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The inefficiency of generating induced pluripotent somatic cells (iPS) engendered two contending models, namely the Stochastic model and Elite model. Although the former is more favorable to explain the inherent inefficiencies, it may be fallible to extrapolate the same working model to reprogramming of cancer cells. Indeed, tumor cells are known to be inherently heterogeneous with respect to distinctive characteristics thus providing a suitable platform to test whether the reprogramming process of cancer cells is biased. Here, we report our observations that all randomly picked induced pluripotent cancer cells (iPCs) established previously do not possess mutations known in the parental population. This unanticipated observation is most parsimoniously explained by the Elite model, whereby putative early tumor progenies were selected during induction to pluripotency.

The inefficiency of generating induced pluripotent somatic cells (iPS) engendered two contending models, namely the Stochastic model and Elite model. Although the former is more favorable to explain the inherent inefficiencies, it may be fallible to extrapolate the same working model to reprogramming of cancer cells. Indeed, tumor cells are known to be inherently heterogeneous with respect to distinctive characteristics thus providing a suitable platform to test whether the reprogramming process of cancer cells is biased. Here, we report our observations that all randomly picked induced pluripotent cancer cells (iPCs) established previously do not possess mutations known in the parental population. This unanticipated observation is most parsimoniously explained by the Elite model, whereby putative early tumor progenies were selected during induction to pluripotency. [ABSTRACT FROM AUTHOR]

An attractive approach to replace the destroyed insulin-producing cells (IPCs) is the generation of functional β cells from stem cells. Embryonal carcinoma (EC) stem cells are pluripotent cells which can differentiate into all cell types. The present study was carried out to establish a simple nonselective inductive culture system for generation of IPCs from P19 EC cells by 1–2 weeks old mouse pancreas extract (MPE). Since, mouse pancreatic islets undergo further remodeling and maturation for 2–3 weeks after birth, we hypothesized that the mouse neonatal MPE contains essential factors to induce in vitro differentiation of pancreatic lineages. Pluripotency of P19 cells were first confirmed by expression analysis of stem cell markers, Oct3/4, Sox-2 and Nanog. In order to induce differentiation, the cells were cultured in a medium supplemented by different concentrations of MPE (50, 100, 200 and 300 µg/ml). The results showed that P19 cells could differentiate into IPCs and form dithizone-positive cell clusters. The generated P19-derived IPCs were immunoreactive to proinsulin, insulin and insulin receptor beta. The expression of pancreatic β cell genes including, PDX-1, INS1 and INS2 were also confirmed. The peak response at the 100 µg/ml MPE used for investigation of EP300 and CREB1 gene expression. When stimulated with glucose, these cells synthesized and secreted insulin. Network analysis of the key transcription factors (PDX-1, EP300, CREB1) during the generation of IPCs resulted in introduction of novel regulatory candidates such as MIR17, and VEZF1 transcription factors, as well as MORN1, DKFZp761P0212, and WAC proteins. Altogether, we demonstrated the possibility of generating IPCs from undifferentiated EC cells, with the characteristics of pancreatic β cells. The derivation of pancreatic cells from EC cells which are ES cell siblings would provide a valuable experimental tool in study of pancreatic development and function as well as rapid production of IPCs for transplantation. [ABSTRACT FROM AUTHOR]

Potential trend of regenerative treatment for type I diabetes has been introduced for more than a decade. However, the technologies regarding insulin-producing cell (IPC) production and transplantation are still being developed. Here, we propose the potential IPC production protocol employing mouse gingival fibroblast-derived induced pluripotent stem cells (mGF-iPSCs) as a resource and the pre-clinical approved subcutaneous IPC transplantation platform for further clinical confirmation study. With a multi-step induction protocol, the functional and matured IPCs were generated by 13 days with a long-term survival capability. Further double encapsulation of mGF-iPSC-derived IPCs (mGF-iPSC-IPCs) could preserve the insulin secretion capacity and the transplantation potential of the generated IPCs. To address the potential on IPC transplantation, a 2-step subcutaneous transplantation procedure was established, comprising 1) vascularized subcutaneous pocket formation and 2) encapsulated IPC bead transplantation. The in vivo testing confirmed the safety and efficiency of the platform along with less inflammatory response which may help minimize tissue reaction and graft rejection. Further preliminary in vivo testing on subcutaneous IPC-bead transplantation in an induced type I diabetic mouse model showed beneficial trends on blood glucose control and survival rate sustainability of diabetic mice. Taken together, an established mGF-iPSC-IPC generation protocol in this study will be the potential backbone for developing the iPSC-derived IPC production employing human and animal cell resources. As well as the potential further development of IPC transplantation platform for diabetes treatment in human and veterinary practices using an established subcutaneous encapsulated IPC-bead transplantation platform presented in this study.

International patent classifications (IPCs) are assigned to patent documents; however, since the procedure for assigning classifications is manually done by the patent examiner, it takes a lot of time and effort to select some IPCs from about 70,000 IPCs. Hence, some research has been conducted on patent classification with machine learning. However, patent documents are very voluminous, and learning with all the claims (the part describing the content of the patent) as input would run out of the necessary memory, even if the batch size is set to a very small size. Therefore, most of the existing methods learn by excluding some information, such as using only the first claim as input. In this study, we propose a model that considers the contents of all claims by extracting important information for input. In addition, we focus on the hierarchical structure of the IPC, and propose a new decoder architecture to consider it. Finally, we conducted an experiment using actual patent data to verify the accuracy of the prediction. The results showed a significant improvement in accuracy compared to existing methods, and the actual applicability of the method was also discussed.

Much is known about environmental influences on metabolism and systemic insulin levels. Less is known about how those influences are translated into molecular mechanisms regulating insulin production. To better understand the molecular mechanisms we generated marked cells homozygous for a null mutation in the Drosophila TGF-β signal transducer dSmad2 in unmated adult females. We then conducted side-by-side single cell comparisons of the pixel intensity of two Drosophila insulin-like peptides (dILP2 and dILP5) in dSmad2- mutant and wild type insulin producing cells (IPCs). The analysis revealed multiple features of dSmad2 regulation of dILPs. In addition, we discovered that dILP5 is expressed and regulated by dSmad2 in circadian pacemaker cells (CPCs). Outcomes of regulation by dSmad2 differ between dILP2 and dILP5 within IPCs and differ for dILP5 between IPCs and CPCs. Modes of dSmad2 regulation differ between dILP2 and dILP5. dSmad2 antagonism of dILP2 in IPCs is robust but dSmad2 regulation of dILP5 in IPCs and CPCs toggles between antagonism and agonism depending upon dSmad2 dosage. Companion studies of dILP2 and dILP5 in the IPCs of dCORL mutant (fussel in Flybase and SKOR in mammals) and upd2 mutant unmated adult females showed no significant difference from wild type. Taken together, the data suggest that dSmad2 regulates dILP2 and dILP5 via distinct mechanisms in IPCs (antagonist) and CPCs (agonist) and in unmated adult females that dSmad2 acts independently of dCORL and upd2.

While interest in antimicrobial stewardship programmes (ASPs) is growing in most low- and middle-income countries (LMICs), there is a paucity of information on their adoption or implementation in Africa, particularly Uganda. The study assessed the presence and characteristics of ASPs, implemented antimicrobial stewardship (AMS) strategies and the challenges to their implementation in hospitals in Uganda. We conducted a cross-sectional study among heads of infection prevention committees (IPCs) in regional referral hospitals, general hospitals, and private-not-for-profit (PNFP) hospitals from November 2019 to February 2020. An interviewer-administered questionnaire was used to collect data. We analysed data using descriptive statistics. A total of 32 heads of IPCs were enrolled in the study. Of these, eight were from regional referral hospitals, 21 were from general hospitals, and three were from PNFPs. Most heads of IPC were pharmacists (17/32, 53.1%) with a mean age and standard deviation (sd) of 36.1 (±1.1) years. A formal ASP was adopted or implemented in 14 out of the 32 (44%, 95% CI 26-62) studied hospitals. Thirty out of 32 hospitals implemented at least one type of AMS strategy. Sixty-eight percent (22/32) of the hospitals implemented pre-authorisation and approval as their primary AMS core strategy to optimise antibacterial use. The most commonly reported challenges to the implementation of ASP across all 32 hospitals (with or without ASP) were lack of time for the ASP team (29/32, 90.6%) and lack of allocated funding for antimicrobial stewardship team (29/32, 90.6%). In this study, most hospitals in Uganda implemented at least one AMS strategy despite the low implementation of ASPs in hospitals. The ministry of health needs to sensitise and support the establishment of ASP in hospitals across the country.

ObjectiveStem cell therapy is a promising approach for diabetes via promoting the differentiation of insulin-producing cells (IPCs). This study aimed to screen the differentially expressed miRNAs (DEmiRNAs) during the differentiation of muscle-derived stem cells (MDSCs) into IPCs, and uncover the underlying function and mechanism of a specific DEmiRNA, miR-708-5p.MethodsMDSCs were successfully isolated from the leg muscle of rats, and were induced for IPCs differentiation through a five-stage protocol. miRNA microarray assay was performed for screening DEmiRNAs during differentiation. The features of MDSCs-derived IPCs were identified by qRT-PCR, flow cytometry, and immunofluorescence staining. The targeting of STK4 by miR-708-5p was examined by luciferase assay. The protein expression of STK4, YAP1, and p-YAP1 was determined by Western blot and immunofluorescence staining.ResultsMDSCs were successfully isolated and differentiated into IPCs. A total of 12 common DEmiRNAs were obtained during five-stage differentiation. Among them, miR-708-5p that highly expressed in MDSCs-derived IPCs was selected. Overexpression of miR-708-5p upregulated some key transcription factors (Pdx1, Ngn3, Nkx2.2, Nkx6.1, Gata4, Gata6, Pax4, and Pax6) involving in IPCs differentiation, and increased insulin positive cells. In addition, STK4 was identified as the target gene of miR-708-5p. miR-708-5p overexpression downregulated the expression of STK4 and the downstream phosphorylated YAP1.ConclusionsThere were 12 DEmiRNAs involved in the differentiation of MDSCs into IPCs. miR-708-5p promoted MDSCs differentiation into IPCs probably by targeting STK4-mediated Hippo-YAP1 signaling pathway.

This research was undertaken to investigate the global role of the plant inositol phosphorylceramide synthase (IPCS), a non-mammalian enzyme previously shown to be associated with the pathogen response. RNA-Seq analyses demonstrated that over-expression of inositol phosphorylceramide synthase isoforms AtIPCS1, 2 or 3 in Arabidopsis thaliana resulted in the down-regulation of genes involved in plant response to pathogens. In addition, genes associated with the abiotic stress response to salinity, cold and drought were found to be similarly down-regulated. Detailed analyses of transgenic lines over-expressing AtIPCS1-3 at various levels revealed that the degree of down-regulation is specifically correlated with the level of IPCS expression. Singular enrichment analysis of these down-regulated genes showed that AtIPCS1-3 expression affects biological signaling pathways involved in plant response to biotic and abiotic stress. The up-regulation of genes involved in photosynthesis and lipid localization was also observed in the over-expressing lines.

BACKGROUND:The two major obstacles in the successful transplantation of islets for diabetes treatment are inadequate supply of insulin-producing tissue and immune rejection. Induction of the differentiation of human bone marrow-derived mesenchymal stem cells (hMSCs) into insulin-producing cells (IPCs) for autologous transplantation may alleviate those limitations. METHODS:hMSCs were isolated and induced to differentiate into IPCs through a three-stage differentiation protocol in a defined media with high glucose, nicotinamide, and exendin-4. The physiological characteristics and functions of IPCs were then evaluated. Next, about 3 × 10(6) differentiated cells were transplanted into the renal sub-capsular space of streptozotocin (STZ)-induced diabetic nude mice. Graft survival and function were assessed by immunohistochemistry, TUNEL staining and measurements of blood glucose levels in the mice. RESULTS:The differentiated IPCs were characterized by Dithizone (DTZ) positive staining, expression of pancreatic β-cell markers, and human insulin secretion in response to glucose stimulation. Moreover, 43% of the IPCs showed L-type Ca2+ channel activity and similar changes in intracellular Ca2+ in response to glucose stimulation as that seen in pancreatic β-cells in the process of glucose-stimulated insulin secretion. Transplantation of functional IPCs into the renal subcapsular space of STZ-induced diabetic nude mice ameliorated the hyperglycemia. Immunofluorescence staining revealed that transplanted IPCs sustainably expressed insulin, c-peptide, and PDX-1 without apparent apoptosis in vivo. CONCLUSIONS:IPCs derived from hMSCs in vitro can ameliorate STZ-induced diabetic hyperglycemia, which indicates that these hMSCs may be a promising approach to overcome the limitations of islet transplantation.

Type 1 diabetes (T1D) is caused by autoimmune disease that leads to the destruction of pancreatic β-cells. Transplantation of cadaveric pancreatic organs or pancreatic islets can restore normal physiology. However, there is a chronic shortage of cadaveric organs, limiting the treatment of the majority of patients on the pancreas transplantation waiting list. Here, we hypothesized that human iPS cells can be directly differentiated into insulin producing cells (IPCs) capable of secreting insulin. Using a series of pancreatic growth factors, we successfully generated iPS cells derived IPCs. Furthermore, to investigate the capability of these cells to secrete insulin in vivo, the differentiated cells were transplanted under the kidney capsules of diabetic immunodeficient mice. Serum glucose levels gradually declined to either normal or near normal levels over 150 days, suggesting that the IPCs were secreting insulin. In addition, using MRI, a 3D organoid appeared as a white patch on the transplanted kidneys but not on the control kidneys. These organoids showed neo-vascularization and stained positive for insulin and glucagon. All together, these data show that a pancreatic organ can be created in vivo providing evidence that iPS cells might be a novel option for the treatment of T1D.

MicroRNAs (miRNAs) are a group of endogenous small non-coding RNAs that regulate gene expression at the post-transcriptional level. A number of studies have led to the notion that some miRNAs have key roles in control of pancreatic islet development and insulin secretion. Based on some studies on miRNAs pattern, the researchers in this paper investigated the pancreatic differentiation of human bone marrow mesenchymal stem cells (hBM-MSCs) by up-regulation of miR-375 and down-regulation of miR-9 by lentiviruses containing miR-375 and anti-miR-9.After 21 days of induction, islet-like clusters containing insulin producing cells (IPCs) were confirmed by dithizone (DTZ) staining. The IPCs and β cell specific related genes and proteins were detected using qRT-PCR and immunofluorescence on days 7, 14 and 21 of differentiation. Glucose challenge test was performed at different concentrations of glucose so extracellular and intracellular insulin and C-peptide were assayed using ELISA kit. Although derived IPCs by miR-375 alone were capable to express insulin and other endocrine specific transcription factors, the cells lacked the machinery to respond to glucose.It was found that over-expression of miR-375 led to a reduction in levels of Mtpn protein in derived IPCs, while treatment with anti-miR-9 following miR-375 over-expression had synergistic effects on MSCs differentiation and insulin secretion in a glucose-regulated manner. The researchers reported that silencing of miR-9 increased OC-2 protein in IPCs that may contribute to the observed glucose-regulated insulin secretion. Although the roles of miR-375 and miR-9 are well known in pancreatic development and insulin secretion, the use of these miRNAs in transdifferentiation was never demonstrated. These findings highlight miRNAs functions in stem cells differentiation and suggest that they could be used as therapeutic tools for gene-based therapy in diabetes mellitus.

A set of 14 insulin-producing cells (IPCs) in the Drosophila brain produces three insulin-like peptides (DILP2, 3 and 5). Activity in IPCs and release of DILPs is nutrient dependent and controlled by multiple factors such as fat body-derived proteins, neurotransmitters, and neuropeptides. Two monoamine receptors, the octopamine receptor OAMB and the serotonin receptor 5-HT1A, are expressed by the IPCs. These receptors may act antagonistically on adenylate cyclase. Here we investigate the action of the two receptors on activity in and output from the IPCs. Knockdown of OAMB by targeted RNAi led to elevated Dilp3 transcript levels in the brain, whereas 5-HT1A knockdown resulted in increases of Dilp2 and 5. OAMB-RNAi in IPCs leads to extended survival of starved flies and increased food intake, whereas 5-HT1A-RNAi produces the opposite phenotypes. However, knockdown of either OAMB or 5-HT1A in IPCs both lead to increased resistance to oxidative stress. In assays of carbohydrate levels we found that 5-HT1A knockdown in IPCs resulted in elevated hemolymph glucose, body glycogen and body trehalose levels, while no effects were seen after OAMB knockdown. We also found that manipulations of the two receptors in IPCs affected male aggressive behavior in different ways and 5-HT1A-RNAi reduced courtship latency. Our observations suggest that activation of 5-HT1A and OAMB signaling in IPCs generates differential effects on Dilp transcription, fly physiology, metabolism and social interactions. However the findings do not support an antagonistic action of the two monoamines and their receptors in this particular system.

In a variety of animal species, females hold a leading position in evaluating potential mating partners. The decision of virgin females to accept or reject a courting male is one of the most critical steps for mating success. In the fruitfly Drosophila melanogaster, however, the molecular and neuronal mechanisms underlying female receptivity are still poorly understood, particularly for virgin females. The Drosophila painless (pain) gene encodes a transient receptor potential (TRP) ion channel. We previously demonstrated that mutations in pain significantly enhance the sexual receptivity of virgin females and that pain expression in pain(GAL4) -positive neurons is necessary and sufficient for pain-mediated regulation of the virgin receptivity. Among the pain(GAL4) -positive neurons in the adult female brain, here we have found that insulin-producing cells (IPCs), a neuronal subset in the pars intercerebralis, are essential in virgin females for the regulation of sexual receptivity through Pain TRP channels. IPC-specific knockdown of pain expression or IPC ablation strongly enhanced female sexual receptivity as was observed in pain mutant females. When pain expression or neuronal activity was conditionally suppressed in adult IPCs, female sexual receptivity was similarly enhanced. Furthermore, both pain mutations and the conditional knockdown of pain expression in IPCs depressed female rejection behaviors toward courting males. Taken together, our results indicate that the Pain TRP channel in IPCs plays an important role in controlling the sexual receptivity of Drosophila virgin females by positively regulating female rejection behaviors during courtship.

BACKGROUND:Human pancreatic islet transplantation is a prospective curative treatment for diabetes. However, the lack of donor pancreases greatly limits this approach. One approach to overcome the limited supply of donor pancreases is to generate functional islets from human embryonic stem cells (hESCs), a cell line with unlimited proliferative capacity, through rapid directed differentiation. This study investigated whether pancreatic insulin-producing cells (IPCs) differentiated from hESCs could correct hyperglycemia in severe combined immunodeficient (SCID)/non-obese diabetic (NOD) mice, an animal model of diabetes. METHODS:We generated pancreatic IPCs from two hESC lines, YT1 and YT2, using an optimized four-stage differentiation protocol in a chemically defined culture system. Then, about 5-7 × 10(6) differentiated cells were transplanted into the epididymal fat pad of SCID/NOD mice (n = 20). The control group were transplanted with undifferentiated hESCs (n = 6). Graft survival and function were assessed using immunohistochemistry, and measuring serum human C-peptide and blood glucose levels. RESULTS:The pancreatic IPCs were generated by the four-stage differentiation protocol using hESCs. About 17.1% of differentiated cells expressed insulin, as determined by flow cytometry. These cells secreted insulin/C-peptide following glucose stimulation, similarly to adult human islets. Most of these IPCs co-expressed mature β cell-specific markers, including human C-peptide, GLUT2, PDX1, insulin, and glucagon. After implantation into the epididymal fat pad of SCID/NOD mice, the hESC-derived pancreatic IPCs corrected hyperglycemia for ≥ 8 weeks. None of the animals transplanted with pancreatic IPCs developed tumors during the time. The mean survival of recipients was increased by implanted IPCs as compared to implanted undifferentiated hESCs (P

Human embryonic stem cells (hESCs) are pluripotent and capable of undergoing multilineage differentiation into highly specialized cells including pancreatic islet cells. Thus, they represent a novel alternative source for targeted therapies and regenerative medicine for diabetes. Significant progress has been made in differentiating hESCs toward pancreatic lineages. One approach is based on the similarities of pancreatic β cell and neuroepithelial development. Nestin-positive cells are selected as pancreatic β cell precursors and further differentiated to secrete insulin. The other approach is based on our knowledge of developmental biology in which the differentiation protocol sequentially reproduces the individual steps that are known in normal β cell ontogenesis during fetal pancreatic development. In the present study, the hESC cell line PKU1.1 was induced to differentiate into insulin-producing cells (IPCs) using both protocols. The differentiation process was dynamically investigated and the similarities and differences between both strategies were explored. Our results show that IPCs can be successfully induced with both differentiation strategies. The resulting IPCs from both protocols shared many similar features with pancreatic islet cells, but not mature, functional β cells. However, these differently-derived IPC cell types displayed specific morphologies and different expression levels of pancreatic islet development-related markers. These data not only broaden our outlook on hESC differentiation into IPCs, but also extend the full potential of these processes for regenerative medicine in diabetes.

Mesenchymal stem cells (MSCs) can be successfully induced to differentiate into insulin-producing cells (IPCs) by a variety of small molecules and cytokines in vitro. However, problems remain, such as low transdifferentiation efficiency and poor maturity of trans-differentiated cells. The damaged pancreatic cells secreted a large amount of soluble proteins, which were able to promote pancreative islet regeneration and MSCs differentiation. In this study, we utilized the rat injured pancreatic tissue extract to modulate rat bone marrow-derived MSCs differentiation into IPCs by the traditional two-step induction. Our results showed that injured pancreatic tissue extract could effectively promote the trans-differentiation efficiency and maturity of IPCs by the traditional induction. Moreover, IPCs were able to release more insulin in a glucose-dependent manner and ameliorate better the diabetic conditions of streptozotocin (STZ)-treated rats. Our study provides a new strategy to induce an efficient and directional differentiation of MSCs into IPCs.

Adipokines secreted from adipose tissue are key regulators of metabolism in animals. Adiponectin, one of the adipokines, modulates pancreatic beta cell function to maintain energy homeostasis. Recently, significant conservation between Drosophila melanogaster and mammalian metabolism has been discovered. Drosophila insulin like peptides (Dilps) regulate energy metabolism similarly to mammalian insulin. However, in Drosophila, the regulatory mechanism of insulin producing cells (IPCs) by adipokine signaling is largely unknown. Here, we describe the discovery of the Drosophila adiponectin receptor and its function in IPCs. Drosophila adiponectin receptor (dAdipoR) has high homology with the human adiponectin receptor 1. The dAdipoR antibody staining revealed that dAdipoR was expressed in IPCs of larval and adult brains. IPC- specific dAdipoR inhibition (Dilp2>dAdipoR-Ri) showed the increased sugar level in the hemolymph and the elevated triglyceride level in whole body. Dilps mRNA levels in the Dilp2>dAdipoR-Ri flies were similar with those of controls. However, in the Dilp2>dAdipoR-Ri flies, Dilp2 protein was accumulated in IPCs, the level of circulating Dilp2 was decreased, and insulin signaling was reduced in the fat body. In ex vivo fly brain culture with the human adiponectin, Dilp2 was secreted from IPCs. These results indicate that adiponectin receptor in insulin producing cells regulates insulin secretion and controls glucose and lipid metabolism in Drosophila melanogaster. This study demonstrates a new adipokine signaling in Drosophila and provides insights for the mammalian adiponectin receptor function in pancreatic beta cells, which could be useful for therapeutic application.

Insulin-like peptides (ILPs) regulate growth, reproduction, metabolic homeostasis, life span and stress resistance in worms, flies and mammals. A set of insulin producing cells (IPCs) in the Drosophila brain that express three ILPs (DILP2, 3 and 5) have been the main focus of interest in hormonal DILP signaling. Little is, however, known about factors that regulate DILP production and release by these IPCs. Here we show that the IPCs express the metabotropic GABA(B) receptor (GBR), but not the ionotropic GABA(A) receptor subunit RDL. Diminishing the GBR expression on these cells by targeted RNA interference abbreviates life span, decreases metabolic stress resistance and alters carbohydrate and lipid metabolism at stress, but not growth in Drosophila. A direct effect of diminishing GBR on IPCs is an increase in DILP immunofluorescence in these cells, an effect that is accentuated at starvation. Knockdown of irk3, possibly part of a G protein-activated inwardly rectifying K(+) channel that may link to GBRs, phenocopies GBR knockdown in starvation experiments. Our experiments suggest that the GBR is involved in inhibitory control of DILP production and release in adult flies at metabolic stress and that this receptor mediates a GABA signal from brain interneurons that may convey nutritional signals. This is the first demonstration of a neurotransmitter that inhibits insulin signaling in its regulation of metabolism, stress and life span in an invertebrate brain.

The Inositol 1,4,5- trisphosphate receptor (InsP(3)R) is an intracellular ligand gated channel that releases calcium from intracellular stores in response to extracellular signals. To identify and understand physiological processes and behavior that depends on the InsP(3) signaling pathway at a systemic level, we are studying Drosophila mutants for the InsP(3)R (itpr) gene. Here, we show that growth defects precede larval lethality and both are a consequence of the inability to feed normally. Moreover, restoring InsP(3)R function in insulin producing cells (IPCs) in the larval brain rescues the feeding deficit, growth and lethality in the itpr mutants to a significant extent. We have previously demonstrated a critical requirement for InsP(3)R activity in neuronal cells, specifically in aminergic interneurons, for larval viability. Processes from the IPCs and aminergic domain are closely apposed in the third instar larval brain with no visible cellular overlap. Ubiquitous depletion of itpr by dsRNA results in feeding deficits leading to larval lethality similar to the itpr mutant phenotype. However, when itpr is depleted specifically in IPCs or aminergic neurons, the larvae are viable. These data support a model where InsP(3)R activity in non-overlapping neuronal domains independently rescues larval itpr phenotypes by non-cell autonomous mechanisms.

Mechanisms by which attainment of specific body sizes trigger developmental transitions to adulthood (e.g. puberty or metamorphosis) are incompletely understood. In Drosophila, metamorphosis is triggered by ecdysone synthesis from the prothoracic gland (PG), whereas growth rate is increased by insulin/insulin growth factor signalling (IIS). Transgene-induced activation of PI3K, the major effector of IIS, within the PG advances the onset of metamorphosis via precocious ecdysone synthesis, raising the possibility that IIS triggers metamorphosis via PI3K activation in the PG. Here we show that blocking the protein kinase A (PKA) pathway in the insulin producing cells (IPCs) increases IIS. This increased IIS increases larval growth rate and also advances the onset of metamorphosis, which is accompanied by precocious ecdysone synthesis and increased transcription of at least one ecdysone biosynthetic gene. Our observations suggest that IIS is regulated by PKA pathway activity in the IPCs. In addition, taken together with previous findings, our observations are consistent with the possibility that, in Drosophila, attainment of a specific body size triggers metamorphosis via the IIS-mediated activation of PI3K and hence ecdysone synthesis in the PG.

Botanical primary metabolites extensively exist in herbal medicine injections (HMIs), but often were ignored to control. With the limitation of bias towards hydrophilic substances, the primary metabolites with strong polarity, such as saccharides, amino acids and organic acids, are usually difficult to detect by the routinely applied reversed-phase chromatographic fingerprint technology. In this study, a proton nuclear magnetic resonance (1H NMR) profiling method was developed for efficient identification and quantification of small polar molecules, mostly primary metabolites in HMIs. A commonly used medicine, Danhong injection (DHI), was employed as a model. With the developed method, 23 primary metabolites together with 7 polyphenolic acids were simultaneously identified, of which 13 metabolites with fully separated proton signals were quantified and employed for further multivariate quality control assay. The quantitative 1H NMR method was validated with good linearity, precision, repeatability, stability and accuracy. Based on independence principal component analysis (IPCA), the contents of 13 metabolites were characterized and dimensionally reduced into the first two independence principal components (IPCs). IPC1 and IPC2 were then used to calculate the upper control limits (with 99% confidence ellipsoids) of χ2 and Hotelling T2 control charts. Through the constructed upper control limits, the proposed method was successfully applied to 36 batches of DHI to examine the out-of control sample with the perturbed levels of succinate, malonate, glucose, fructose, salvianic acid and protocatechuic aldehyde. The integrated strategy has provided a reliable approach to identify and quantify multiple polar metabolites of DHI in one fingerprinting spectrum, and it has also assisted in the establishment of IPCA models for the multivariate statistical evaluation of HMIs.

Extracellular microRNAs (miRNAs) are under investigation as minimally-invasive biomarkers for a wide range of disease conditions. We have recently shown in a mouse model of the progressive muscle-wasting condition Duchenne muscular dystrophy (DMD) that a set of highly elevated serum miRNAs reflects the regenerative status of muscle. These miRNAs are promising biomarkers for monitoring DMD disease progression and the response to experimental therapies. The gold standard miRNA detection methodology is Reverse Transcriptase-quantitative Polymerase Chain Reaction (RT-qPCR), which typically exhibits high sensitivity and wide dynamic range. Accurate determination of miRNA levels is affected by RT-qPCR normalization method and therefore selection of the optimal strategy is of critical importance. Serum miRNA abundance was measured by RT-qPCR array in 14 week old mice, and by individual RT-qPCR assays in a time course experiment spanning 48 weeks. Here we utilize these two datasets to assess the validity of three miRNA normalization strategies (a) normalization to the average of all Cq values from array experiments, (b) normalization to a stably expressed endogenous reference miRNA, and (c) normalization to an external spike-in synthetic oligonucleotide. Normalization approaches based on endogenous control miRNAs result in an under-estimation of miRNA levels by a factor of ∼2. An increase in total RNA and total miRNA was observed in dystrophic serum which may account for this systematic bias. We conclude that the optimal strategy for this model system is to normalize to a synthetic spike-in control oligonucleotide. [ABSTRACT FROM AUTHOR]

Introduction: IFNA1 (interferon alpha) is a key cytokine regulating the activity of numerous immune cells. Plasmacytoid dendritic cells (pDCs) as natural interferon-producing cells play critical roles as sensors of pathogens and link innate to adaptive immunity. CpG motifs within DNA sequences activating toll-like receptor 9 (TLR9) are the main stimuli eliciting IFNA1 secretion from pDCs. Adrenergic substances are capable of differentially modulating the response from various immune cells. Hence, the aim of this study was to examine how adrenoceptor stimulation influences TLR9-induced IFNA1 secretion from human pDCs. Methods: PBMCs generated from human whole blood and pDCs enriched from buffy coats were stimulated with LPS and CpG-ODN 2336 in the presence or absence of epinephrine and different adrenoceptor antagonists. Secretion of TNF and IFNA1 was measured by ELISA. Flow cytometry was used to determine efficacy of pDC enrichment and adrenoceptor expression of PBMC subsets. The influence of modified IFNA1 secretion on NK cell activity was evaluated using a colorimetric tumor cell lysis assay. Results: TLR9-induced IFNA1 secretion as well as TLR4-induced TNF secretion from PBMCs was dose-dependently attenuated by coincubation with epinephrine. Combination with different specific adrenoceptor antagonists revealed that this effect was mediated by the adrenoceptor β2 (ADRB2). Since flow cytometric analysis could exclude the presence of ADRB2 on pDCs, highly enriched pDCs lacked any visible impact of adrenoceptor stimulation on TLR9-induced IFNA1 release. Combination of pDCs with PBMCs restored the effect, even when they were separated by a permeable membrane. Suppression of TLR9-mediated IFNA1 secretion from PBMCs by adrenoceptor stimulation reduced the lytic activity of NK cells on K562 tumor cells. Conclusion: We provide insights into the underlying mechanisms of the interrelation between immune responses and pharmacological agents widely used in clinical practice. Our results have implications for the future treatment of human patients, in which the endogenous immune response plays a pivotal role, such as during viral infections, inflammatory diseases and cancers. [ABSTRACT FROM AUTHOR]

Cadmium (Cd), one of well-known highly toxic environmental and industrial pollutants, causes a number of adverse health effects and diseases in humans. The growing epidemiological studies have suggested a possible link between Cd exposure and diabetes mellitus (DM). However, the toxicological effects and underlying mechanisms of Cd-induced pancreatic β-cell injury are still unknown. In this study, we found that Cd significantly decreased cell viability, and increased sub-G1 hypodiploid cells and annexin V-Cy3 binding in pancreatic β-cell-derived RIN-m5F cells. Cd also increased intracellular reactive oxygen species (ROS) generation and malondialdehyde (MDA) production and induced mitochondrial dysfunction (the loss of mitochondrial membrane potential (MMP) and the increase of cytosolic cytochrome c release), the decreased Bcl-2 expression, increased p53 expression, poly (ADP-ribose) polymerase (PARP) cleavage, and caspase cascades, which accompanied with intracellular Cd accumulation. Pretreatment with the antioxidant N-acetylcysteine (NAC) effectively reversed these Cd-induced events. Furthermore, exposure to Cd induced the phosphorylations of c-jun N-terminal kinases (JNK), extracellular signal-regulated kinases (ERK)1/2, and p38-mitogen-activated protein kinase (MAPK), which was prevented by NAC. Additionally, the specific JNK inhibitor SP600125 or JNK-specific small interference RNA (si-RNA) transfection suppressed Cd-induced β-cell apoptosis and related signals, but not ERK1/2 and p38-MAPK inhibitors (PD98059 and SB203580) did not. However, the JNK inhibitor or JNK-specific si-RNA did not suppress ROS generation in Cd-treated cells. These results indicate that Cd induces pancreatic β-cell death via an oxidative stress downstream-mediated JNK activation-triggered mitochondria-regulated apoptotic pathway. [ABSTRACT FROM AUTHOR]