER to the Golgi in S2KO INS1 cells. Moreover, pancreas sections from βS2KO mice showed increased co-localization of proinsulin and ProPC2 in the early compartments of the secretory pathway. Taken together, these data suggest that loss of SERCA2 activity and ER Ca2+ loss in the pancreatic β cell leads to impaired proinsulin processing via reduced maturation and trafficking of proinsulin processing enzymes. Disclosure T. Kono: None. H. Iida: None. T.S. Jarvela: None. C. Lee: None. X. tong: None. P. Arvan: None. I. Lindberg: None. C. Evans-Molina: Consultant; Self; Bristol-Myers Squibb. Funding National Institutes of Health (R01DK093954); U.S. Department of Veterans Affairs (2-I01BX001733)

Alternative splicing (AS) is a tightly regulated mechanism whereby pre-mRNA transcripts are processed to generate structurally distinct mRNA and protein isoforms. AS within the β cell has been proposed as a potential mechanism that may exacerbate autoimmunity and unmask novel immunogenic epitopes in type 1 diabetes (T1D). Here, we employed a computational strategy to catalog AS events in human islets treated with IL-1β + IFN-γ, as an ex vivo model of T1D. In total, 970 events were identified in cytokine-treated islets, with the majority (44.8%) involving a skipped exon. To identify potentially pathogenic AS events, we utilized ExonImpact, which is a validated machine learning based method, that derives 31 curated features of protein structure from RNA sequencing results. ExonImpact identified 129 AS events predicted to impact protein structure; differentially spliced mRNAs were enriched in gene ontology pathways that included “actin filament-based movement” and “post-Golgi vesicle-mediated transport.” Whereas variants in MHC Class II genes are strongly associated with T1D risk, expression of Class II genes in the β-cell has been controversial. Notably, we identified 98 AS events in MHC Class II genes. Targeted qPCR was used to validate reduced inclusion of Exon5 in HLA-DMB, which is a Class II gene involved in shaping the immunopeptidome. RNA-FISH demonstrated increased abundance of the splice variant in pancreatic sections from human T1D donors. Finally, a k-mer based approach was used to identify RNA binding proteins associated with skipped-exon events. Serine and Arginine Rich Splicing Factor 2 exhibited increased expression in response to IL-1β + IFN-γ and was implicated in 37.2% of potentially pathogenic events, including HLA-DMB Exon5 exclusion. Together, these findings suggest that stimulus specific isoform expression may control how β-cells respond to inflammatory signals in T1D and potentially contribute to amplification of the autoimmune attack against the β-cells. Disclosure W. Wu: None. F. Syed: None. E. Simpson: None. C. Lee: None. D.L. Eizirik: None. R. Mirmira: Advisory Panel; Self; Hibercell, Sigilon Therapeutics, Veralox Therapeutics. Employee; Spouse/Partner; Eli Lilly and Company. Y. Liu: None. C. Evans-Molina: Consultant; Self; Bristol-Myers Squibb. Funding National Institute of Diabetes and Digestive and Kidney Diseases (R01DK093954, UC4-DK-104166); U.S. Department of Veterans Affairs (I01BX001733); Sigma Beta Sorority; Ball Brothers Foundation; George and Frances Ball Foundation; Indiana Diabetes Research Center (P30DK097512)

Store-operated calcium entry (SOCE) is a mechanism that functions to refill endoplasmic reticulum (ER) Ca2+ stores in response to ER Ca2+ depletion, through gating of plasmallemal Ora channels by the ER Ca2+ sensor STIM1. Dysfunctional β cell SOCE has been observed in response to cytokine-mediated stress and in models of diabetes. To identify compounds capable of modulating SOCE, a screening was performed in INS-1 cells treated with interleukin-1β (IL-1β). Two HDAC inhibitors (HDACi), sodium butyrate (NaB) and MS275, rescued SOCE in a dose-dependent manner in IL-1β-treated INS-1 cells. While dysregulation of HDACs has been linked with metabolic disease, the relationship between HDACs and SOCE has not been tested in the pancreatic β cell. To this end, we explored the effect of HDACi on Ca2+ signaling, insulin secretion, and gene expression in cytokine-treated islets and β cells. In mouse islets treated with IL-1 β and interferon-𝛾;, NaB and MS275 increased first phase Ca2+ responses in response to glucose, while NaB restored glucose-induced Ca2+ oscillations. Consistent with this, NaB, but not MS275, restored GSIS in IL-1β-treated INS-1 cells and in human islets from donors with type 2 diabetes. Chronic (24 hrs) but not acute (8 hrs) HDACi treatment restored cytokine-mediated reductions in SOCE, raising the possibility that changes in gene expression may underlie these effects. To test this, qRT-PCR for SOCE molecular components was performed. Both NaB and MS275 restored STIM1 expression but had no effect on Orai1, Orai2 and Orai3 levels. Consistent with this, HDACi were unable to restore SOCE in IL-1β-treated STIM1 knockout INS-1 cells. Taken together, these data suggest that HDAC inhibition restores insulin secretion under pro-inflammatory conditions through improved SOCE and upregulated STIM1 expression. Thus, our findings identify a novel pathway through which HDAC inhibition exerts protective effects in the diabetic β cell. Disclosure C. Lee: None. T. Kono: None. S.A. Weaver: None. P. Sohn: None. C. Evans-Molina: Consultant; Self; Bristol-Myers Squibb. Funding National Institute of Diabetes and Digestive and Kidney Diseases (2R01DK093954)

Store-operated Ca2+ entry (SOCE) is a dynamic process that leads to refilling of endoplasmic reticulum (ER) Ca2+ stores through reversible gating of plasma membrane Ca2+ channels by the ER Ca2+ sensor Stromal Interaction Molecule 1 (STIM1). Pathogenic reductions in β-cell ER Ca2+ have been observed in diabetes. However, a role for impaired SOCE in this phenotype has not been tested. We measured the expression of SOCE molecular components in human and rodent models of diabetes and found a specific reduction in STIM1 mRNA and protein levels in human islets from donors with type 2 diabetes (T2D), islets from hyperglycemic streptozotocin-treated mice, and INS-1 cells (rat insulinoma cells) treated with proinflammatory cytokines and palmitate. Pharmacologic SOCE inhibitors led to impaired islet Ca2+ oscillations and insulin secretion, and these effects were phenocopied by β-cell STIM1 deletion. STIM1 deletion also led to reduced ER Ca2+ storage and increased ER stress, whereas STIM1 gain of function rescued β-cell survival under proinflammatory conditions and improved insulin secretion in human islets from donors with T2D. Taken together, these data suggest that the loss of STIM1 and impaired SOCE contribute to ER Ca2+ dyshomeostasis under diabetic conditions, whereas efforts to restore SOCE-mediated Ca2+ transients may have the potential to improve β-cell health and function.

ObjectivesEpidemiological studies indicate that first- and second-hand cigarette smoke (CS) exposure are important risk factors for the development of type 2 diabetes (T2D). Additionally, elevated diabetes risk has been reported to occur within a short period of time after smoking cessation, and health risks associated with smoking are increased when combined with obesity. At present, the mechanisms underlying these associations remain incompletely understood. The objective of this study was to test the impact of CS exposure on pancreatic β-cell function using rodent and in vitro models.MethodsBeginning at 8 weeks of age, C57BL/6J mice were concurrently fed high fat-diet (HFD) and exposed to CS for 11 weeks, followed by an additional 11 weeks of smoking cessation with continued HFD exposure. Glucose tolerance testing was performed during CS exposure and during the cessation period. Cultured β-cells (INS-1) and primary islets were exposed ex vivo to CS extract (CSE), and β-cell function and viability were tested. Since CS increases ceramide in lungs cells and these bioactive sphingolipids have been implicated in pancreatic β-cell dysfunction in diabetes, islet and β-cell sphingolipid levels were measured in islets from CS-exposed mice and in CSE-treated islets and INS-1 cells using liquid chromatography-tandem mass spectrometry.ResultsCompared to HFD-fed ambient air-exposed mice, HFD-fed and CS- exposed mice had reduced weight gain and better glucose tolerance during the active smoking period. Following smoking cessation, CS-mice exhibited rapid weight gain and a significantly greater increase in glucose intolerance compared to non-smoking control mice. CS-exposed mice had higher serum proinsulin/insulin ratios, indicative of β-cell dysfunction, significantly lower β-cell mass (p=0.02), and reduced β-cell proliferation (p=0.006), and increased islet ceramide accumulation. Ex vivo exposure of isolated islets to CSE was sufficient to increase islet ceramide accumulation, reduce insulin gene expression and glucose-stimulated insulin secretion, and increase β-cell oxidative and ER stress. Treatment with the antioxidant N-acetylcysteine, markedly attenuated the effects of CSE on ceramide levels, restored β-cell function and survival, and increased cyclin D2 expression, while also reducing activation of β-cell ER and oxidative stress.ConclusionsOur results indicate that CS exposure inhibits insulin production, processing, and secretion and reduced β-cell viability and proliferation. These effects were linked to increased β-cell oxidative and ER stress and ceramide accumulation. Mice fed HFD continued to experience detrimental effects of CS exposure even during smoking cessation. Elucidation of mechanisms by which CS exposure impairs β-cell function in synergy with obesity will help design therapeutic and preventive interventions for both active and former smokers.

Background and Hypothesis: In diabetic mice, intermittent fasting (IF) prevents diabetic complications by temporarily reducing inflammation during the fasting period. As calcium homeostasis is tightly connected to the inflammatory response, we hypothesize that IF regulates macrophage response by altering store-operated calcium entry (SOCE). SOCE, mediated by the STIM and ORAI families, is the primary form of calcium entry into the cell. Experimental Design or Project Methods: We assessed SOCE levels in control and diabetic (INS2Akita) mouse primary macrophages stimulated with a metabolic stimulus (MET: insulin, high glucose, palmitate) to mimic feeding and a starvation stimulus (STARVE: low glucose, low FBS, oleate) to mimic fasting. SOCE levels were measured by a Flexstation using Calcium-6 dye. We also assessed gene expression of SOCE components by RT-PCR and STIM1 and ORAI1 proteins by western blot. Results: A decrease in SOCE was observed with MET stimuli and an increase with STARVE stimuli in control macrophages. Interestingly, gene expression and protein levels of all major SOCE components, including STIM1 and ORAI1, were increased in the MET and decreased with STARVE. While diabetic macrophages had similar SOCE function than control under basal and MET conditions, they showed significantly downregulated SOCE under starvation conditions. Conclusion and Potential Impact: These data show that diabetic macrophages have altered SOCE function in response to fasting. This could have potential impact on how diabetic individuals respond to nutritional interventions such as IF that are recently proposed for prevention of diabetic complications.

Early detection of lung cancer offers an important opportunity to decrease mortality while it is still treatable and curable. Thirteen secretory proteins that are Stat3 downstream gene products were identified as a panel of biomarkers for lung cancer detection in human sera. This panel of biomarkers potentially differentiates different types of lung cancer for classification. Among them, the transthyretin (TTR) concentration was highly increased in human serum of lung cancer patients. TTR concentration was also induced in the serum, bronchoalveolar lavage fluid, alveolar type II epithelial cells, and alveolar myeloid cells of the CCSP-rtTA/(tetO)7-Stat3C lung tumor mouse model. Recombinant TTR stimulated lung tumor cell proliferation and growth, which were mediated by activation of mitogenic and oncogenic molecules. TTR possesses cytokine functions to stimulate myeloid cell differentiation, which are known to play roles in tumor environment. Further analyses showed that TTR treatment enhanced the reactive oxygen species production in myeloid cells and enabled them to become functional myeloid-derived suppressive cells. TTR demonstrated a great influence on a wide spectrum of endothelial cell functions to control tumor and immune cell migration and infiltration. TTR-treated endothelial cells suppressed T cell proliferation. Taken together, these 13 Stat3 downstream inducible secretory protein biomarkers potentially can be used for lung cancer diagnosis, classification, and as clinical targets for lung cancer personalized treatment if their expression levels are increased in a given lung cancer patient in the blood.

The objective of the present study was to determine if chicken melanoma-differentiation-associated gene 5 (MDA5) senses infectious bursal disease virus infection to induce innate immunity that bridges to adaptive immunity. During IBDV infection in HD11 cells, IBDV titers and RNA loads increased up to 3.4 × 10(7) plaque-forming units (PFU)/mL and 1114 ng/µL, respectively, at 24 hours postinfection (hpi). IBDV infection in HD11 cells induced significantly upregulated (p < 0.05) expression levels of chicken MDA5 (59-fold), interferon-β (IFN-β) (693-fold), dsRNA-dependent protein kinase (PKR) (4-fold), 2', 5'-oligoadenylate synthetase (OAS) (286-fold), myxovirus resistance gene (Mx) (22-fold), interleukin-1β (IL-1β) (5-fold), IL-6 (146-fold), IL-8 (4-fold), IL-10 (4-fold), inducible nitric oxide synthase (iNOS) (15-fold), and major histocompatibility complex class I (MHC class I) (4-fold). Nitric oxide production in the culture supernatants increased significantly (p < 0.05) up to 6.5 μM at 24 hpi. The expressed chMDA5 and IBDV-derived dsRNA were localized in the cytoplasm of HD11 cells during IBDV infection. ChMDA5-knockdown HD11 cells had significantly higher (p < 0.05) IBDV RNA loads at 24 hpi and significantly lower (p < 0.05) nitric oxide production and expression levels of chicken MDA5, IFN-β, PKR, OAS, Mx, IL-1β, IL-6, IL-8, IL-12(p40), IL-18, IL-10, iNOS, MHC class I and CD86 at 24 hpi. In addition, chMDA5 overexpression in HD11 cells resulted in significantly reduced (p < 0.05) IBDV titers and RNA loads and significantly increased (p < 0.05) nitric oxide production at 16 and 24 hpi. It also resulted in significantly higher (p < 0.05) expression levels of chicken MDA5, IFN-β, PKR, OAS, Mx, IL-1β, IL-6, IL-8, IL-12(p40), IL-10 and iNOS at 2 hpi. In conclusion, the results indicate that chMDA5 senses IBDV infection in chicken macrophages, and this is associated with IBDV-induced expression of IFN-β and initiation of an innate immune response that in turn activates the adaptive immune response and limits IBDV replication.

Krüppel-like factor 4 (KLF4) plays important roles in development, stemness and tumorigenesis; however limited information is available on the detailed function of KLF4 in hepatocellular carcinoma (HCC). The objective of the present study was to examine the functional roles of KLF4 in the metastasis of HCC cells. KLF4 was overexpressed and knocked down by lentiviral transduction method in highly metastatic HCC cells. KLF4 overexpression in HCC cells led to inhibition of cell migration and invasion. These inhibitory effects were associated with the upregulation of tissue inhibitors of metalloproteinase (TIMP)-1 and TIMP-2 by KLF4. Treatment with recombinant TIMP-1 decreased the migratory ability of HCC cells. Moreover, myeloperoxidase (MPO)-TIMP-1/TIMP-2 inactivator counteracted the KLF4-induced inhibition of cell migration/invasion. Consistently, KLF4 knockdown in HCC cells downregulated TIMP-1 and TIMP-2 expression, consequently promoting cell migration and invasion. Furthermore, we found that KLF4 regulated E-cadherin and epithelial-mesenchymal transition (EMT)-related proteins such as snail, vimentin and Bmi1 to modulate the cell migration ability. These results together demonstrated for the first time that KLF4 plays an important role in inhibiting the aggressiveness of HCC cells via upregulation of TIMP-1 and TIMP-2.

The objective of the present study was to determine if chicken melanoma differentiation-associated gene 5 (MDA5) senses infectious bursal disease virus (IBDV) infection to initiate and amplify an innate immune response in the chicken MDA5 (chMDA5) signaling pathway. Chicken embryo fibroblast DF-1 cells were infected with IBDV LP1 at a multiplicity of infection (MOI) of 0.5 or 10. In addition, knockdown and overexpression of chMDA5 were performed by transfecting DF-1 cells with chMDA5-targeting small interfering RNA (siRNA) or chMDA5-expressing DNA. The transfected cells were infected with IBDV LP1 at an MOI of 10. Cell culture supernatants and lysates were collected at 2, 8, 16 and 24 hours postinfection (hpi) for IBDV titer determination and RNA extraction, respectively. IBDV RNA loads and mRNA expression levels of chicken MDA5, interferon-β (IFN-β) promoter stimulator 1 (IPS-1), interferon regulatory factor-3 (IRF-3), IFN-β, double-stranded RNA-dependent protein kinase (PKR), 2′,5′-oligoadenylate synthetase (OAS), myxovirus resistance gene (Mx), and major histocompatibility complex class I (MHC class I) were determined by real-time RT-PCR. The IBDV titer increased up to 1.4 × 107 plaque-forming units (PFU)/mL at 24 hpi, and the IBDV RNA load reached 464 ng/μL at 24 hpi. The mRNA expression levels of chicken MDA5, IRF-3, IFN-β, PKR, OAS, Mx and MHC class I in IBDV-infected DF-1 cells exhibited significant (p

Peroxisome proliferator-activated receptor γ (PPARγ) is a ligand-activated nuclear receptor that regulates cellular lipid and glucose metabolism and also plays an inhibitory role in various cancers. However, the role of PPARγ in hepatocellular carcinoma (HCC) remains controversial. This study aimed to investigate the prognostic value of PPARγ in HCC and its role in inhibiting tumor progression, namely, HCC cell growth, migration, and angiogenesis. Immunohistochemical PPARγ staining was examined in 83 HCC specimens to investigate the clinicopathological correlations between PPARγ expression and various parameters. The functional role of PPARγ was determined via PPARγ overexpression and knockdown in HCC cells. Patients with low HCC tissue PPARγ expression were significantly younger (p = 0.006), and exhibited more tumor numbers (p = 0.038), more macroscopic vascular invasion (MVI) (p = 0.008), and more advanced TNM (size of primary tumor, number of regional lymph nodes, and distant metastasis) stages at diagnosis (p = 0.013) than patients with high HCC tissue PPARγ expression. PPARγ knockdown increased HCC cell growth, migration, and angiogenesis, while PPARγ overexpression reduced HCC cell growth, migration, and angiogenesis. These results suggest that low PPARγ expression is an independent predictor of more MVI in HCC patients. PPARγ contributes to the suppression of HCC cell growth, migration, and angiogenesis. Therefore, PPARγ may be a therapeutic target in HCC patients.

Functional dependency is concerned with rewriting a Boolean function f as a function h over a set of base functions {g1,?,gn}, i.e., f = h(g1,?,gn). It plays an important role in many aspects of electronic design automation (EDA). Prior approaches to the exploration of functional dependency are based on binary decision diagrams (BDDs), which may not be easily scalable to large designs. This paper formulates both single-output and multiple-output functional dependencies as satisfiability (SAT) solving and exploits extensively the capability of a modern SAT solver. Thereby, functional dependency can be detected effectively through incremental SAT solving, and the dependency function h, if it exists, is obtained through Craig interpolation. The proposed method enables (1) scalable detection of functional dependency, (2) fast enumeration of dependency function under a large set of candidate base functions, and (3) potential application to large-scale logic synthesis and formal verification. Experimental results show that the proposed method is far superior to prior work and scales well in dealing with the largest ISCAS and ITC benchmark circuits with up to 200 K gates.

Although Escherichia coli does not have a natural transformation process, strains of E. coli can incorporate extracellular plasmids into cytoplasm 'naturally' at low frequencies. A standard method was developed in which stationary phase cells were concentrated, mixed with plasmids, and then plated on agar plates with nutrients which allowed cells to grow. Transformed cells could then be selected by harvesting cells and plating again on selective agar plates. Competence developed in the lag phase, but disappeared during exponential growth. As more plasmids were added to the cell suspension, the number of transformants increased, eventually reaching a plateau. Supercoiled monomeric or linear concatemeric DNA could transform cells, while linear monomeric DNA could not. Plasmid transformation was not related to conjugation and was recA-independent. Most of the E. coli strains surveyed had this process. All tested plasmids, except pACYC184, could transform E. coli. Insertion of a DNA fragment containing the ampicillin resistance gene into pACYC184 made the plasmid transformable. By inserting random 20-base-pair oligonucleotides into pACYC184 and selecting for transformable plasmids, a most frequent sequence was identified. This sequence resembled the bacterial interspersed medium repetitive sequence of E. coli, suggesting the existence of a recognition sequence. We conclude that plasmid natural transformation exists in E. coli.

Cigarette butts (CBs) littering is not just an unsightly nuisance but also a public health problem, because chemicals contained in cigarettes can leach into aquatic environments and pose a risk to the health of humans and wildlife. However, this risk is largely unrecognized or ignored by the public, and toxicological evidence of CBs is scarce. Therefore, we used medaka embryos (Oryzias latipes) to explore developmental toxicity of CBs. The embryos were exposed to various concentrations of leachates from smoked and unsmoked cigarette tobacco (ST and UST) and filters (SF and USF), and observed from 1 to 3 days post-fertilization. The images were recorded and several developmental endpoints analyzed. The values from these endpoints were then used to calculate the Integrated Biomarker Response and evaluate overall effects of the leachates. Some of the embryos were allowed to hatch, and the hatchlings were tested for anxiety-like behavior. Our results showed that low concentrations of the leachates from ST, UST, and SF raised the heart rate, accelerated development, and changed behavior, while high concentrations lowered the heart rate, suppressed development, and increased mortality. The lowest observed effect concentration for the leachates was ≤0.2piece (pc)/L. The USF leachate had no effect at the concentration of 20pc/L. Developmental toxicity of the leachates was ranked as: ST>UST>SF>USF. This study has demonstrated for the first time that CB leachates affect fish development, and provided toxicological evidence to better assess ecological impacts of CBs.

Infectious bursal disease virus (IBDV) infection destroys the bursa of Fabricius, causing immunosuppression and rendering chickens susceptible to secondary bacterial or viral infections. IBDV large-segment-protein-expressing DNA has been shown to confer complete protection of chickens from infectious bursal disease (IBD). The purpose of the present study was to compare DNA-vaccinated chickens and unvaccinated chickens upon IBDV challenge by transcriptomic analysis of bursa regarding innate immunity, inflammation, immune cell regulation, apoptosis and glucose transport. One-day-old specific-pathogen-free chickens were vaccinated intramuscularly three times at weekly intervals with IBDV large-segment-protein-expressing DNA. Chickens were challenged orally with 8.2 × 102 times the egg infective dose (EID)50 of IBDV strain variant E (VE) one week after the last vaccination. Bursae collected at 0.5, 1, 3, 5, 7, and 10 days post-challenge (dpc) were subjected to real-time RT-PCR quantification of bursal transcripts related to innate immunity, inflammation, immune cell regulation, apoptosis and glucose transport. The expression levels of granzyme K and CD8 in DNA-vaccinated chickens were significantly (p

This paper discusses some notes on the vibration design for the piezoelectric cooling fan. After reviewing the fundamental formulas of the cantilever Euler beam, it is not easy to find the optimal design of the piezoelectric fan. The experiments also show the complicated results of the vibration behavior and air flow., {"references":["","M. Toda and S. Osaka, \"Vibrational fan using the piezoelectric polymer PVF2,\"Proceedings of the IEEE, vol. 67, pp. 1171–1173, 1979.","J.H. Yoo, J.I. Hong and W. Cao, \"Piezoelectric ceramic bimorph coupled to thin metal plate as cooling fan for electronic devices,\" Sensors and Actuators, vol. 79, pp. 8–12, 2000.","T. Acikalin, S.V. Garimella, A. Raman and J. Petroski, \"Characterization and optimization of the thermal performance of miniature piezoelectric fans,\" International Journal of Heat and Fluid Flow, vol. 28, pp. 806–820, 2007.","M. Kimber and S.V. Garimella, \"Measurement and prediction of the cooling characteristics of a generalized vibrating piezoelectric fan,\" International Journal of Heat and Mass Transfer, vol. 52, pp. 4470–4478, 2009.","W.J. Sheu, R.T. Huang and C.C. Wang, \"Influence of bonding glues on the vibration of piezoelectric fans,\" Sensors and Actuators A: Physical, vol. 148, pp. 115–121, 2008.","H.K. Ma, H.C. Su, C.L. Liu and W.H. Ho, \"Investigation of a piezoelectric fan embedded in a heat sink,\" International Communications in Heat and Mass Transfer, vol. 39, pp. 603609, 2012.","J. Yang, Analysis of Piezoelectric Devices. Singapore: World Scientific Publishing, 2006.","T.J.C. Liu, \"Fracture mechanics and crack contact analyses of the active repair of multi-layered piezoelectric patches bonded on cracked structures,\" Theoretical and Applied Fracture Mechanics, vol. 47, pp.120-132, 2007.","J.M. Gere and B.J. Goodno, Mechanics of Materials. 8th Ed., Singapore: Cengage Learning, 2013.\n[10] L. Meirovitch, Fundamentals of Vibrations. New York: McGraw-Hill, 2001."]}

This study presents a novel design and fabrication process to realize an on-chip micro ball bearing. The ball bearing consists of a polymer-Si-polymer outer-race (stator), and a suspended polymer-Si-polymer inner-disk (rotor) supported by polymer micro-balls. The outer-race and inner-disk are demolded from silicon mold, which is patterned by wet isotropic etching on double-polished 525 μm-thick silicon wafer. The liquid phase photocurable polymer balls are dispensed by commercial dispenser with a hundred micrometer diameter tip and in-situ self-assembled in buffer liquid. After photocurable polymer solidified by UV-curing, the polymer balls are confined between the outer race and inner disk. Note the ball bearing is fabricated on a single wafer with no assembly required.

This study presents a novel approach to implement Ultraviolet (UV) sensor using low temperature process. The UV-sensitive TiO 2 /Glycerol is embedded inside a cavity on silicon chip and then encapsulated by CVD (chemical vapor deposition) parylene C film. In addition, the percentage weight of TiO 2 in TiO 2 /Glycerol can be easily changed to improve the performance of UV sensor. The characteristics of photo-response measured from the presented photo sensor of 0∼30 wt% TiO 2 . Typical measurements show the photo-dark current ratio ranging 2.04–5.70, response time ranging 40–48 sec, and recovery time: ranging 5–10 sec. Moreover, the UV sensor with 20 wt% TiO 2 content shows a higher UV sensitivity. The performances of presented sensor with various electrode gap designs (1581–335μm) under 20wt % TiO 2 have also been characterized. Typical measurements show the photo-dark current ratio ranging 11.4–5.85, response time ranging 19–23 sec, and recovery time: ranging 1.5–6 sec. Moreover, the UV sensor of with 335 μm gap design show a higher UV sensitivity.

This study presents an unique process to integrate the temperature sensing/heating elements, multi-layer through-hole vias (THVs), and electrical routings on parylene substrate to implement a multi-functional flexible temperature sensor array. Moreover, the polymer-staking processes with 3D electrical routings enable the further integration of various devices. In application, this study also demonstrates the implementation and integration of flexible temperature sensor array by: (1) temperature distribution and contour measurement using 4×4 temperature sensor array, (2) temperature monitoring and control using the integration of Pt-sensor and Pt-heater, (3) heterogeneous integration of temperature sensor array with LED-chip for LED temperature monitoring, and (4) 3D integration of temperature sensor array with LED and micro-lens and micro chamber.

This study presents a novel design and fabrication process to realize a miniature polymer ball bearing slide table (slider) with long linear travel range. The linear slide table consists of a pair of V-shape silicon (111) crystal plane rails, and a suspended slider supported by four polymer ball bearings. The ball-housing on slider is also designed to confine the bearing. The slider, rails, and ball-housings are patterned by DRIE and wet anisotropic etching on double-polished (100) wafer. The ball bearings are fabricated and in-situ self-assembled using the liquid polymer formation technique in buffer liquid. After polymer solidified by UV-curing, the ball bearing is confined in ball-housing. The device is successfully implemented and the traveling test is also demonstrated using magnetic force.