The document describes research identifying a small molecule called BRD7389 that induces insulin expression in pancreatic α-cells. Key findings include:
1) BRD7389 was identified in a high-throughput screen as inducing insulin expression in mouse α-cells. It also increased expression of the β-cell marker genes Pdx1, Pax4, Iapp, and Npy.
2) Treatment with BRD7389 caused α-cells to take on a clustered morphology resembling β-cells and to express low levels of insulin protein.
3) Profiling and biochemical assays identified BRD7389 as an inhibitor of RSK kinases, and knockdown experiments supported a role for RSK kin
Systemic analysis of data combined from genetic qtl's and gene expression dat...Laurence Dawkins-Hall
Elucidating changes in gene expression by Micro array genomic sweeps of genetic QTLs linked to Tryp resistance in WT cattle to identify putative candidates underpinning pathophysiology
This study examined the role of the insulin signaling pathway in regulating pancreatic beta-cell numbers in zebrafish. The researchers determined beta-cell counts in zebrafish larvae that were mutant for the insulin receptor genes insra and insrb, under normal and metabolically stressed conditions induced by nutrient deprivation or overfeeding. Preliminary results showed a significant reduction in beta-cell number in insra and insrb mutants under normal conditions. However, no increase in beta-cell count was observed in wild-type larvae under metabolic stress, requiring the data to be repeated. The goal was to establish zebrafish mutants as a model for studying insulin resistance and type 2 diabetes.
SSR 2015-poster-A Hypoxia-HIF-Kdm3a Pathway Controls Trophoblast Stem Cell Li...Wei Cui
This document contains abstracts from two studies:
1. The first study identifies a hypoxia/HIF/Kdm3a pathway that controls trophoblast stem cell differentiation and organization of the hemochorial placenta. This pathway regulates genes involved in trophoblast invasion and vascular remodeling during pregnancy.
2. The second study examines endometrial gene expression in lactating cows, dry cows, and heifers on day 19 of pregnancy. The study finds 135 differentially expressed genes between lactating cows and heifers, but only 17 between dry cows and heifers, suggesting lactation impacts the endometrial response to the developing embryo. Functional analysis found effects on vesicle transport and cytokine signaling
The document analyzes antigenic variation in lngA, the pilin subunit of the CS21 pilus found in enterotoxigenic E. coli (ETEC). It screened 158 ETEC strains isolated from the Middle East, finding 126 to be ETEC-positive. Of these, 41 strains were CS21-positive. Sequence analysis of lngA alleles revealed four groups with variation clustered in the αβ-loop and D-region. Further screening of additional strains is needed to characterize the antigenic variation and evaluate lngA's potential as a vaccine target.
This document describes an experiment examining the effect of the PI3K/AKT pathway on HOXA1 expression in prostate cancer stem-like cells induced by diepoxybutane (DEB). DU145 prostate cancer cells were treated with DEB to generate cancer stem cells. RNA and protein were extracted from treated and untreated cells. PCR and gel electrophoresis showed expression of HOXA1, p38, cMYC, and JNK-2 genes in DEB-treated cells. Western blot and immunofluorescence were used to analyze protein expression and localization of HOXA1, p53, and pro-caspase 9. Results indicate DEB activates the PI3K/AKT pathway, leading to changes in
1. This study analyzed clinical isolates from 2014 in Indiana for the presence of KPC carbapenemase genes. Of 56 total isolates screened, 41 were found to produce KPC variants, with the majority (28 isolates) producing KPC-3 and a smaller number (3 isolates) producing KPC-2.
2. The study observed that KPC-producing organisms were decreasing compared to an upward trend seen from 2009-2013 based on previous data. However, the reasons for the decreasing prevalence are unclear but may involve improved infection control or antibiotic usage changes.
3. Future work will continue screening 2014 isolates for other beta-lactamase genes like TEM, SHV, and CTX-M, and
This paper describes a novel method for generating large libraries for directed evolution of proteins using error-prone PCR and a Kunkel-like mutagenesis reaction. The method uses error-prone PCR to generate a mutated DNA fragment, with one primer containing phosphorothioate modifications. Treatment with exonuclease preferentially removes the non-modified strand, generating a single-stranded "megaprimer". This megaprimer is then used in a Kunkel-like reaction with a uracilated template to introduce mutations efficiently without subcloning. This approach enables generation of libraries with over 108 clones from a single E. coli transformation, which is more efficient than conventional error-prone PCR
1. The study examined the role of microRNA 146a (miR146a) in regulating plasma cholesterol levels in a mouse model of hypercholesterolemia induced by a gain-of-function mutation in proprotein convertase subtilisin/kexin type 9 (PCSK9).
2. Mice deficient in miR146a showed decreased total plasma cholesterol, including HDL-cholesterol, compared to wild-type mice when injected with an adeno-associated virus expressing the mutated form of PCSK9.
3. miR146a is thought to play a protective role against atherosclerosis by suppressing NF-kB signaling and endothelial cell activation, and the study aimed to better understand how miR
Systemic analysis of data combined from genetic qtl's and gene expression dat...Laurence Dawkins-Hall
Elucidating changes in gene expression by Micro array genomic sweeps of genetic QTLs linked to Tryp resistance in WT cattle to identify putative candidates underpinning pathophysiology
This study examined the role of the insulin signaling pathway in regulating pancreatic beta-cell numbers in zebrafish. The researchers determined beta-cell counts in zebrafish larvae that were mutant for the insulin receptor genes insra and insrb, under normal and metabolically stressed conditions induced by nutrient deprivation or overfeeding. Preliminary results showed a significant reduction in beta-cell number in insra and insrb mutants under normal conditions. However, no increase in beta-cell count was observed in wild-type larvae under metabolic stress, requiring the data to be repeated. The goal was to establish zebrafish mutants as a model for studying insulin resistance and type 2 diabetes.
SSR 2015-poster-A Hypoxia-HIF-Kdm3a Pathway Controls Trophoblast Stem Cell Li...Wei Cui
This document contains abstracts from two studies:
1. The first study identifies a hypoxia/HIF/Kdm3a pathway that controls trophoblast stem cell differentiation and organization of the hemochorial placenta. This pathway regulates genes involved in trophoblast invasion and vascular remodeling during pregnancy.
2. The second study examines endometrial gene expression in lactating cows, dry cows, and heifers on day 19 of pregnancy. The study finds 135 differentially expressed genes between lactating cows and heifers, but only 17 between dry cows and heifers, suggesting lactation impacts the endometrial response to the developing embryo. Functional analysis found effects on vesicle transport and cytokine signaling
The document analyzes antigenic variation in lngA, the pilin subunit of the CS21 pilus found in enterotoxigenic E. coli (ETEC). It screened 158 ETEC strains isolated from the Middle East, finding 126 to be ETEC-positive. Of these, 41 strains were CS21-positive. Sequence analysis of lngA alleles revealed four groups with variation clustered in the αβ-loop and D-region. Further screening of additional strains is needed to characterize the antigenic variation and evaluate lngA's potential as a vaccine target.
This document describes an experiment examining the effect of the PI3K/AKT pathway on HOXA1 expression in prostate cancer stem-like cells induced by diepoxybutane (DEB). DU145 prostate cancer cells were treated with DEB to generate cancer stem cells. RNA and protein were extracted from treated and untreated cells. PCR and gel electrophoresis showed expression of HOXA1, p38, cMYC, and JNK-2 genes in DEB-treated cells. Western blot and immunofluorescence were used to analyze protein expression and localization of HOXA1, p53, and pro-caspase 9. Results indicate DEB activates the PI3K/AKT pathway, leading to changes in
1. This study analyzed clinical isolates from 2014 in Indiana for the presence of KPC carbapenemase genes. Of 56 total isolates screened, 41 were found to produce KPC variants, with the majority (28 isolates) producing KPC-3 and a smaller number (3 isolates) producing KPC-2.
2. The study observed that KPC-producing organisms were decreasing compared to an upward trend seen from 2009-2013 based on previous data. However, the reasons for the decreasing prevalence are unclear but may involve improved infection control or antibiotic usage changes.
3. Future work will continue screening 2014 isolates for other beta-lactamase genes like TEM, SHV, and CTX-M, and
This paper describes a novel method for generating large libraries for directed evolution of proteins using error-prone PCR and a Kunkel-like mutagenesis reaction. The method uses error-prone PCR to generate a mutated DNA fragment, with one primer containing phosphorothioate modifications. Treatment with exonuclease preferentially removes the non-modified strand, generating a single-stranded "megaprimer". This megaprimer is then used in a Kunkel-like reaction with a uracilated template to introduce mutations efficiently without subcloning. This approach enables generation of libraries with over 108 clones from a single E. coli transformation, which is more efficient than conventional error-prone PCR
1. The study examined the role of microRNA 146a (miR146a) in regulating plasma cholesterol levels in a mouse model of hypercholesterolemia induced by a gain-of-function mutation in proprotein convertase subtilisin/kexin type 9 (PCSK9).
2. Mice deficient in miR146a showed decreased total plasma cholesterol, including HDL-cholesterol, compared to wild-type mice when injected with an adeno-associated virus expressing the mutated form of PCSK9.
3. miR146a is thought to play a protective role against atherosclerosis by suppressing NF-kB signaling and endothelial cell activation, and the study aimed to better understand how miR
This document discusses research into the effects of black cohosh (Cimicifuga racemosa) on breast cancer cells. It finds that extracts of black cohosh inhibit the growth of both estrogen-dependent and estrogen-independent breast cancer cell lines. The active compounds in black cohosh, such as actein and cinnamic acid esters, induce apoptosis in breast cancer cells through multiple pathways including interaction with Na-K-ATPase, HER2 receptors, and reduction of reactive oxygen species. Synergy between compounds contributes to the anti-cancer activity of black cohosh.
Antioxidant-mediated up-regulation of OGG1 via NRF2 induction is associated ...Enrique Moreno Gonzalez
Estrogen metabolism-mediated oxidative stress is suggested to play an important role in estrogen-induced breast carcinogenesis. We have earlier demonstrated that antioxidants,
vitamin C (Vit C) and butylated hydroxyanisole (BHA) inhibit 17β-estradiol (E2)-mediated oxidative stress and oxidative DNA damage, and breast carcinogenesis in female August
Copenhagen Irish (ACI) rats. The objective of the present study was to characterize the mechanism by which above antioxidants prevent DNA damage during breast carcinogenesis.
This document describes a study comparing placental malaria pathology caused by three different Plasmodium berghei parasite lines (K173, NK65, and ANKAΔpm4) in pregnant C57BL/6 mice. The parasites were able to induce severe placental malaria features like poor pregnancy outcomes and placental damage, though infection with the ANKAΔpm4 line resulted in milder disease associated with a strong innate immune response. The study establishes these parasite lines as experimental systems to identify host factors involved in placental malaria pathogenesis using the C57BL/6 mouse model.
OSU-03012 sensitizes breast cancers to lapatinib-induced cell killing: a role...Enrique Moreno Gonzalez
Lapatinib is characterized as an ErbB1/ErbB2 dual inhibitor and has recently been approved for the treatment of metastatic breast cancer. In this study, we examined mechanisms
associated with enhancing the activity of lapatinib via combination with other therapies.
This paper describes a novel vector system called pMINERVA that allows for the direct conversion of single-chain variable fragments (scFvs) selected from phage display into full-length immunoglobulin G (IgG) molecules without subcloning. The pMINERVA system takes advantage of site-specific bacteriophage integrases expressed in E. coli and intron splicing that occurs in mammalian cells. Using this system, a phage display vector contains regulatory elements to support antibody expression in both E. coli and mammalian cells. The scFv is expressed on phage as a fusion to coat protein III and can be converted to an IgG expressed in mammalian cells through recombination in E. coli. This eliminates the
Lack of induced mutagenesis-Seminario Biología Molecular-Juan Restrepo RuizJuan Restrepo Ruiz
This document summarizes an experiment evaluating the effect of irradiated meat samples on E. coli and human lymphoblast cell lines. Various assays were conducted including measuring mutation frequency in E. coli, detecting antibiotic resistance plasmids, genome sequencing, and analyzing human lymphoblast mutation rates. The results found no evidence of mutagenesis from the irradiated meat samples in any of the assays. The conclusion is that food irradiation is a safe technology for food preservation and addresses misconceptions around safety.
CETP overexpression in diabetic and obese db/db mice prevented the formation of diet-induced atherosclerotic plaques in the aorta. Mice overexpressing CETP had lower total cholesterol concentrations and less cholesterol in the VLDL and IDL/LDL subfractions compared to db/db mice. This study suggests CETP plays an anti-atherogenic role in the context of diabetic obesity by lowering atherogenic lipids and preventing plaque formation.
The Effects of Genetic Alteration on Reprogramming of Fibroblasts into Induc...remedypublications2
Induced Pluripotent Stem Cells (iPSCs) can be generated from somatic cells by ectopic expression of
Yamanaka factors (
Oct4
,
Sox2
,
Klf4
and
c-Myc
) or combination of other factors. Genetic alteration
of fibroblasts exhibits an effect on reprogramming efficiency through multiple signaling pathways,
including epigenetic modifications, metabolic shifts, Mesenchymal-To-Epithelial Transition
(MET) and cell proliferation. In order to better understand the underlying mechanisms in cell fate
determination, in this review we will summarize several genetic alterations involved in the regulation
of reprogramming fibroblasts into iPSCs.
Search for atoxic cereals: a single blind, cross-over study on the safety of...Enrique Moreno Gonzalez
Cereals of baking quality with absent or reduced toxicity are actively sought as alternative therapy to a gluten-free diet (GFD) for patients with coeliac disease (CD). Triticum monococcum, an ancient wheat, is a potential candidate having no toxicity in in-vitro and exvivo studies. The aim of our study was to investigate on the safety of administration of a single dose of gluten of Tm in patients with CD on GFD.
Genetics of fetal hemoglobin in tribal Indian patients sickle cell anemiaSujata Singh
This study investigated the association between genetic variants known to influence fetal hemoglobin (HbF) levels and HbF levels in 240 Indian patients with sickle cell anemia and 60 with sickle cell trait. Genotyping was performed for variants in the BCL11A, HMIP, and HBB genes. All three quantitative trait loci were associated with HbF levels, with the strongest association seen for the HBB Xmn1 variant. The BCL11A and HMIP variants were also associated with HbF levels and explained a percentage of trait variance. This is the first such study in India and indicates these genetic factors influence HbF levels and likely disease severity in this population.
ADAR2 editing activity in newly diagnosed versus relapsed pediatric high-grad...Enrique Moreno Gonzalez
High-grade (WHO grade III and IV) astrocytomas are aggressive malignant brain tumors affecting humans with a high risk of recurrence in both children and adults. To date, limited information is available on the genetic and molecular alterations important in the onset and progression of pediatric high-grade astrocytomas and, even less, on the prognostic factors that influence long-term outcome in children with recurrence. A-to-I RNA editing is an essential post-transcriptional mechanism that can alter the nucleotide sequence of several RNAs and is
mediated by the ADAR enzymes. ADAR2 editing activity is particularly important in mammalian brain and is impaired in both adult and pediatric high-grade astrocytomas.
Moreover, we have recently shown that the recovered ADAR2 activity in high-grade astrocytomas inhibits in vivo tumor growth. The aim of the present study is to investigate whether changes may occur in ADAR2-mediated RNA editing profiles of relapsed highgrade astrocytomas compared to their respective specimens collected at diagnosis, in four pediatric patients.
This study examined how reduced expression of the integrin β3 subunit gene (Itgb3) in mice affects serotonin transporter (SERT) function and the effectiveness of selective serotonin reuptake inhibitors (SSRIs). The researchers found that αvβ3 integrin receptors and SERTs are enriched in presynaptic membranes in brain regions involved in mood regulation. Loss of one Itgb3 allele decreased serotonin uptake by SERT in midbrain synaptosomes. Pharmacological assays suggest this is due to fewer active SERT molecules when integrin β3 levels decrease. Mice with one less Itgb3 allele required a lower SSRI dose to show antidepressant effects. The researchers also identified a human ITGB3 variant associated
This document summarizes a mathematical model of glioma (brain cancer) growth that includes explicit tumor cell birth and death rates.
The model treats tumor growth as a reaction-diffusion process, tracking the densities of proliferating tumor cells, necrotic cells, and available nutrient/space over time. Birth and death rates depend on nutrient availability.
The model predicts a traveling wave solution that matches MRI image data of the layered tumor structure. Parameter values like growth rate and wave speed can be estimated by fitting the model wave profile to measurements of tumor widths from images taken at different times.
Simplified analysis shows the traveling wave form and allows some parameter identifiability. Numerical simulations validate the approximate analytical solution.
This study developed a system using fruit flies (Drosophila) to model tumor growth induced by the oncogene Ras. Cells containing an inducible Ras gene were injected into flies. Flies injected with cells and fed a drug (RU486) developed tumors that killed the hosts, while flies injected without the drug did not develop tumors and survived, demonstrating controlled tumor formation. Optimal conditions for injecting cells and inducing tumor growth were determined. This system allows examination of oncogene-driven tumorigenesis in a controllable manner.
Genomic evaluation of low-heritability traits: dairy cattle health as a modelJohn B. Cole, Ph.D.
Genetic selection has been very successful when applied to traits of moderate to high heritability, but progress has been slow for traits with low heritabilities. The problem is further compounded when novel traits are considered because data needed to calculate high-reliability PTA generally are not available. A combination of producer-recorded health event data and SNP genotypes may permit the routine calculation of PTA with reasonable reliabilities for health traits.
Poster presented at the 5th International Symposium on Animal Functional Genetics in Guaruja, Brazil, in 2014.
Differences in the endometrial transcript profile during the receptive period between women who were refractory to implantation and those who achieved pregnancy.
By Luis Alberto Velásquez Cumplido
This study examined the efficacy of a gonadotropin-releasing hormone (GnRH) vaccine in suppressing reproductive function in female vervet monkeys on the island of St. Kitts. Six experimental monkeys were given the GnRH vaccine using two different adjuvants and blood samples were collected over time to analyze hormone levels. Adjuvant 1 suppressed cycling in monkeys for 32 to 52+ weeks, while adjuvant 2 suppressed cycling for 17 to 30 weeks. Both adjuvants caused moderate to severe reactions at the injection site. The study concludes that while the vaccine can suppress cycling, the side effects of currently available adjuvants limit its suitability for use in wild monkey populations on St. Kitts.
DOI: 10.21276/ijlssr.2016.2.3.5
ABSTRACT- Purpose: Multidrug resistant organisms are on rise. Various enzymes present in the organisms are
responsible for this resistance. Detection of these enzymes become challenging if organisms harbor multiple enzymes.
This study was done to find the prevalence of various enzymes at our tertiary care hospital.
Materials and methods: Extended spectrum beta lactamases (ESBL) detection was done by screening method followed
by two phenotypic confirmatory methods (double disc synergy and disc potentiation method). Carbapenems (imipenem,
meropenem) resistant strain were analyzed for metallo beta lactamases (MBL) and carbapenemases (KPC) using
combined disc test and modified Hodge test. Amp C detection was done by using cefoxitin disc on heavy lawn of E. coli
ATCC 25922. Distortion of the zone size on the streaked line of test was taken as positive for Amp C.
Results: 87.15% were screened positive for ESBL and confirmed cases were 36.80%. Carbapenem resistant was 31.86%,
MBL was 7.52%, KPC was 0.82 %, Amp C in 0.23%.
Conclusions: There is high prevalence of ESBL. Detection of these enzymes is important in routine diagnostics for
treatment. Co-expression of multiple enzymes was detected in this study. Judicious and rational use of antibiotics is
required which might lead to decrease in emergence of resistance. Also knowledge of the prevalence of these enzymes
helps in empirical antibiotic therapy and in infection control purpose.
Key-words- Multidrug resistant, ESBL, MBL, KPC, Amp C
Participation of the oviductal s100 calcium binding protein G in the genomic effect of estradiol that accelerates oviductal embryo transport in mated rats
Mariana Ríos1, Alexis Parada-Bustamante1, Luis A Velásquez2,3, Horacio B Croxatto2,3,4 and Pedro A Orihuela2,3*
By Luis Alberto Velasquez Cumplido
GW8510 increases insulin expression in pancreatic alpha cells through activation of p53 transcriptional activity. Treatment of mouse alpha cells and human islet cells with GW8510 upregulates insulin expression and secretion. Gene expression profiling reveals upregulation of p53 target genes in alpha cells treated with GW8510. Further experiments show that GW8510 enhances p53 transcriptional activity and increases p53 phosphorylation and acetylation associated with increased activity. GW8510 treatment arrests alpha cell cycle progression, consistent with activation of p53. These results suggest that GW8510 induces insulin expression by activating the p53 signaling pathway in alpha cells.
This document summarizes a study investigating the epigenetic changes that occur during the transdifferentiation of pancreatic acinar cells (HPACs) to hepatocyte-like cells. The study found that DNA methylation in HPACs peaks at 24 hours after treatment with dexamethasone, and the cells display phenotypic changes associated with hepatocytes after 7 days. The study also examined the promoter sequence of the SGK1 gene, which is involved in the transdifferentiation process. The results suggest therapeutic potential for producing hepatocytes from pancreatic cells to treat liver disease in a more cost-effective manner than current alternatives.
This document discusses research into the effects of black cohosh (Cimicifuga racemosa) on breast cancer cells. It finds that extracts of black cohosh inhibit the growth of both estrogen-dependent and estrogen-independent breast cancer cell lines. The active compounds in black cohosh, such as actein and cinnamic acid esters, induce apoptosis in breast cancer cells through multiple pathways including interaction with Na-K-ATPase, HER2 receptors, and reduction of reactive oxygen species. Synergy between compounds contributes to the anti-cancer activity of black cohosh.
Antioxidant-mediated up-regulation of OGG1 via NRF2 induction is associated ...Enrique Moreno Gonzalez
Estrogen metabolism-mediated oxidative stress is suggested to play an important role in estrogen-induced breast carcinogenesis. We have earlier demonstrated that antioxidants,
vitamin C (Vit C) and butylated hydroxyanisole (BHA) inhibit 17β-estradiol (E2)-mediated oxidative stress and oxidative DNA damage, and breast carcinogenesis in female August
Copenhagen Irish (ACI) rats. The objective of the present study was to characterize the mechanism by which above antioxidants prevent DNA damage during breast carcinogenesis.
This document describes a study comparing placental malaria pathology caused by three different Plasmodium berghei parasite lines (K173, NK65, and ANKAΔpm4) in pregnant C57BL/6 mice. The parasites were able to induce severe placental malaria features like poor pregnancy outcomes and placental damage, though infection with the ANKAΔpm4 line resulted in milder disease associated with a strong innate immune response. The study establishes these parasite lines as experimental systems to identify host factors involved in placental malaria pathogenesis using the C57BL/6 mouse model.
OSU-03012 sensitizes breast cancers to lapatinib-induced cell killing: a role...Enrique Moreno Gonzalez
Lapatinib is characterized as an ErbB1/ErbB2 dual inhibitor and has recently been approved for the treatment of metastatic breast cancer. In this study, we examined mechanisms
associated with enhancing the activity of lapatinib via combination with other therapies.
This paper describes a novel vector system called pMINERVA that allows for the direct conversion of single-chain variable fragments (scFvs) selected from phage display into full-length immunoglobulin G (IgG) molecules without subcloning. The pMINERVA system takes advantage of site-specific bacteriophage integrases expressed in E. coli and intron splicing that occurs in mammalian cells. Using this system, a phage display vector contains regulatory elements to support antibody expression in both E. coli and mammalian cells. The scFv is expressed on phage as a fusion to coat protein III and can be converted to an IgG expressed in mammalian cells through recombination in E. coli. This eliminates the
Lack of induced mutagenesis-Seminario Biología Molecular-Juan Restrepo RuizJuan Restrepo Ruiz
This document summarizes an experiment evaluating the effect of irradiated meat samples on E. coli and human lymphoblast cell lines. Various assays were conducted including measuring mutation frequency in E. coli, detecting antibiotic resistance plasmids, genome sequencing, and analyzing human lymphoblast mutation rates. The results found no evidence of mutagenesis from the irradiated meat samples in any of the assays. The conclusion is that food irradiation is a safe technology for food preservation and addresses misconceptions around safety.
CETP overexpression in diabetic and obese db/db mice prevented the formation of diet-induced atherosclerotic plaques in the aorta. Mice overexpressing CETP had lower total cholesterol concentrations and less cholesterol in the VLDL and IDL/LDL subfractions compared to db/db mice. This study suggests CETP plays an anti-atherogenic role in the context of diabetic obesity by lowering atherogenic lipids and preventing plaque formation.
The Effects of Genetic Alteration on Reprogramming of Fibroblasts into Induc...remedypublications2
Induced Pluripotent Stem Cells (iPSCs) can be generated from somatic cells by ectopic expression of
Yamanaka factors (
Oct4
,
Sox2
,
Klf4
and
c-Myc
) or combination of other factors. Genetic alteration
of fibroblasts exhibits an effect on reprogramming efficiency through multiple signaling pathways,
including epigenetic modifications, metabolic shifts, Mesenchymal-To-Epithelial Transition
(MET) and cell proliferation. In order to better understand the underlying mechanisms in cell fate
determination, in this review we will summarize several genetic alterations involved in the regulation
of reprogramming fibroblasts into iPSCs.
Search for atoxic cereals: a single blind, cross-over study on the safety of...Enrique Moreno Gonzalez
Cereals of baking quality with absent or reduced toxicity are actively sought as alternative therapy to a gluten-free diet (GFD) for patients with coeliac disease (CD). Triticum monococcum, an ancient wheat, is a potential candidate having no toxicity in in-vitro and exvivo studies. The aim of our study was to investigate on the safety of administration of a single dose of gluten of Tm in patients with CD on GFD.
Genetics of fetal hemoglobin in tribal Indian patients sickle cell anemiaSujata Singh
This study investigated the association between genetic variants known to influence fetal hemoglobin (HbF) levels and HbF levels in 240 Indian patients with sickle cell anemia and 60 with sickle cell trait. Genotyping was performed for variants in the BCL11A, HMIP, and HBB genes. All three quantitative trait loci were associated with HbF levels, with the strongest association seen for the HBB Xmn1 variant. The BCL11A and HMIP variants were also associated with HbF levels and explained a percentage of trait variance. This is the first such study in India and indicates these genetic factors influence HbF levels and likely disease severity in this population.
ADAR2 editing activity in newly diagnosed versus relapsed pediatric high-grad...Enrique Moreno Gonzalez
High-grade (WHO grade III and IV) astrocytomas are aggressive malignant brain tumors affecting humans with a high risk of recurrence in both children and adults. To date, limited information is available on the genetic and molecular alterations important in the onset and progression of pediatric high-grade astrocytomas and, even less, on the prognostic factors that influence long-term outcome in children with recurrence. A-to-I RNA editing is an essential post-transcriptional mechanism that can alter the nucleotide sequence of several RNAs and is
mediated by the ADAR enzymes. ADAR2 editing activity is particularly important in mammalian brain and is impaired in both adult and pediatric high-grade astrocytomas.
Moreover, we have recently shown that the recovered ADAR2 activity in high-grade astrocytomas inhibits in vivo tumor growth. The aim of the present study is to investigate whether changes may occur in ADAR2-mediated RNA editing profiles of relapsed highgrade astrocytomas compared to their respective specimens collected at diagnosis, in four pediatric patients.
This study examined how reduced expression of the integrin β3 subunit gene (Itgb3) in mice affects serotonin transporter (SERT) function and the effectiveness of selective serotonin reuptake inhibitors (SSRIs). The researchers found that αvβ3 integrin receptors and SERTs are enriched in presynaptic membranes in brain regions involved in mood regulation. Loss of one Itgb3 allele decreased serotonin uptake by SERT in midbrain synaptosomes. Pharmacological assays suggest this is due to fewer active SERT molecules when integrin β3 levels decrease. Mice with one less Itgb3 allele required a lower SSRI dose to show antidepressant effects. The researchers also identified a human ITGB3 variant associated
This document summarizes a mathematical model of glioma (brain cancer) growth that includes explicit tumor cell birth and death rates.
The model treats tumor growth as a reaction-diffusion process, tracking the densities of proliferating tumor cells, necrotic cells, and available nutrient/space over time. Birth and death rates depend on nutrient availability.
The model predicts a traveling wave solution that matches MRI image data of the layered tumor structure. Parameter values like growth rate and wave speed can be estimated by fitting the model wave profile to measurements of tumor widths from images taken at different times.
Simplified analysis shows the traveling wave form and allows some parameter identifiability. Numerical simulations validate the approximate analytical solution.
This study developed a system using fruit flies (Drosophila) to model tumor growth induced by the oncogene Ras. Cells containing an inducible Ras gene were injected into flies. Flies injected with cells and fed a drug (RU486) developed tumors that killed the hosts, while flies injected without the drug did not develop tumors and survived, demonstrating controlled tumor formation. Optimal conditions for injecting cells and inducing tumor growth were determined. This system allows examination of oncogene-driven tumorigenesis in a controllable manner.
Genomic evaluation of low-heritability traits: dairy cattle health as a modelJohn B. Cole, Ph.D.
Genetic selection has been very successful when applied to traits of moderate to high heritability, but progress has been slow for traits with low heritabilities. The problem is further compounded when novel traits are considered because data needed to calculate high-reliability PTA generally are not available. A combination of producer-recorded health event data and SNP genotypes may permit the routine calculation of PTA with reasonable reliabilities for health traits.
Poster presented at the 5th International Symposium on Animal Functional Genetics in Guaruja, Brazil, in 2014.
Differences in the endometrial transcript profile during the receptive period between women who were refractory to implantation and those who achieved pregnancy.
By Luis Alberto Velásquez Cumplido
This study examined the efficacy of a gonadotropin-releasing hormone (GnRH) vaccine in suppressing reproductive function in female vervet monkeys on the island of St. Kitts. Six experimental monkeys were given the GnRH vaccine using two different adjuvants and blood samples were collected over time to analyze hormone levels. Adjuvant 1 suppressed cycling in monkeys for 32 to 52+ weeks, while adjuvant 2 suppressed cycling for 17 to 30 weeks. Both adjuvants caused moderate to severe reactions at the injection site. The study concludes that while the vaccine can suppress cycling, the side effects of currently available adjuvants limit its suitability for use in wild monkey populations on St. Kitts.
DOI: 10.21276/ijlssr.2016.2.3.5
ABSTRACT- Purpose: Multidrug resistant organisms are on rise. Various enzymes present in the organisms are
responsible for this resistance. Detection of these enzymes become challenging if organisms harbor multiple enzymes.
This study was done to find the prevalence of various enzymes at our tertiary care hospital.
Materials and methods: Extended spectrum beta lactamases (ESBL) detection was done by screening method followed
by two phenotypic confirmatory methods (double disc synergy and disc potentiation method). Carbapenems (imipenem,
meropenem) resistant strain were analyzed for metallo beta lactamases (MBL) and carbapenemases (KPC) using
combined disc test and modified Hodge test. Amp C detection was done by using cefoxitin disc on heavy lawn of E. coli
ATCC 25922. Distortion of the zone size on the streaked line of test was taken as positive for Amp C.
Results: 87.15% were screened positive for ESBL and confirmed cases were 36.80%. Carbapenem resistant was 31.86%,
MBL was 7.52%, KPC was 0.82 %, Amp C in 0.23%.
Conclusions: There is high prevalence of ESBL. Detection of these enzymes is important in routine diagnostics for
treatment. Co-expression of multiple enzymes was detected in this study. Judicious and rational use of antibiotics is
required which might lead to decrease in emergence of resistance. Also knowledge of the prevalence of these enzymes
helps in empirical antibiotic therapy and in infection control purpose.
Key-words- Multidrug resistant, ESBL, MBL, KPC, Amp C
Participation of the oviductal s100 calcium binding protein G in the genomic effect of estradiol that accelerates oviductal embryo transport in mated rats
Mariana Ríos1, Alexis Parada-Bustamante1, Luis A Velásquez2,3, Horacio B Croxatto2,3,4 and Pedro A Orihuela2,3*
By Luis Alberto Velasquez Cumplido
GW8510 increases insulin expression in pancreatic alpha cells through activation of p53 transcriptional activity. Treatment of mouse alpha cells and human islet cells with GW8510 upregulates insulin expression and secretion. Gene expression profiling reveals upregulation of p53 target genes in alpha cells treated with GW8510. Further experiments show that GW8510 enhances p53 transcriptional activity and increases p53 phosphorylation and acetylation associated with increased activity. GW8510 treatment arrests alpha cell cycle progression, consistent with activation of p53. These results suggest that GW8510 induces insulin expression by activating the p53 signaling pathway in alpha cells.
This document summarizes a study investigating the epigenetic changes that occur during the transdifferentiation of pancreatic acinar cells (HPACs) to hepatocyte-like cells. The study found that DNA methylation in HPACs peaks at 24 hours after treatment with dexamethasone, and the cells display phenotypic changes associated with hepatocytes after 7 days. The study also examined the promoter sequence of the SGK1 gene, which is involved in the transdifferentiation process. The results suggest therapeutic potential for producing hepatocytes from pancreatic cells to treat liver disease in a more cost-effective manner than current alternatives.
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1. Small-molecule inducers of insulin expression in
pancreatic α-cells
Dina Fomina-Yadlina,b,c,1
, Stefan Kubiceka,b,1
, Deepika Walpitab
, Vlado Dancikb,d
, Jacob Hecksher-Sørensenb,2
,
Joshua A. Bittkerb
, Tanaz Sharifniab,e
, Alykhan Shamjib
, Paul A. Clemonsb
, Bridget K. Wagnerb
, and
Stuart L. Schreibera,b,f,3
a
Howard Hughes Medical Institute, b
Broad Institute of Harvard and Massachusetts Institute of Technology, Cambridge, MA 02142; c
Department of Molecular
and Cellular Biology, Harvard University, Cambridge, MA 02138; d
Mathematical Institute, Slovak Academy of Sciences, Košice, 040 01, Slovakia; e
Department
Biological and Biomedical Sciences, Harvard Medical School, Boston, MA 02115; and f
Department of Chemistry and Chemical Biology, Harvard University,
Cambridge, MA 02138
Contributed by Stuart L. Schreiber, July 15, 2010 (sent for review June 22, 2010)
High-content screening for small-molecule inducers of insulin
expression identified the compound BRD7389, which caused α-cells
to adopt several morphological and gene expression features of
a β-cell state. Assay-performance profile analysis suggests kinase
inhibition as a mechanism of action, and we show that biochemical
and cellular inhibition of the RSK kinase family by BRD7389 is likely
related to its ability induce a β-cell-like state. BRD7389 also in-
creases the endocrine cell content and function of donor human
pancreatic islets in culture.
BRD7389 | pancreatic islets | Rsk kinase | transdifferentiation | beta cells
Type 1 diabetes is an autoimmune disease characterized by the
loss of insulin-producing β-cells in pancreatic islets of Lang-
erhans. Islet transplantation into the liver can effectively cure the
disease (1), but is not an ideal treatment due to limited donor
material and immunological complications. An alternative ap-
proach, not yet feasible, is to create new β-cells (2), either by
stepwise differentiation of undifferentiated stem or stem-like cells
(3), or by transdifferentiation (4), the heritable change of cell
identity to an insulin-producing (β-like) cell. The latter approach
could result in a replacement source for the deficient cell type
directly from patient material (either in vivo or ex vivo). Increasing
β-cell mass by small-molecule drug-induced transdifferentiation is
a speculative but exciting approach to treating diabetes—one that
is significantly different from currently available small-molecule
drugs that increase insulin secretion in existing β-cells and are
therefore ineffective in the later stages of type 1 diabetes, in which
most β-cell mass has been lost.
Cell-type specification in the pancreas is regulated by a set of
master regulatory transcription factors that control the transition
from one progenitor cell state to the next, ultimately yielding
mature endocrine cell types in islets (5). Recently, it has been
shown that misexpression of these master regulatory transcription
factors causes direct transdifferentiation between cell types. For
example, ectopic overexpression of a single transcription factor
(Arx) is sufficient for in vivo conversion of β-cells to α-cells in the
adult mouse pancreas (6). Similarly, viral delivery of three tran-
scription factors (Pdx1, Ngn3, MafA) to an adult mouse pancreas
causes the transdifferentiation of acinar cells to β-cells (7). Finally,
in vivo conversion of α-cells to β-cells has recently been achieved in
mature mouse α-cells by ectopic overexpression of Pax4 (8).
Results
Because a single gene is sufficient to induce transdifferentiation of
α-cells to β-cells, we sought to determine whether a small molecule
could have the same effect. Possible readouts for induction of a β-
cell state include insulin production and insulin secretion. We
chose to target the production of insulin protein because we
imagined that this would be more feasible to achieve in the course
of a 3-d small-molecule treatment than insulin secretion. To that
end, we developed a high-content, cell-based assay to detect in-
sulin protein expression in the mouse α-cell line αTC1. Normal
mouse α-cells are insulin negative, but have the ability to adopt
a β-cell phenotype after extreme β-cell loss (9). Similarly, the α-cell
line we used spontaneously reexpressed small but detectable levels
of insulin, despite being a subclone selected for low insulin protein
(10). During assay development and optimization, we could show,
by spiking in β-cells and by antibody competition, that our assay
was sensitive enough to reliably detect insulin levels in as few as
3% of cells, and at 15-fold lower levels than in β-cells (Fig. S1).
We screened 30,710 compounds for induction of insulin pro-
duction using this assay and identified a molecule, BRD7389 (Fig.
1A), that after 3-d treatment induced insulin expression in mouse
α-cells. BRD7389 induced a dose-dependent up-regulation of
Ins2 mRNA, peaking at ≈0.85 μM; 5-d treatment with BRD7389
resulted in greater induction of insulin gene expression, about 50-
fold at 0.85 μM (Fig. 1B), which could not be further increased by
longer treatments up to 21 d. This compound appears to be spe-
cific to α-cells, because a pancreatic ductal cell line (PANC-1)
showed no induction, and a mouse β-cell line (βTC3) no further
increase of insulin expression. In addition to insulin expression,
BRD7389 significantly up-regulated expression of Pdx1 (Fig. 1C),
a master regulatory transcription factor that specifies pancreatic
progenitors and directly activates the insulin promoter (11). We
also observed a dose-dependent increase in the expression of
other β-cell markers, including Pax4, Iapp, and Npy, after a 5-d
treatment with BRD7389 (Fig. S2).
Treatment with BRD7389 caused a stable change in cell shape
from a fibroblast-like morphology, characteristic of α-cells, to
a clustered state resembling β-cells in culture (Fig. 1 D–F, Left).
Finally, we detected low levels of insulin protein in compound-
treated α-cells by immunofluorescence (Fig. 1 D–F, Right). Rela-
tive to background fluorescence in DMSO-treated α-cells, insulin
staining is induced 1.5-fold following 5-d treatment with BRD7389,
compared with 4-fold higher levels in β-cells. Both insulin mRNA
and protein levels are significantly increased from a basal α-cell
state in compound-treated cells, but do no reach levels detected in
mature β-cells. Therefore, although these cells have not achieved
a β-cell state, they have adopted several features of β-cells.
To identify the mechanism of action of BRD7389, we used
screening data in ChemBank (12) to compare assay performance
Author contributions: D.F.-Y., S.K., J.H.-S., B.K.W., and S.L.S. designed research; D.F.-Y.,
S.K., and T.S. performed research; D.W. and V.D. contributed new reagents/analytic tools;
D.F.-Y., S.K., J.A.B., and P.A.C. analyzed data; and D.F.-Y., S.K., A.S., B.K.W., and S.L.S wrote
the paper.
The authors declare no conflict of interest.
Freely available online through the PNAS open access option.
1
D.F.-Y. and S.K. contributed equally to this work.
2
Present address: Hagedorn Research Institute, DK-2820 Gentofte, Denmark.
3
To whom correspondence should be addressed. E-mail: stuart_schreiber@harvard.edu.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1010018107/-/DCSupplemental.
www.pnas.org/cgi/doi/10.1073/pnas.1010018107 PNAS | August 24, 2010 | vol. 107 | no. 34 | 15099–15104
CELLBIOLOGY
2. of BRD7389 with 9,995 other small molecules in a total of 32 assays
involving both BRD7389 and other compounds. This computa-
tional method looks for similarity of biological assay-performance
profiles among a diverse set of compounds, including many known
“bioactives.” We uncovered multiple connections of BRD7389 to
known kinase inhibitors. Accordingly, we profiled this compound
at 10 μM against a panel of 219 kinases, selected to represent
a diverse subset of the human kinome (13). We observed signifi-
cant inhibition of a number of kinases, including FLT3, DRAK2,
and the RSK family (Fig. 2A and Table S1). To validate these
profiling results, we obtained dose–response curves and deter-
mined half-maximal inhibitory concentration (IC50) values for
BRD7389 and the most potently inhibited kinases (Fig. S3). The
compound was most active against the entire RSK family of ki-
nases, with IC50 values of 1.5 μM, 2.4 μM, and 1.2 μM for RSK1,
RSK2, and RSK3, respectively (Fig. 2B). Therefore, we focused on
investigating the role of RSK kinases in α-cells.
In addition to measuring the biochemical in vitro inhibition
of RSKs, we also determined the functional consequences of
BRD7389 on Rsk activity in mouse α-cells. All Rsk kinases consist
of two functional domains, which are activated through a series
of consecutive phosphorylation events (14). Kinase activity was
measured using pan- and phospho-specific antibodies to detect
total and active Rsk protein in αTC1 cells. Western blot analy-
sis revealed a 50% decrease in kinase activity, as measured by
autophosphorylation of both N-terminal and C-terminal domains,
at concentrations above 3.4 μM (Fig. 2 C and E). Phosphorylation
of ribosomal protein S6 at serines 235 and 236, direct targets of
the Rsk kinases (15), was reduced by a similar amount after
compound treatment (Fig. 2 D and F). These findings confirm that
BRD7389 has activity as an Rsk family kinase inhibitor in vitro
and in cell culture.
We then sought to determine whether knockdown of Rsk
family members would have an effect on insulin production in
α-cells. We observed 2- to 4-fold increases in insulin expres-
sion upon RNAi of individual Rsk proteins, especially Rsk2 and
Rsk3, but the effect is not as strong as compound treatment with
BRD7389 (Fig. 2G). The knockdown efficiency was at least 50%
for all constructs (Fig. 2H), and better knockdown did not cor-
relate with stronger induction of insulin expression. Similar to
compound treatment, which causes maximum induction of insulin
expression at concentrations around the biochemical IC50 for
Rsks, only partial knockdown of the enzymes seems optimal for
insulin induction.
Though mouse α-cells are useful for screening, species differ-
ences and potential microenvironmental factors make testing
compounds in human pancreatic cells essential. Using human
donor-derived pancreatic islets, we tested BRD7389 in dissociated
islet cells cultured on an extracellular matrix (16) designed to
preserve the functional characteristics of β-cells. Though we did
observe donor-to-donor variability in the response to BRD7389,
some observations were shared among islets from donors with
a low body-mass index (BMI) (Fig. 3 and Figs. S4–S8). For ex-
ample, 5-d treatment with BRD7389 enhanced glucose-stimulated
insulin secretion (GSIS) in both high- (16.7 mM) and low-glucose
(1.67 mM) conditions (Fig. 3A), as well as glucose-stimulated
glucagon secretion (GSGS) in low-glucose (1.67 mM) conditions
(Fig. 3B). Moreover, we detected a dose-dependent increase in the
expression of endocrine hormones and transcription factors fol-
lowing 5-d compound treatment (Fig. 3C). Microscopy revealed
Fig. 1. BRD7389 stimulates insulin production in a mouse α-cell line. (A) Structure of BRD7389. Quantitative PCR analysis of (B) insulin (Ins2) and (C) Pdx1
expression following 3- and 5-d treatment with the indicated concentrations of BRD7389. Gene expression was normalized by actin (Actb) expression, and
scored relative to the DMSO-treated controls. Data represent the mean ± SDs of three independent experiments. Bright field microscopy and immunoflu-
orescence for insulin protein in the Cy5 channel was performed on (D) DMSO-treated αTC1 α-cells (average Cy5 intensity per cell: 212 ± 43), (E) αTC1 cells
treated for 5 d with 3.4 μM BRD7389 (average Cy5 intensity per cell: 320 ± 67), and (F) DMSO-treated βTC3 β-cells (average Cy5 intensity per cell: 865 ± 177).
(Scale bar: 50 μm.)
15100 | www.pnas.org/cgi/doi/10.1073/pnas.1010018107 Fomina-Yadlin et al.
3. Fig. 2. BRD7389 inhibits the RSK family of kinases. (A) Inhibition of the AGC family of kinases (figure modified from ref. 13). Biochemical inhibition of
selected kinases by 10 μM BRD7389 was tested at an ATP concentration within 15 μM of the apparent KM, resulting in 75% activity remaining (gray), 51–75%
remaining (yellow), 25–50% remaining (orange), or 25% remaining (maroon). (B) Dose-dependent inhibition of RSK kinases by BRD7389. Each protein was
incubated with the indicated concentration of BRD7389, and kinase activity was determined in a radiometric filter-binding assay. Activity was scored relative
to DMSO. (C) Western blot analysis for levels and activity of intracellular RSKs upon 5-d compound treatment. Each blot was simultaneously probed with the
indicated primary antibody (Rsk1/2/3, Rsk pT359/pS363, or Rsk pT573) and β-actin antibody, followed by incubation with IRDye-labeled secondary antibodies.
Blots were scanned on an infrared imaging system. (D) Western blot analysis for levels and phosphorylation of intracellular ribosomal protein S6 upon
compound treatment. Each blot was simultaneously probed with the indicated primary antibody (S6rp, S6rp pS235/pS236, S6rp pS240/pS244) and β-actin
antibody, followed by incubation with IRDye-labeled secondary antibodies. Blots were scanned on an infrared imaging system. (E) Fraction of active Rsk by
quantification of Western blots in C. Each specific band was quantified using Odyssey software, normalized to the β-actin signal, and phosphorylation was
plotted as the ratio of normalized phospho-specific to normalized pan-specific antibody signal. (F) Fraction of phosphorylated ribosomal protein S6 by
quantification of Western blots in D as described. (G) Insulin (Ins2) mRNA levels following treatment with BRD7389 or knockdown of the indicated Rsk kinases.
For knockdowns, αTC1 cells were infected with lentiviruses carrying expression cassettes that encode short hairpin RNAs directed against the indicated Rsk.
The following day, infected cells were selected with puromycin, and RNA was prepared after 4 additional d. Significant difference from the average of empty
vector controls, **P 0.01. (H) Knockdown efficiency of Rsk hairpins. Remaining mRNA levels following knockdown of individual Rsk enzymes assayed by
qPCR with corresponding primer sets.
Fomina-Yadlin et al. PNAS | August 24, 2010 | vol. 107 | no. 34 | 15101
CELLBIOLOGY
4. that the total number of cells in culture decreased, with ≈50% of
cells remaining at 3.4 μM BRD7389 (Fig. 3 D and E). Nonetheless,
the β-cell population remained essentially unchanged, decreasing
only slightly at higher compound concentrations, whereas the
α-cell population decreased dramatically at high concentrations
(Fig. 3D). Staining for cleaved caspase 3, an indicator of apoptosis,
revealed an increase in the fraction of total cells undergoing ap-
optosis (Fig. S9). Whereas other cell types start undergoing apo-
ptosis at 1.7 μM BRD7389, β-cells are only marginally affected at
the highest concentration tested. These differences in viability and
the resulting changes in the ratios of cell types are likely too small
to account for the increases in expression of β-cell-specific genes,
suggesting that treatment with BRD7389 either induces β-cell-like
characteristics in non–β-cells, or enhances existing β-cell function
in human pancreatic islet culture.
Discussion
In summary, we have identified a unique small molecule that up-
regulates insulin expression, normally a defining property of
pancreatic β-cells, in terminally differentiated α-cells. A mecha-
nism potentially involving the inhibition of RSK kinases is sup-
ported by the increase in insulin expression following knockdown
of individual RSK kinases. Our findings raise the possibility that
BRD7389 functions by inhibiting multiple RSK family members
simultaneously. Interestingly, previously described RSK inhib-
itors (17) FMK and BI-D1870 did not induce insulin expression in
α-cells. These compounds inhibit not only RSK enzymes, but also
members of several other kinase families (18). These data suggest
that a tight specificity profile for different kinases might be nec-
essary for optimal induction of insulin expression in α-cells.
Therefore, a systematic evaluation of the entire kinome by both
small-molecule and knockdown approaches will better define the
roles of on- and off-target effects and may lead to the identifica-
tion of conditions for complete transdifferentiation to β-cells.
BRD7389 also increases β-cell–specific gene expression in
primary human islet cells. These experiments could in principle be
confounded by differences in donor age, sex, BMI, and the purity
and viability of islet batches. We found that differences in BMI
appear to influence compound effects; there was an increase in
endocrine hormone secretion in islets from lower BMI donors,
whereas islets from high-BMI donors had attenuated responses.
Interestingly, primary human islet cells tolerate higher concen-
trations of BRD7389 than the mouse α-cell line used here. Al-
though we observed pronounced compound effects on endocrine
cell numbers and function, it is not clear whether these effects are
mediated through effects on α-cells or other cell types in this
Fig. 3. BRD7389 affects primary human islets. (A) Glucose-stimulated insulin secretion after 5-d treatment with the indicated concentration of BRD7389.
Data represent mean ± SD of four replicates. Single asterisk indicates significant difference from DMSO-treated control (*P 0.05 and **P 0.01).
(B) Glucose-stimulated glucagon secretion after 5-d treatment with the indicated concentration of BRD7389. Data represent mean ± SD of four replicates.
Significant difference from DMSO-treated control, **P 0.01. (C) Relative gene-expression changes of endocrine specific (colored lines) and control (gray
lines) genes following 5-d treatment with the indicated concentration of BRD7389. Data represent mean ± SD of four replicates. (D) Quantification of relative
cell numbers compared with DMSO-treated controls from immunofluorescence samples. Data represent mean ± SD of four replicates. (E) Representative
images of dissociated human-islet cells treated for 5 d with the indicated concentrations of BRD7389. Immunofluorescence staining was performed with
insulin and glucagon antibodies; DNA was stained with Hoechst 33342. (Scale bar: 50 μm.)
15102 | www.pnas.org/cgi/doi/10.1073/pnas.1010018107 Fomina-Yadlin et al.
5. culture system. Future experiments involving in vivo β-cell abla-
tion, lineage tracing in animal models, and purified human α-cells
will help illuminate the effects of BRD7389 in greater detail.
These findings show the feasibility of identifying compounds that
induce insulin expression in α-cells and suggest small-molecule
approaches to increase β-cell mass by transdifferentiation in vivo.
Materials and Methods
Reagents. Compound BRD7389 (kbsa-0113758) was obtained from Aurora
Fine Chemicals Ltd. All other reagents were obtained from Sigma Aldrich
unless otherwise stated. Primers were bought from Eurofins MWG Operon,
except for Rsk2 and Rsk3 primers, which were ordered from Applied Bio-
systems. Antibodies used in this study were insulin (Sigma I8510), glucagon
(Sigma G2654), RSK1/RSK2/RSK3 (32D7; Cell Signaling Technology, CST 9355),
phospho-p90RSK (Thr359/Ser363; CST 9344), phospho-p90RSK (Thr573; CST
9346), S6 ribosomal protein (CST 2217), phospho-S6 ribosomal protein (Ser235/
236; CST 2211), phospho-S6 ribosomal protein (Ser240/244; CST 2215), β-actin
(Sigma A1978), and cleaved-caspase 3 (Abcam, ab13847). Fluorescently la-
beled secondary antibodies were purchased from Jackson ImmunoResearch.
IRDye antibodies for Western blots were purchased from Odyssey.
Cell and Human Islet Culture. Mouse pancreatic cell lines αTC1 and βTC3 were
grown in low-glucose DMEM supplemented with 10% FBS, 50 U/mL peni-
cillin, and 50 μg/mL streptomycin.
Human islets were obtained through the Islet Cell Resource Consortium
(http://icr.coh.org/) and through the National Disease Research Interchange
(http://www.ndriresource.org/). The purity and viability of human islets are
reported to be 70–93% and 70–98%, respectively, and the average age of
cadaveric donors was 40.7 ± 9.0 y (range 32–57 y; n = 6). Specific data on
individual donors is reported in Table S2. Islets were washed with PBS and
incubated in CMRL medium supplemented with 10% FBS, 2 mM glutamine,
100 U/mL penicillin, and 100 μg/mL streptomycin. Islets were gently dissoci-
ated into a cell suspension by incubating in Accutase (37 °C, 10 min), and
seeded in 96-well plates containing extracellular matrix secreted by the
HTB9 human bladder carcinoma cell line [adapted from Beattie et al. (16)].
Compound treatments for both cell lines and primary human islet cultures
were performed as follows: cells were plated and allowed to adhere over-
night, after which compound solutions in DMSO were added to achieve the
indicated concentrations in 0.1% DMSO. For 5-d treatment, media was
changed and new compound added on day 3.
High-Content Screening. A total of 10,000 αTC1 cells per well were plated in
50 μL media in black, optical bottom, tissue-culture-treated 384-well plates
(Corning) and allowed to attach overnight. Compounds (100 nL per well)
were pin-transferred from concentrated DMSO stocks. Three days after the
beginning of compound treatment, cells were fixed with 1% formaldehyde
in PBS for 30 min at room temperature. Following one wash with PBS, cells
were permeabilized by addition of 50 μL PBS-T (PBS supplemented with
0.1% Triton X-100) for 60 min at room temperature and blocked with 2%
BSA/PBS-T for 60 min. Twenty microliters of primary antiinsulin antibody,
diluted 1:4,000 in 2% BSA/PBS-T, was added per well and incubated over-
night at 4 °C. Following two PBS-T washes, 20 μL Cy-2–labeled donkey-
α-guinea pig antibody diluted 1:500 in 2% BSA, 10 μg/mL Hoechst 33342/
PBS-T was added per well and incubated for 1 h at room temperature in the
dark. After two washes with 50 μL PBS-T, plates were stored in PBS in the
dark at 4 °C until analysis.
Images were acquired on an ImageXpress Micro automated microscope
(Molecular Devices) using a 4× objective (binning 2, gain 2), with laser- and
image-based focusing (offset −130 μm, range ±50 μm, step 25 μm). Images
were exposed for 10 ms in the DAPI channel (Hoechst) and 500 ms in the GFP
channel (insulin). Image analysis was performed using the cell-scoring mod-
ule of MetaXpress software (Molecular Devices). All nuclei were detected
with a minimum width of 1 pixel, maximum width of 3 pixels, and an intensity
of 200 gray levels above background. Cytoplasmic regions around these
nuclei were evaluated for Cy2 staining in the green GFP channel (minimum
width of 5 pixels, maximum width of 30 pixels, intensity 200 gray levels
above background, 10 μm minimum stained area). In total, 75,264 wells were
screened, corresponding to 30,710 unique compounds in duplicate plus
control wells. The compounds screened were selected from a number of su-
blibraries in the Broad Institute compound collection. The screening set was
comprised of 1,920 molecules with previously annotated biological activity,
purchased from commercial vendors Biomol International Inc., Calbiochem,
EMD Biosciences, Microsource Discovery Systems Inc., Prestwick Chemical Inc.,
and Sigma-Aldrich; 1,280 purified natural products from Analyticon Discov-
ery; 15,356 commercial drug-like compounds from ChemDiv Inc., Maybridge,
and TimTec LLC; and 12,154 diversity-oriented synthetic (DOS) compounds
generated at the Broad Institute. The commercial drug-like compounds were
prefiltered by the suppliers to avoid undesired reactive functional groups and
meet physical property filters based on Lipinski’s rule of five. The DOS com-
pounds consisted of a series of stereochemically diverse eight- and nine-
membered macrocycles ranging in molecular mass from 307 to 727 Da, with
an average molecular mass of 572 Da.
Compound purity and identity were determined by UPLC-MS (Waters).
Purity was measured by UV absorbance at 210 nm. Identity was determined
on a SQ mass spectrometer by positive electrospray ionization. Mobile phase
A consisted of 0.1% ammonium hydroxide; mobile phase B consisted of 0.1%
ammonium hydroxide in acetonitrile. The gradient ran from 5% to 95%
mobile phase B over 0.8 min at 0.45 mL/min. An Acquity BEH C18, 1.7 μm, 1.0 ×
50-mm column was used with column temperature maintained at 65 °C.
Compounds were dissolved in DMSO at a nominal concentration of 1 mg/mL,
and 0.25 μL of this solution was injected.
Hits were selected based on the intensity of staining in the Cy2 channel and
the number of Cy2 positive cells, and counterscreened in the same assay
without the use of primary antibody and with Cy3-labeled secondary anti-
body to remove inactive autofluorescent compounds.
In all subsequent immunofluorescence experiments, Cy3 and Cy5 sec-
ondary antibodies were used to avoid effects of compound autofluorescence
in the Cy2 channel.
Gene Expression Analysis. Following compound treatment, cells were lysed
and RNA isolated using the RNeasy Mini Kit (Qiagen) according to the
manufacturer’s protocol. RNA was reverse transcribed with random primers
using the High Capacity cDNA Reverse Transcription Kit with RNase inhibitor
(Applied Biosystems).
Quantitative PCR was performed with Power SYBR Green PCR Master Mix
(Applied Biosystems) on an Applied Biosystems 7900HT real-time PCR ma-
chine using primers in Table S3.
Kinase Profiling. Kinase profiling and dose–response curves were performed
at Millipore’s KinaseProfiler according to the manufacturer’s protocols. ATP
concentrations were within 15 μM of the apparent KM for each enzyme.
Western Blot Analysis. Cell extracts were generated by lysing cells in modified
RIPA buffer containing 1% Nonidet P-40, 0.1% Na deoxycholate, 150 mM
NaCl, 1 mM EDTA, and 50 mM Tris (pH 7.5), and supplemented with protease
and phosphatase inhibitors. A total of 20 μg of each sample were run on E-
Page 48 gels (Invitrogen) and transferred to PVDF membranes using Invi-
trogen iBlot technology. Each blot was simultaneously probed with indicated
primary antibody (all at 1:1,000) and 1:10,000 β-actin antibody, following by
incubation with 1:5,000 IRDye-labeled secondary antibody. Blots were scan-
ned on LI-COR Odyssey Infrared Imaging System and analyzed using Odyssey
software. Each specific band was normalized to the β-actin signal, and
phosphorylation was plotted as a ratio of normalized phospho-specific to
normalized pan-antibody signal.
RNAi Experiments. Lentiviruses resulting in the expression of shRNAs against
RSK family members were obtained from the RNAi Consortion (TRC) (19). The
following hairpins were used: Rsk1 shRNA1: NM_009097.1-559s1c1, Rsk1
shRNA2: NM_009097.1-685s1c1, Rsk2 shRNA1: NM_148945.1-269s1c1, Rsk2
shRNA2: NM_148945.1-1345s1c1, Rsk2 shRNA3: NM_148945.1-1833s1c1,
Rsk3 shRNA1: NM_011299.3-384s1c1, Rsk3 shRNA2: NM_011299.3-627s1c1,
Rsk3 shRNA3: NM_011299.3-2351s1c1. Mouse αTC1 cells were plated in 96-
well plates at 15,000 cells per well in 200 μL of DMEM. The next day, poly-
brene was added to each well (8 μg/mL), and cells were spin-infected with
8 μL virus at 2,250 rpm for 30 min at 30 °C. Media was changed the following
day to fresh, low-glucose DMEM containing 1 μg/mL puromycin and cultured
for 4 additional d. Cells were lysed in RLT buffer and mRNA extracted using
Qiagen RNeasy 96 Kit.
Hormone Secretion in Human Islets. Dissociated human isletscultured in 96-well
plates were washed once with 100 μL per well of PBS and incubated for 1 h in
100 μL low-glucose (1.67 mM) KRB buffer (138 mM NaCl, 5.4 mM KCl, 2.6 mM
MgCl2, 2.6 mM CaCl2, 5 mM NaHCO3, 0.1% BSA), and for an additional hour in
either high-glucose (16.7 mM) or low-glucose KRB buffer. Supernatant from
the first hour was used for glucose-stimulated glucagon secretion using ALPCO
Glucagon (human, mouse, rat) ELISA (following manufacturer’s protocol for
50 μL of sample). Supernatant from the second hour was used to measure
glucose-stimulated insulin secretion using ALPCO Insulin ELISA (human).
Fomina-Yadlin et al. PNAS | August 24, 2010 | vol. 107 | no. 34 | 15103
CELLBIOLOGY
6. ACKNOWLEDGMENTS. We thank Andrew Stern, Michelle Palmer, Lynn
Verplank, and the entire Chemical Biology Platform at the Broad Institute for
helpful suggestions in assay development and with high-content screening;
Thomas Nieland, Serena Silver, and David Root from the Broad RNAi platform
for lentiviral knockdown constructs and advice for optimization of the infection
protocol; Jack Taunton (University of California, San Francisco) for a sample of
the RSK inhibitor FMK and advice on RSK biology; Yuan Yuan (Chemistry and
Chemical Biology Department, Harvard University) for expression primers;
Robert Gould and the entire CB/NT Diabetes Team for helpful discussion and
advice; and Alejandro Wolf Yadlin (Chemistry and Chemical Biology Depart-
ment, Harvard University) for performing Western blot quantification. Funding
for this project was provided by the Juvenile Diabetes Research Foundation and
National Institute for General Medical Sciences Grant GM38627 (to S.L.S.); Na-
tional Institutes of Health Grant RL1-HG004671 for computational work toward
target-hypothesis generation (to V.D. and P.A.C.); Ernst Schering Research Foun-
dation and European Union FP7 Marie Curie Program Grant PIOF-GA-2008-
221135 (to S.K.); an MCO training grant from Harvard University (to D.F.); and
Type 1 Diabetes Pathfinder Award DP2-DK083048 from the National Institutes
of Health–National Institute of Diabetes and Digestive and Kidney Diseases (to
B.K.W.). S.L.S. is an Investigator at the Howard Hughes Medical Institute.
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15104 | www.pnas.org/cgi/doi/10.1073/pnas.1010018107 Fomina-Yadlin et al.
7. Supporting Information
Fomina-Yadlin et al. 10.1073/pnas.1010018107
Fig. S1. High-content screen for small-molecule inducers of insulin production in mouse α-cells. (A) Assay setup. (B) Detection of spiked-in β-cells (cell line βTC3)
in a population of α-cells (cell line αTC1) by automated image analysis following immunofluorescence detection of insulin. (C) Competition experiment sim-
ulating lower insulin levels compared with β-cells.In a population of α-cells, 5% spiked-in β-cells were stained with insulin primary antibody and Cy2-labeled
α-guinea pig secondary antibody and increasing amounts of competing Cy3-labeled α -guinea pig antibody. Staining intensity of insulin positive and negative
cells was measured in the green (Cy2) channel. (D) Representative images from spiked-in and antibody competition experiments. (Scale bar: 50 μm.)
Fomina-Yadlin et al. www.pnas.org/cgi/content/short/1010018107 1 of 6
8. Fig. S2. Induction of β-cell markers in α-cells and comparison with β-cell levels. Five-day treatment with BRD7389 causes dose-dependent up-regulation of Ins2
(A), Pdx1 (B), Pax4 (C), Npy (D), and Iapp (E) mRNA in mouse α-cells. Levels of expression in mature mouse β-cells are plotted for comparison. Data represent the
mean ± SDs of three independent experiments.
Fomina-Yadlin et al. www.pnas.org/cgi/content/short/1010018107 2 of 6
9. Fig. S3. Dose-responsive kinase inhibition by BRD7389. Human kinases CDK5/p35 (A), DRAK1 (B), FLT3 (C), PIM1 (D), PKG1α (E), RSK1 (F), RSK2 (G), RSK3 (H),
and SGK (I) were incubated with the indicated concentrations of BRD7389, and kinase activity was determined in a radiometric filter-binding assay. Activity was
scored relative to DMSO. Two replicate experiments were used to fit curves and calculate IC50 values using logistic regression implemented in Spotfire.
Fig. S4. Effects of BRD7389 treatment in primary human islets from donor 1. (A) Glucose-stimulated insulin secretion after 5-d treatment with BRD7389.
(B) Relative gene-expression changes of endocrine-specific (colored) and housekeeping (gray) genes following 5-d treatment with BRD7389. (C) Quantification
of relative cell numbers compared with DMSO-treated controls from immunofluorescence samples. (D) Representative images of compound-treated dissoci-
ated islets. (Scale bar: 50 μm.)
Fomina-Yadlin et al. www.pnas.org/cgi/content/short/1010018107 3 of 6
10. Fig. S5. Effects of BRD7389 treatment in primary human islets from donor 3. (A) Glucose-stimulated insulin secretion after 5-d treatment with BRD7389. (B)
Quantification of relative cell numbers compared with DMSO-treated controls from immunofluorescence samples. (C) Representative images of compound-
treated dissociated islets. (Scale bar: 50 μm.)
Fig. S6. Effects of BRD7389 treatment in primary human islets from donor 4. (A) Glucose-stimulated insulin secretion after 5-d treatment with BRD7389.
(B) Glucose-stimulated glucagon secretion after 5-d treatment with BRD7389. (C) Quantification of relative cell numbers compared with DMSO-treated
controls from immunofluorescence samples. (D) Representative images of compound-treated dissociated islets. (Scale bar: 50 μm.)
Fomina-Yadlin et al. www.pnas.org/cgi/content/short/1010018107 4 of 6
11. Fig. S7. Effects of BRD7389 treatment in primary human islets from donor 5. (A) Glucose-stimulated insulin secretion after 5-d treatment with BRD7389.
(B) Relative gene-expression changes of endocrine-specific (colored) and housekeeping (gray) genes following 5-d treatment with BRD7389. (C) Quantification
of relative cell numbers compared with DMSO-treated controls from immunofluorescence samples. (D) Representative images of compound-treated dissoci-
ated islets. (Scale bar: 50 μm.)
Fig. S8. Effects of BRD7389 treatment in primary human islets from donor 6. (A) Glucose-stimulated insulin secretion after 5-d treatment with BRD7389.
(B) Glucose-stimulated glucagon secretion after 5-d treatment with BRD7389. (C) Relative gene-expression changes of endocrine specific (colored) and
housekeeping (gray) genes following 5-d treatment with BRD7389. (D) Quantification of relative cell numbers compared with DMSO-treated controls from
immunofluorescence samples. (E) Representative images of compound-treated dissociated islets. (Scale bar: 50 μm.)
Fomina-Yadlin et al. www.pnas.org/cgi/content/short/1010018107 5 of 6
12. Fig. S9. Activation of caspase 3 in dissociated human islets after 5-d treatment with BRD7389. (A) Quantification of insulin-positive cells and cleaved
caspase 3-positive cells. (B) Quantification of costaining for insulin and cleaved caspase 3, indicating insulin-positive β-cells undergoing apoptosis and insulin-
negative non–β-cells undergoing apoptosis. Significant difference from DMSO-treated control, *P 0.05 and **P 0.01. (C) Representative images of
cleaved caspase 3 staining in compound-treated dissociated islets. (Scale bar: 50 μm.)
Table S1. Inhibition profiling across a panel of 253 kinases with compounds BRD7389 and
BRD6447
Table S1 (DOC)
Values indicate the percent remaining activity for the indicated human (h), mouse (m), rat (r), and yeast (y)
kinases. Assays were performed using 10 μM BRD7389 and BRD6447, and at an ATP concentration within 15 μM of
the apparent Km of each kinase.
Table S2. Donor information for human islet samples analyzed in Fig. 3 and Figs. S3–S7
Table S2 (DOC)
Table S3. Primers used for quantitative RT-PCR gene expression measurements
Table S3 (DOC)
Table lists mouse (mm) and human (hs) primers used, their target transcript, orientation, and sequence.
Fomina-Yadlin et al. www.pnas.org/cgi/content/short/1010018107 6 of 6