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Aims/hypothesis: Mutations in Isl1, encoding the insulin enhancer-binding protein islet-1 (ISL1), may contribute to attenuated insulin secretion in type 2 diabetes mellitus. We made an Isl1E283D mouse model to investigate the disease-causing mechanism of diabetes mellitus. Methods: The ISL1E283D mutation (c. 849A>T) was identified by whole exome sequencing on an early-onset type 2 diabetes family and then the Isl1E283D knockin (KI) mouse model was created and an IPGTT and IPITT were conducted. Glucose-stimulated insulin secretion (GSIS), expression of Ins2 and other ISL1 target genes and interacting proteins were evaluated in isolated pancreas islets. Transcriptional activity of Isl1E283D was evaluated by cell-based luciferase reporter assay and electrophoretic mobility shift assay, and the expression levels of Ins2 driven by Isl1 wild-type (Isl1WT) and Isl1E283D mutation in rat INS-1 cells were determined by RT-PCR and western blotting. Results: Impaired GSIS and elevated glucose level were observed in Isl1E283D KI mice while expression of Ins2 and other ISL1 target genes Mafa, Pdx1, Slc2a2 and the interacting protein NeuroD1 were downregulated in isolated islets. Transcriptional activity of the Isl1E283D mutation for Ins2 was reduced by 59.3%, and resulted in a marked downregulation of Ins2 expression when it was overexpressed in INS-1 cells, while overexpression of Isl1WT led to an upregulation of Ins2 expression. Conclusions/interpretation: Isl1E283D mutation reduces insulin expression and secretion by regulating insulin and other target genes, as well as its interacting proteins such as NeuroD1, leading to the development of glucose intolerance in the KI mice, which recapitulated the human diabetic phenotype. This study identified and highlighted the Isl1E283D mutation as a novel causative factor for type 2 diabetes, and suggested that targeting transcription factor ISL1 could offer an innovative avenue for the precise treatment of human type 2 diabetes. [ABSTRACT FROM AUTHOR]

Precise differentiation of glucokinase (GCK) monogenic diabetes from gestational diabetes mellitus (GDM) is critical for accurate management of the pregnancy outcome. We screened GCK-MODY complicating pregnancies in Chinese GDM patients, explored the pathogenesis of novel GCK mutations, and evaluated the patients' pregnancy outcome and management. The GCK gene from 411 GDM patients was screened with PCR–direct sequencing and multiplex ligation-dependent probe amplification (MLPA) and 15 GCK mutations were identified. We also retrospectively analyzed a total of 65 pregnancies from 21 GCK-MODY families, wherein 41 were from 15 maternal families and 24 were from six paternal families. Bioinformatic analysis and biochemical functional study were conducted to identify novel GCK mutations. In total, we identified 21 GCK mutations: 15 from the 411 GDM patients and six from 24 fathers. Of th Asp78Asn (GAC → AAC), Met87Arg (ATG → AGG), Leu451Val (CTT → GTT), Leu451Pro (CTG → CCG) and 1019 + 20G > A e mutations, five, i.e., were novel and deleterious, with markedly decreased enzyme activity and thermal stability. The unaffected offspring of GCK mutation-affected mothers were heavier than affected offspring (p < 0.001). Of 21 insulin-treated affected mothers, 10 had maternal hypoglycemia (47.6%) and seven had perinatal complications (33.3%), and the affected offspring of the insulin-treated affected mothers had significantly lower birth weights than that of the 20 diet-control affected mothers (p = 0.031). In this study, the prevalence of GCK-MODY complicating pregnancy in Chinese GDM patients was 3.6% (15/411). The defective GCK may contribute to the hyperglycemia in GCK-MODY. Insulin therapy is not beneficial for GCK-MODY complicating pregnancy and therefore should not be recommended. [ABSTRACT FROM AUTHOR]

More than 80% of maturity-onset diabetes of the young (MODY) in Chinese is genetically unexplained. To investigate whether the insulin gene (INS) mutation is responsible for some Chinese MODY, we screened INS mutations causing MODY10 in MODY pedigrees and explored the potential pathogenic mechanisms. INS mutations were screened in 56 MODY familial probands. Structure–function characterization and clinical profiling of identified INS mutations were conducted. An INS mutation, at the position 2 alanine-to-threonine substitution (A2T), was identified and co-segregated with hyperglycemia in a MODY pedigree. The A2T mutation converted an α-helix into a β-sheet at the N-terminal of the signal peptide (SP) of preproinsulin. The A2T mutation did not affect preproinsulin translocation across endoplasmic reticulum (ER) membrane, but impaired its SP cleavage within the ER. In INS-1 cells transfected with an A2T mutant, glucose-stimulated insulin secretion (GSIS) was significantly decreased, while BiP luciferase activities were significantly increased compared to that of wild type (WT). We identified an INS-A2T mutation cosegregating with diabetes in a Chinese MODY pedigree. This mutation severely impaired SP cleavage and thus blocked the formation of proinsulin, resulting in enhanced ER stress, which may be responsible for decreased insulin secretion and subsequently, the onset of MODY10. [ABSTRACT FROM AUTHOR]

The mechanic performance of ejecting pneumatic fire extinguisher is determined by the following parameters: air speed at funnel exit, outfire range and ejecting stability; and the second and third parameters depend on the fire extinguishing agent pellet's velocity on funnel exit and the amount of fire extinguishing agent pellets detained in the funnel when extinguisher is working. In this research, the CFD-DEM coupling model was adopted to simulate the working process of ejecting pneumatic fire extinguisher, and the type of 6MF-30 pneumatic extinguisher was used to test the reliability of the coupling model. The funnel throat angle X1, the adding position of fire extinguishing agent X2, and the funnel length X3 were selected as the influencing factors, and total 23 experiments were conducted by the simulation model under the quadratic orthogonal rotation design. The virtual tunnel was designed according to the tunnel equipped to 6MF-30 pneumatic extinguisher. The virtual fire extinguishing agent pellets were found on the EDEM interface, coupled to the original CFD by Lagrangian model. The pretest results showed that this model had strong convergence which was proved by its stable airflow performance when running at the time of 0.2-0.25 s, and stable pellet number when running at 0.34 s. The air velocity at exit calculated by the CFD-DEM coupling model was similar to the theoretical value. It could be found that the number of aggregating virtual pellets detained in the region between the air tunnel and the fire extinguishing agent's channel was consistent with the result of research by other scholars. The simulating experiment data were exported for analyzing. The average velocity in axial direction on each point at funnel exit was read by Fluent, and after subtracting the average value 197.550, the deviation data of the 23 experiments were got and selected as the outlet wind velocity (index 71). The max virtual pellet velocities in axial direction monitored by EDEM within 0-0.4 s of all experiments were selected as the spouting velocity (index Y2). The vertical coordinates of every virtual pellet at 0.37, 0.38, 0.39, 0.4 s in all experiments were read by EDEM, and 'countif' function was used to calculate the number of virtual pellet located in the tunnel (as assessment index Y3) when working. After the significant test and the variance analysis, the regression equation building was done by using SAS9.1. The result showed that the fire extinguishing agent pellet's velocity on funnel exit and the amount of fire extinguishing agent pellets detained in the funnel were significantly correlated to the three factors (P<0.0001, P<0.001 respectively), but the air speed at funnel exit had no correlation with these factors. By using response surface method combined with weighting method, the optimal aggregative index could be obtained under the condition that the funnel throat angle was 20°, the adding position of fire extinguishing agent was 140 mm and the funnel length was 700 mm. Under this condition, the efficiency of ejecting pneumatic fire extinguisher was mostly improved. The virtual test designed by the optimized parameters showed that the fire extinguishing agent pellet's velocity on funnel exit increased to 41.24 m/s, and the number of fire extinguishing agent pellets detained in the funnel decreased to 209. The verify test of the physical prototype indicated that the optimized parameters could improve the working effect of ejecting pneumatic fire extinguisher, which was manifested in extending the outfire range by 2.15 m, reducing the possibility of reburning and shortening the outfire time by 1.7 s. [ABSTRACT FROM AUTHOR]

ABSTRACT The p. Arg116 His mutation in the heat shock transcription factor-4 ( HSF4) has been associated with age-related cataracts, but it is also seen in 2% of the normal population, indicating either reduced penetrance or that the normal subjects were not old enough to express the phenotype. Based on the proximity of p. Arg116 His to two known mutations in the DNA-binding domain of HSF4, namely, p. Leu114 Pro and p. Arg119 Cys, which segregate with childhood lamellar cataract, we tested the possibility that this phenotype may have been missed by the ophthalmologist and/or that it did not spread to the visual axis so as to affect vision significantly. Here, we demonstrate via BAC (bacterial artificial chromosome) transgenesis that p. Arg116 His recreates the childhood lamellar cataract in mice suggesting that incomplete penetrance associated with early cataracts may not be an absence but a limitation of the detection of the phenotype. [ABSTRACT FROM AUTHOR]

The prereceptor activation of glucocorticoid production in adipose tissue by NADPH-dependent 11 hydroxysteroid dehydrogenase type 1 (11β-HSD1) has emerged as a potential mechanism in the pathogenesis of visceral obesity and metabolic syndrome. Hexose-6-phosphate dehydrogenase (H6PDH) is an endoplasmic reticulum lumen-resident enzyme that generates cofactor NADPH and thus mediates 11β-HSD1 activity. To determine the role of adipose H6PDH in the prereceptor modulation of 11β- HSD1 and metabolic phenotypes, we generated a transgenic (Tg) mouse model overexpressing H6PDH under the control of the enhancerpromoter region of the adipocyte fatty acid-binding protein (aP2) gene (aP2/H6PDH Tg mice). Transgenic aP2/H6PDH mice exhibited relatively high expression of H6PDH and elevated corticosterone production with induction of 11β-HSD1 activity in adipose tissue. This increase in corticosterone production in aP2-H6PDH Tg mice resulted in mild abdominal fat accumulation with induction of C/EBP mRNA expression and slight weight gain. Transgenic aP2/H6PDH mice also exhibited fasting hyperglycemia and glucose intolerance with insulin resistance. In addition, the aP2/H6PDH Tg mice have elevated circulating free fatty acid levels with a concomitant increased adipose lipolytic action associated with elevated HSL mRNA and Ser660 HSL phosphorylation within abdominal fat. These results suggest that increased H6PDH expression specifically in adipose tissue is sufficient to cause intra-adipose glucocorticoid production and adverse metabolic phenotypes. These findings suggest that the aP2/H6PDH Tg mice may provide a favorable model for studying the potential impact of H6PDH in the pathogenesis of human metabolic syndrome [ABSTRACT FROM AUTHOR]

A portable pneumatic extinguisher is an effective device that has been widely used for forest and grassland fire extinguishing in China. In order to enhance the effective range and rate of discharge of portable pneumatic extinguishers, a new method to weaken air velocity attenuation by increasing the flow rate using an air ejector was proposed and investigated in this study. This research belongs to the category of subsonic air ejector. First, the turbulivity of air jet "c" has been updated to the value of 0.106 by experiments. It is necessary to use this factor for the calculation of air velocity where 2.5 m downstream from the centrifugal fan which defined as u2.5. Experimental and the CFD (Computational Fluid Dynamics) methods are applied to investigate the influence on the performance of the air ejector. Three parameters were characterized: converging angle of entraining chamber θ; diameter of the mixing chamber Dm and the nozzle position (NXP) S. Up to 240 different models were established and meshed by Gambit 2.3, and then simulated and calculated by Fluent 6.3 with the turbulence model of RNG k-epsilon. Consequently, 240 different results containing the flow rate of nozzle outlet cross-section mp, the entrainment flow rate me, the flow rate of the mixing chamber outlet cross-section mc, and the air velocity of a mixing chamber outlet cross-section uc were acquired. Based on this data, the response surfaces for u2.5 and entrainment ratio that are used to investigate the interaction between Dm, θ and S to u2.5 and entrainment ratio were established. The results indicated that the parameters Dm and θ have a great influence on u2.5 and entrainment ratio. It was also demonstrated that the interaction between Dm and θ is significant. However, parameter S gave a relatively delicate influence on u2.5 and entrainment ratio, and also the interaction with the other parameters was weak. In addition, to find out the mechanism of interaction between Dm and θ, three groups of different pressure fields have been investigated and compared as well. It indicates that a high value of θ (when θ < 36.25°) could create a relatively significant negative pressure zone in the mixing chamber, which requires a larger Dm to provide sufficient air input, and as a consequence u2.5 and entrainment ratio increase accordingly. Because of the negative pressure zone nearby the nozzle outlet, the effective power of the centrifugal fan and engine are both increased. The optimum value of θ is 36.25°, and when Dm equals to 144 mm, u2.5 could reach the maximum value. The maximum u2.5, which was gained by experiments using an ejecting pneumatic extinguisher, was 36.4 m/s. Meanwhile, the value of u2.5 could keep adding up as Dm continued to increase, but the range of Dm has been limited by the design and practical applicability of the portable pneumatic extinguisher. [ABSTRACT FROM AUTHOR]

Purpose: Ocular Albinism type 1 (OA1) is a disease caused by mutations in the OA1 gene and characterized by the presence of macromelanosomes in the retinal pigment epithelium (RPE) as well as abnormal crossing of the optic axons at the optic chiasm. We showed in our previous studies in mice that Oa1 activates specifically Gαi3 in its signaling pathway and thus, hypothesized that a constitutively active Gαi3 in the RPE of Oa1−/− mice might keep on the Oa1 signaling cascade and prevent the formation of macromelanosomes. To test this hypothesis, we have generated transgenic mice that carry the constitutively active Gαi3 (Q204L) protein in the RPE of Oa1−/− mice and are now reporting the effects that the transgene produced on the Oa1−/− RPE phenotype. Methods: Transgenic mice carrying RPE-specific expression of the constitutively active Gαi3 (Q204L) were generated by injecting fertilized eggs of Oa1−/− females with a lentivirus containing the Gαi3 (Q204L) cDNA. PCR, Southern blots, Western blots and confocal microscopy were used to confirm the presence of the transgene in the RPE of positive transgenic mice. Morphometrical analyses were performed using electron microscopy to compare the size and number of melanosomes per RPE area in putative Oa1−/−, Gαi3 (Q204L) transgenic mice with those of wild-type NCrl and Oa1−/− mice. Results: We found a correlation between the presence of the constitutively active Gαi3 (Q204L) transgene and the rescue of the normal phenotype of RPE melanosomes in Oa1−/−, Gαi3 (Q204L) mice. These mice have higher density of melanosomes per RPE area and a larger number of small melanosomes than Oa1−/− mice, and their RPE phenotype is similar to that of wild-type mice. Conclusions: Our results show that a constitutively active Gαi3 protein can by-pass the lack of Oa1 protein in Oa1−/− mice and consequently rescue the RPE melanosomal phenotype. [ABSTRACT FROM AUTHOR]

Background: Ocular albinism type 1, an X-linked disease characterized by the presence of enlarged melanosomes in the retinal pigment epithelium (RPE) and abnormal crossing of axons at the optic chiasm, is caused by mutations in the OA1 gene. The protein product of this gene is a G-protein-coupled receptor (GPCR) localized in RPE melanosomes. The Oa1-/- mouse model of ocular albinism reproduces the human disease. Oa1 has been shown to immunoprecipitate with the Gαi subunit of heterotrimeric G proteins from human skin melanocytes. However, the Gαi subfamily has three highly homologous members, Gαi1, Gαi2 and Gαi3 and it is possible that one or more of them partners with Oa1. We had previously shown by in-vivo studies that Gαi3-/- and Oa1-/- mice have similar RPE phenotype and decussation patterns. In this paper we analyze the specificity of the Oa1-Gαi interaction. Methodology: By using the genetic mouse models Gαi1-/-, Gαi2-/-, Gαi3-/- and the double knockout Gαi1-/-, Gαi3-/- that lack functional Gαi1, Gαi2, Gαi3, or both Gαi1 and Gαi3 proteins, respectively, we show that Gαi3 is critical for the maintenance of a normal melanosomal phenotype and that its absence is associated with changes in melanosomal size and density. GSTpull- down and immunoprecipitation assays conclusively demonstrate that Gαi3 is the only Gαi that binds to Oa1. Western blots show that Gαi3 expression is barely detectable in the Oa1-/- RPE, strongly supporting a previously unsuspected role for Gαi3 in melanosomal biogenesis. Conclusion: Our results identify the Oa1 transducer Gai3 as the first downstream component in the Oa1 signaling pathway. [ABSTRACT FROM AUTHOR]

OBJECTIVE--Pertussis toxin uncoupling-based studies have shown that Gαi and Gαo can inhibit insulin secretion in pancreatic B-cells. Yet it is unclear whether Gαi and Gαo operate through identical mechanisms and how these G-protein-mediated signals inhibit insulin secretion in vivo. Our objective is to examine whether/how Gαo regulates islet development and insulin secretion in p-cells. RESEARCH DESIGN AND METHODS--Immunoassays were used to analyze the Gαo expression in mouse pancreatic cells. Gαo was specifically inactivated in pancreatic progenitor cells by pancreatic cell-specific gene deletion. Hormone expression and insulin secretion in response to different stimuli were assayed in vivo and in vitro. Electron microscope and total internal reflection fluorescence-based assays were used to evaluate how Gαo regulates insulin vesicle docking and secretion in response to glucose stimulation. RESULTS--Islet cells differentiate properly in Gαo[sup -/-] mutant mice. Gαo inactivation significantly enhances insulin secretion both in vivo and in isolation. Gαo nullizygous β-cells contain an increased number of insulin granules docked on the cell plasma membrane, although the total number of vesicles per β-cell remains unchanged. CONCLUSIONS--Gαo is not required for endocrine islet cell differentiation, but it regulates the number of insulin vesicles docked on the p-cell membrane. Diabetes 59:2522-2529, 2010 [ABSTRACT FROM AUTHOR]

Thymosin alpha 1 (Tα1) has therapeutic potential in the treatment of infectious diseases and cancer. However, the exact molecular pathways for Tα1 action are not fully understood. We found that Tα1 induces the production of interleukin-6 (IL-6), IL-10, and IL-12 in murine bone marrow-derived macrophages (BMDMs) through IKK and MAPK pathways. Tα1 triggers the activation of AP-1 and the phosphorylation of JNK and p38. Inhibition of p38 impairs IL-6 production in response to Tα1. Further, TRAF6 is involved in the activation of JNK and IRAK4 is involved for the activation of IKK and PKCζ in a Tα1-induced system. Loss of IRAK4 largely blocked induction of IL-6. Thus, our studies define early signal events that are critical for the Tα1-induced immune responses. [ABSTRACT FROM AUTHOR]

Presents information on a study which examined the role of G[sub i2] alpha protein in the regulation of cardiac calcium channels. Methodology; Results of the study; Discussion.

• Background: Intraocular transplantation of genetically modified cells that release a particular substance could have a major impact on the treatment of various ocular diseases. We studied the expression of the reporter gene β-galactosidase ( lacZ) in transplanted retinal pigment epithelial (RPE) cells in vivo • Methods: RPE cells from pigmented rabbits were transduced with the β-galactosidase gene in a retroviral vector. Cells were then assayed for gene expression and transplanted subretinally into the eyes of New Zealand White rabbits. RPE cells that were transduced with a similar vector without the β-galactosidase gene were used as controls. Rabbits were killed on days 1, 7, and 21 and the eyes processed for transmission electron microscopy • Results: Neomycin-resistant rabbit RPE cells that showed β-galactosidase activity were generated within 2-5 weeks. After transplantation, viable RPE cells that expressed the transgene and that phagocytosed rod outer segments were observed on days 1, 7, and 21 • Conclusions: The results show that generation of genetically modified RPE cells is feasible and that the transplanted cells remain viable and continue to express the transgene in the subretinal space of the host animal for at least 21 days. Transplantation of such genetically modified RPE cells could provide a new tool for studying retinal diseases and, potentially, for correcting metabolic abnormalities in retinal degenerations and dystrophies. [ABSTRACT FROM AUTHOR]

Focuses on the roles that the G protein Go may play in intact cells and whole body homeostatis in relation to multiple neurological abnormalities. Materials and methods used; Background information on Go; Results and discussion of the study.

Excessive glucocorticoids (GCs) production in adipose tissue promotes the development of visceral obesity and the metabolic syndrome. 11β-Hydroxysteroid dehydrogenase type 1 (11 β-HSD1) is critical for controlling intracellular GC production in adipose tissue, and this process is also regulated by hexose-6-phosphate dehydrogenase (H6PDH) that provides cofactor NADPH for 11β-HSD1 amplifying local GC production. To better understand the integrated molecular and physiological role of adipose H6PDH, we created a tissue specific knockout of H6PDH gene mouse model in adipocytes (H6PDHAcKO mice) and characterized the metabolic related phenotypes. H6PDHAcKO mice had decreased levels of intra-adipose corticosterone with reduction of 11β-HSD1 activity in response to H6PDH deletion in adipose tissue. Moreover, H6PDHAcKO mice also exhibited reduced fasting blood glucose levels and increased glucose tolerance with increased insulin sensitivity. In addition, plasma FFA levels were decreased with a concomitant decrease in the expression of lipase ATGL and HSL in the abdominal adipose tissue. These results indicate that inactivation of H6PDH expression in adipose tissue is sufficient to cause intra-adipose GC inactivation leading to the favorable pattern of metabolic phenotype. These data suggest that H6PDHAcKO mice may provide a good model for study the potential contributions of fat-specific H6PDH inhibition in the metabolic phenotype and other pathological disorders in vivo. Disclosure: J. Wang: None. X. Wang: None. K. Lutfy: None. T. Friedman: None. M. Jiang: None. Y. Liu: None. Funding: National Institute of Diabetes and Digestive and Kidney Diseases (SC1DK104821) [ABSTRACT FROM AUTHOR]