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A hallmark of pancreatic ductal adenocarcinoma (PDAC) is an exuberant stroma comprised of diverse cell types that enable or suppress tumor progression. Here, we explored the role of oncogenic KRAS in protumorigenic signaling interactions between cancer cells and host cells. We show that KRAS mutation (KRAS*) drives cell-autonomous expression of type I cytokine receptor complexes (IL2rγ–IL4rα and IL2rγ–IL13rα1) in cancer cells that in turn are capable of receiving cytokine growth signals (IL4 or IL13) provided by invading Th2 cells in the microenvironment. Early neoplastic lesions show close proximity of cancer cells harboring KRAS* and Th2 cells producing IL4 and IL13. Activated IL2rγ–IL4rα and IL2rγ–IL13rα1 receptors signal primarily via JAK1–STAT6. Integrated transcriptomic, chromatin occupancy, and metabolomic studies identified MYC as a direct target of activated STAT6 and that MYC drives glycolysis. Thus, paracrine signaling in the tumor microenvironment plays a key role in the KRAS*-driven metabolic reprogramming of PDAC.Significance:Type II cytokines, secreted by Th2 cells in the tumor microenvironment, can stimulate cancer cell–intrinsic MYC transcriptional upregulation to drive glycolysis. This KRAS*-driven heterotypic signaling circuit in the early and advanced tumor microenvironment enables cooperative protumorigenic interactions, providing candidate therapeutic targets in the KRAS* pathway for this intractable disease.

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Crop diversification in the farming sectors has been accepted to be a wise strategy for augmenting the farmers’ income level as well as for mitigating the challenges for climatic change worldwide. Considering the importance of crop diversification both from the view points of farmers and environmental aspects, this paper using the secondary data tries to find out the pattern of growth of area, production, and yield of the major crops in Nepal, and at the same time, it makes an attempt to examine the crop pattern changes in Nepal along with its determining factors including both demand and supply-side factors. The growth analysis reveals that for most of the crops the second sub-period of 1986 – 2007 was the best in terms of the growth of production as compared to the other two-subperiods (1961 – 1985 and 2008 – 2017). The crop pattern of Nepal over the years shows that though the food grain crops still dominate the crop mix of the economy, there is clear evidence of necessary intercrop substitution where the traditional food grain crops are being replaced by commercial cash crops. Tea has come up with a big way in Nepal commercial cash crop production. Also, the crop diversification indices measured by the Herfindahl index and Shannon index, show a rising trend in diversification pattern during the nineties. Urbanization, better infrastructural supports in terms of better technology, irrigation coverage has been found to provide a significant positive impact on the diversification process. However, the trade openness is not found to be beneficial for Nepal’s agricultural sector diversification process as does the policy reforms in the post-political reforms.

Crop diversification is considered as a means to promote agricultural development while lowering its environmental implications. Diversified food production must substantially increase to fulfil the needs of food security and sustainability for the world’s future population. This study identified the finer details of diversification in India’s 20 agriculturally prosperous states through analyzing the regional trends in agricultural diversification. The analysis, which covered the period from 1971–1972 to 2017–2018, showed that agricultural diversification has taken place in several states of India. This study revealed that, due to the significant heterogeneity in agro-climatic and socio-economic conditions, the type of diversification varies among the regions. In these periods, cash- crop percentage in the gross-cropped area has increased from 18 percent to 37 percent, reflecting a major impact on the Shannon-Wiener diversification index. Crop diversification has been influenced by rural literacy, irrigation intensity, road infrastructure, per-capita, gross-state-domestic products, regional-rural-branch intensity, rainfall deviation, credit intensity, and India’s growing urban population, whereas cropping intensity has not affected crop diversification in India.

Adeno-associated virus' (AAV) relatively simple structure makes it accommodating for engineering into controllable delivery platforms. Cancer, such as pancreatic ductal adenocarcinoma (PDAC), are often characterized by upregulation of membrane-bound proteins, such as MMP-14, that propagate survival integrin signaling. In order to target tumors, we have engineered an MMP-14 protease-activatable AAV vector that responds to both membrane-bound and extracellularly active MMPs. This "provector" was generated by inserting a tetra-aspartic acid inactivating motif flanked by the MMP-14 cleavage sequence IPESLRAG into the capsid subunits. The MMP-14 provector shows lower background transduction than previously developed provectors, leading to a 9.5-fold increase in transduction ability. In a murine model of PDAC, the MMP-14 provector shows increased delivery to an allograft tumor. This proof-of-concept study illustrates the possibilities of membrane-bound protease-activatable gene therapies to target tumors.

BACKGROUND: RNF43 is an E3 ubiquitin ligase that is recurrently mutated in pancreatic ductal adenocarcinoma (PDAC) and precursor cystic neoplasms of the pancreas. The impact of RNF43 mutations on PDAC is poorly understood and autochthonous models have not been sufficiently characterized. In this study we describe a genetically engineered mouse model (GEMM) of PDAC with conditional expression of oncogenic Kras and deletion of the catalytic domain of Rnf43 in exocrine cells. METHODS: We generated Ptf1a-Cre;LSL-KrasG12D;Rnf43(flox/flox) (KRC) and Ptf1a-Cre; LSL-KrasG12D (KC) mice and animal survival was assessed. KRC mice were sacrificed at 2 months, 4 months and at moribund status followed by analysis of pancreata by single cell RNA sequencing (scRNAseq). Comparative analyses between moribund KRC and a moribund Kras/Tp53 driven PDAC GEMM (KPC) was performed. Cell lines were isolated from KRC and KC tumors and interrogated by cytokine array analyses, ATAC-seq and in vitro drug assays. KRC GEMMs were also treated with an anti-CTLA4 neutralizing antibody with treatment response measured by magnetic response imaging. RESULTS: We demonstrate that KRC mice display a marked increase in incidence of high-grade cystic lesions of the pancreas and PDAC compared to KC. Importantly, KRC mice have a significantly decreased survival compared to KC mice. By use of scRNAseq we demonstrated that KRC tumor progression is accompanied by a decrease in macrophages, as well as an increase in T and B lymphocytes with evidence of increased immune checkpoint molecule expression and affinity maturation, respectively. This was in stark contrast to the tumor immune microenvironment observed in the KPC PDAC GEMM. Furthermore, expression of the chemokine, CXCL5, was found to be specifically decreased in KRC cancer cells by means of epigenetic regulation and emerged as a putative candidate for mediating the unique KRC immune landscape. CONCLUSIONS: The KRC GEMM establishes RNF43 as a bona fide tumor suppressor gene in PDAC. This GEMM features a markedly different immune microenvironment compared to previously reported PDAC GEMMs and puts forth a rationale for an immunotherapy approach in this subset of PDAC cases.

Intrahepatic cholangiocarcinoma (ICC) is a primary biliary malignancy that harbors a dismal prognosis. Oncogenic mutations of KRAS and loss-of-function mutations of BRCA1-associated protein 1 (BAP1) have been identified as recurrent somatic alterations in ICC. However, an autochthonous genetically engineered mouse model of ICC that genocopies the co-occurrence of these mutations has never been developed. By crossing Albumin-Cre mice bearing conditional alleles of mutant Kras and/or floxed Bap1, Cre-mediated recombination within the liver was induced. Mice with hepatic expression of mutant KrasG12D alone (KA), bi-allelic loss of hepatic Bap1 (BhomoA), and heterozygous loss of Bap1 in conjunction with mutant KrasG12D expression (BhetKA) developed primary hepatocellular carcinoma (HCC), but no discernible ICC. In contrast, mice with homozygous loss of Bap1 in conjunction with mutant KrasG12D expression (BhomoKA) developed discrete foci of HCC and ICC. Further, the median survival of BhomoKA mice was significantly shorter at 24 weeks when compared to the median survival of ≥40 weeks in BhetKA mice and approximately 50 weeks in BhomoA and KA mice (p &lt, 0.001). Microarray analysis performed on liver tissue from KA and BhomoKA mice identified differentially expressed genes in the setting of BAP1 loss and suggests that deregulation of ferroptosis might be one mechanism by which loss of BAP1 cooperates with oncogenic Ras in hepato-biliary carcinogenesis. Our autochthonous model provides an in vivo platform to further study this lethal class of neoplasm.

RNF43 is an E3 ubiquitin ligase that is recurrently mutated in pancreatic ductal adenocarcinoma (PDAC) and precursor cystic neoplasms of the pancreas. The impact of RNF43 mutations on PDAC is poorly understood and autochthonous models have not been sufficiently characterized. In this study we describe a genetically engineered mouse model (GEMM) of PDAC with conditional expression of oncogenic Kras and deletion of the catalytic domain of Rnf43 (KRC) in exocrine cells. We demonstrate that Rnf43 loss results in an increased incidence of high-grade cystic lesions of the pancreas and PDAC. Importantly, KRC mice have a significantly decreased survival compared to mice containing only an oncogenic Kras mutation. By use of single cell RNA sequencing we demonstrated that KRC tumor progression is accompanied by a decrease in macrophages, as well as an increase in T and B lymphocytes with evidence of increased immune checkpoint molecule expression and affinity maturation, respectively. This was in stark contrast to the tumor immune microenvironment observed in the Kras/Tp53 driven PDAC GEMM. Furthermore, expression of the chemokine, CXCL5, was found to be specifically decreased in KRC cancer cells by means of epigenetic regulation and emerged as a putative candidate for mediating the unique KRC immune landscape. This GEMM establishes RNF43 as a bona fide tumor suppressor gene in PDAC and puts forth a rationale for an immunotherapy approach in this subset of PDAC cases.

Background &amp, Aims: ARID1A is postulated to be a tumor suppressor gene owing to loss-of-function mutations in human pancreatic ductal adenocarcinomas (PDAC). However, its role in pancreatic pathogenesis is not clear despite recent studies using genetically engineered mouse (GEM) models. We aimed at further understanding of its direct functional role in PDAC, using a combination of GEM model and PDAC cell lines. Methods: Pancreas-specific mutant Arid1a-driven GEM model (Ptf1a-Cre, KrasG12D, Arid1af/f or &ldquo, KAC&rdquo, ) was generated by crossing Ptf1a-Cre, KrasG12D (&ldquo, KC&rdquo, ) mice with Arid1af/f mice and characterized histologically with timed necropsies. Arid1a was also deleted using CRISPR-Cas9 system in established human and murine PDAC cell lines to study the immediate effects of Arid1a loss in isogenic models. Cell lines with or without Arid1a expression were developed from respective autochthonous PDAC GEM models, compared functionally using various culture assays, and subjected to RNA-sequencing for comparative gene expression analysis. DNA damage repair was analyzed in cultured cells using immunofluorescence and COMET assay. Results: Retention of Arid1a is critical for early progression of mutant Kras-driven pre-malignant lesions into PDAC, as evident by lower Ki-67 and higher apoptosis staining in &ldquo, as compared to &ldquo, mice. Enforced deletion of Arid1a in established PDAC cell lines caused suppression of cellular growth and migration, accompanied by compromised DNA damage repair. Despite early development of relatively indolent cystic precursor lesions called intraductal papillary mucinous neoplasms (IPMNs), a subset of &ldquo, mice developed aggressive PDAC in later ages. PDAC cells obtained from older autochthonous &ldquo, mice revealed various compensatory (&ldquo, escaper&rdquo, ) mechanisms to overcome the growth suppressive effects of Arid1a loss. Conclusions: Arid1a is an essential survival gene whose loss impairs cellular growth, and thus, its expression is critical during early stages of pancreatic tumorigenesis in mouse models. In tumors that arise in the setting of ARID1A loss, a multitude of &ldquo, mechanisms drive progression.

A hallmark of pancreatic ductal adenocarcinoma (PDAC) is an exuberant stroma comprised of diverse cell types that enable or suppress tumor progression. Here, we explored the role of oncogenic KRAS in protumorigenic signaling interactions between cancer cells and host cells. We show that KRAS mutation (KRAS*) drives cell-autonomous expression of type I cytokine receptor complexes (IL2rγ–IL4rα and IL2rγ–IL13rα1) in cancer cells that in turn are capable of receiving cytokine growth signals (IL4 or IL13) provided by invading Th2 cells in the microenvironment. Early neoplastic lesions show close proximity of cancer cells harboring KRAS* and Th2 cells producing IL4 and IL13. Activated IL2rγ–IL4rα and IL2rγ–IL13rα1 receptors signal primarily via JAK1–STAT6. Integrated transcriptomic, chromatin occupancy, and metabolomic studies identified MYC as a direct target of activated STAT6 and that MYC drives glycolysis. Thus, paracrine signaling in the tumor microenvironment plays a key role in the KRAS*-driven metabolic reprogramming of PDAC. Significance: Type II cytokines, secreted by Th2 cells in the tumor microenvironment, can stimulate cancer cell–intrinsic MYC transcriptional upregulation to drive glycolysis. This KRAS*-driven heterotypic signaling circuit in the early and advanced tumor microenvironment enables cooperative protumorigenic interactions, providing candidate therapeutic targets in the KRAS* pathway for this intractable disease.

Genetically engineered mouse models (GEMMs) that recapitulate the major genetic drivers in pancreatic ductal adenocarcinoma (PDAC) have provided unprecedented insights into the pathogenesis of this lethal neoplasm. Nonetheless, generating an autochthonous model is an expensive, time consuming and labor intensive process, particularly when tissue specific expression or deletion of compound alleles are involved. In addition, many of the current PDAC GEMMs cause embryonic, pancreas-wide activation or loss of driver alleles, neither of which reflects the cognate human disease scenario. The advent of CRISPR/Cas9 based gene editing can potentially circumvent many of the aforementioned shortcomings of conventional breeding schema, but ensuring the efficiency of gene editingin vivoremains a challenge. Here we have developed a pipeline for generating PDAC GEMMs of complex genotypes with high efficiency using a single “workhorse” mouse strain expressing Cas9 in the adult pancreas under ap48promoter. Using adeno-associated virus (AAV) mediated delivery of multiplexed guide RNAs (sgRNAs) to the adult murine pancreas ofp48-Cre; LSL-Cas9mice, we confirm our ability to express an oncogenicKrasG12Dallele through homology-directed repair (HDR), in conjunction with CRISPR-induced disruption of cooperating alleles (Trp53, Lkb1andArid1A). The resulting GEMMs demonstrate a spectrum of precursor lesions (pancreatic intraepithelial neoplasia [PanIN] or Intraductal papillary mucinous neoplasm [IPMN] with eventual progression to PDAC. Next generation sequencing of the resulting murine PDAC confirms HDR of oncogenicKrasG12Dallele at the endogenous locus, and insertion deletion (“indel”) and frameshift mutations of targeted tumor suppressor alleles. By using a single “workhorse” mouse strain and optimal AAV serotype forin vivogene editing with combination of driver alleles, we have created a facile autochthonous platform for interrogation of the PDAC genome.

BACKGROUND & AIMS: Mutations at hot spots in GNAS, which encodes stimulatory G-protein, alpha subunits, are detected in ~60% of intraductal papillary mucinous neoplasms (IPMNs) of the pancreas. We generated mice with KRAS-induced IPMNs that also express a constitutively active form of GNAS in pancreas and studied tumor development. METHODS: We generated p48-Cre; LSL-KrasG12D; Rosa26R-LSL-rtTA-TetO-GnasR201C mice (Kras;Gnas mice); pancreatic tissues of these mice express activated KRAS and also express a mutant form of GNAS (GNAS(R201C)) upon doxycycline administration. Mice that were not given doxycycline were used as controls and survival times were compared by Kaplan-Meier analysis. Pancreasta were collected at different timepoints after doxycycline administration and analyzed by histology. Pancreatic ductal adenocarcinomas (PDACs) were isolated from mice and used to generate cell lines, which were analyzed by reverse transcription PCR, immunoblotting, immunohistochemistry, and colony formation and invasion assays. Full-length and mutant forms of YAP were expressed in PDAC cells. IPMN specimens were obtained from 13 patients with IPMN undergoing surgery and analyzed by immunohistochemistry. RESULTS: All Kras;Gnas mice developed pancreatic cystic lesions that resemble human IPMNs; the grade of epithelial dysplasia increased with time. None of the control mice developed cystic lesions. Approximately one-third of Kras;Gnas mice developed PDACs, at a median of 30 weeks after doxycycline administration, whereas 33% of control mice developed PDACs. Expression of GNAS(R201C) did not accelerate the development of PDACs, compared with control mice. However, the neoplasms observed in Kras;Gnas mice were more differentiated, and expressed more genes associated with ductal phenotypes, than in control mice. PDACs isolated from Kras;Gnas mice had activation of the Hippo pathway; in cells from these tumors, phosphorylated YAP1 was sequestered in the cytoplasm—this was also observed in human IPMNs with GNAS mutations. Sequestration of YAP1 was not observed in PDAC cells from control mice. CONCLUSIONS: In mice that express activated KRAS in the pancreas, we found expression of GNAS(R201C) to cause development of more differentiated tumors, with gene expression pattern associated with the ductal phenotype. Expression of mutant GNAS caused phosphorylated YAP1 to be sequestered in the cytoplasm, altering tumor progression.

Background & AimsSomatic “hotspot” mutations of GNAS, which encodes for the alpha subunit of stimulatory G-protein, are present in ~60% of intraductal papillary mucinous neoplasms (IPMNs) of the pancreas. There are currently no cognate animal models that recapitulate the biology of mutant Gnas-induced IPMNs, and the underlying mechanisms that lead to the cystic pathway of neoplasia in the pancreas remain unknown.MethodsWe generated p48-Cre; LSL-KrasG12D; Rosa26R-LSL-rtTA-TetO-GnasR201C mice (Kras; Gnas mice) where pancreas-specific GnasR201C expression was induced by doxycycline administration. In this model, mutant Kras is constitutively expressed, and control mice were produced through absence of doxycycline. Separate cohorts of mice were utilized for timed necropsies and for Kaplan-Meier survival analysis. Isogenic cell lines (with doxycycline inducible mutant Gnas expression) were propagated from the resulting pancreatic ductal adenocarcinoma (PDAC).ResultsCo-expression of KrasG12D and GnasR201C resulted in the development of pancreatic cystic lesions resembling human IPMNs in 100% of mice, with higher grades of epithelial dysplasia observed over time. Approximately one-third of Kras; Gnas mice developed PDAC at a median of 38 weeks post doxycycline induction. GnasR201C did not accelerate oncogenic transformation with KrasG12D, but rather, reprogrammed Ras-induced neoplasms towards a well-differentiated phenotype. GnasR201C induction led to activation of the inhibitory Hippo kinase cascade and cytoplasmic sequestration of phosphorylated YAP1 protein, a phenomenon that was also observed in human IPMN with GNAS mutations.ConclusionsGNASR201C functions not as a traditional oncogene, but rather as an “oncomodulator” of KRAS-mediated pancreatic neoplasia, through suppression of YAP1 and transcriptional reprogramming towards a differentiated (large ductal) phenotype.

Dicer is an essential ribonuclease involved in the biogenesis of miRNAs. Previous studies have reported downregulation of Dicer in multiple cancers including hepatocellular carcinoma. To identify signaling pathways that are altered upon Dicer depletion, we carried out quantitative phosphotyrosine profiling of liver tissue from Dicer knockout mice. We employed antibody-based enrichment of phosphotyrosine containing peptides coupled with SILAC spike-in approach for quantitation. High resolution mass spectrometry-based analysis identified 349 phosphotyrosine peptides corresponding to 306 unique phosphosites of which 75 were hyperphosphorylated and 78 were hypophosphorylated. Several receptor tyrosine kinases including MET, PDGF receptor alpha, Insulin-like growth factor 1 and Insulin receptor as well as non-receptor tyrosine kinases such as Src family kinases were found to be hyperphosphorylated upon depletion of Dicer. In addition, signaling molecules such as IRS-2 and STAT3 were hyperphosphorylated. Activation of these signaling pathways has been implicated previously in various types of cancers. Interestingly, we observed hypophosphorylation of molecules including focal adhesion kinase and paxillin. Our study profiles the perturbed signaling pathways in response to dysregulated miRNAs resulting from depletion of Dicer. Our findings warrant further studies to investigate oncogenic effects of downregulation of Dicer in cancers.

Background & Aims Mutations at hotspots in GNAS, which encodes stimulatory G-protein, α subunits, are detected in approximately 60% of intraductal papillary mucinous neoplasms (IPMNs) of the pancreas. We generated mice with KRAS-induced IPMNs that also express a constitutively active form of GNAS in pancreas and studied tumor development. Methods We generated p48-Cre; LSL-KrasG12D; Rosa26R-LSL-rtTA-TetO-GnasR201C mice (Kras;Gnas mice); pancreatic tissues of these mice express activated KRAS and also express a mutant form of GNAS (GNASR201C) upon doxycycline administration. Mice that were not given doxycycline were used as controls, and survival times were compared by Kaplan-Meier analysis. Pancreata were collected at different time points after doxycycline administration and analyzed by histology. Pancreatic ductal adenocarcinomas (PDACs) were isolated from mice and used to generate cell lines, which were analyzed by reverse transcription polymerase chain reaction, immunoblotting, immunohistochemistry, and colony formation and invasion assays. Full-length and mutant forms of yes-associated protein (YAP) were expressed in PDAC cells. IPMN specimens were obtained from 13 patients with IPMN undergoing surgery and analyzed by immunohistochemistry. Results All Kras;Gnas mice developed pancreatic cystic lesions that resemble human IPMNs; the grade of epithelial dysplasia increased with time. None of the control mice developed cystic lesions. Approximately one third of Kras;Gnas mice developed PDACs at a median of 30 weeks after doxycycline administration, whereas 33% of control mice developed PDACs. Expression of GNASR201C did not accelerate the development of PDACs compared with control mice. However, the neoplasms observed in Kras;Gnas mice were more differentiated, and expressed more genes associated with ductal phenotypes, than in control mice. PDACs isolated from Kras;Gnas mice had activation of the Hippo pathway; in cells from these tumors, phosphorylated YAP1 was sequestered in the cytoplasm, and this was also observed in human IPMNs with GNAS mutations. Sequestration of YAP1 was not observed in PDAC cells from control mice. Conclusions In mice that express activated KRAS in the pancreas, we found expression of GNASR201C to cause development of more differentiated tumors, with gene expression pattern associated with the ductal phenotype. Expression of mutant GNAS caused phosphorylated YAP1 to be sequestered in the cytoplasm, altering tumor progression.

In the developing pancreas, the onset of exocrine differentiation is driven by the activity of the PTF1 (pancreas transciption factor 1) transcriptional complex, which is comprised of the class II bHLH (basic helix–loop–helix) protein, Ptf1-p48 [also known as Ptf1a (pancreas specific transcription factor 1a)], and a class I E-box binding partner. Activity of the PTF1 complex is normally inhibited by the Notch signalling pathway, a process mediated by Notch effector proteins in the HES (Hairy/Enhancer of Split) family of bHLH transcriptional repressors. In the present study, we show that this inhibitory effect occurs through direct interaction between HES family members and Ptf1-p48. The HES family members Hey1 (hairy/enhancer-of-split related with YRPW motif 1) and Hey2 co-immunoprecipitate with Ptf1-p48, and Ptf1-p48 binding by Hes1 is also evident in yeast two-hybrid and GST (glutathione S-transferase) pull-down assays. The ability of Hes1 to interact with Ptf1-p48 resides within a fragment comprised of the bHLH, Orange and C-terminal domains, and does not require the N-terminal or WRPW elements. The ability of truncated versions of Hes1 to bind Ptf1-p48 correlates with their ability to down-regulate the activity of the PTF1 transcriptional complex, defining Ptf1-p48 binding as the most likely mechanism by which Notch effector proteins delay exocrine pancreatic differentiation.

Mammalian studies have implicated important roles for the basic helix–loop–helix transcription factor Ptf1a-p48 in the development of both exocrine and endocrine pancreas. We have cloned the Ptf1a-p48 ortholog in Danio rerio. Early zebrafish ptf1a expression is observed in developing hindbrain and in endodermal pancreatic precursors. Analysis of ptf1a and insulin expression reveals a population of exocrine precursors that, throughout early development, are temporally and spatially segregated from endocrine elements. Morpholino-mediated knockdown of ptf1a confirms early divergence of these endocrine and exocrine lineages. Ptf1a morphants lack differentiated exocrine pancreas, but maintain normal differentiation and organization of the principal islet. In addition to the exocrine phenotype, ptf1a knockdown also reduces the prevalence of a small population of anterior endocrine cells normally found outside the principal islet. Together, these findings suggest the presence of distinct ptf1a-dependent and ptf1a-independent precursor populations in developing zebrafish pancreas.

Notch signaling regulates cell fate decisions in a wide variety of adult and embryonic tissues. Here we show that Notch pathway components and Notch target genes are upregulated in invasive pancreatic cancer, as well as in pancreatic cancer precursors from both mouse and human. In mouse pancreas, ectopic Notch activation results in accumulation of nestin-positive precursor cells and expansion of metaplastic ductal epithelium, previously identified as a precursor lesion for pancreatic cancer. Notch is also activated as a direct consequence of EGF receptor activation in exocrine pancreas and is required for TGF alpha-induced changes in epithelial differentiation. These findings suggest that Notch mediates the tumor-initiating effects of TG alpha by expanding a population of undifferentiated precursor cells.

Peroxisomal disorders are a newly described group of inherited neurological diseases. In disorders of peroxisomal biogenesis, e.g., Zellweger syndrome, owing to the lack of peroxisomes, catalase, a peroxisomal enzyme, is found to be present in the cytoplasm instead. We observed higher catalase activity (7.59 +/- 0.41 mU/ mg of protein) in cultured skin fibroblasts from Zellweger patients than in control fibroblasts (4.45 +/- 0.29 mU/mg of protein). Moreover, we also found that the majority of the catalase in Zellweger cells was present in the inactive form. The specific activities following reactivation in Zellweger and control cells were 12.1 and 4.9 mU/mg of protein, respectively. To understand the molecular basis of higher levels of catalase in Zellweger than control cells, we examined the rate of synthesis and turnover of catalase and levels of catalase mRNA and protein levels in Zellweger cells as compared with control cells. The initial rates of synthesis of catalase in Zellweger (1.68 +/- 0.15 mU/mg of protein) and control (1.51 +/- 0.14 mU/mg of protein) cells were similar. The rates of turnover of catalase in Zellweger (t1/2 = 47 +/- 8 h) and control (t1/2 = 49 +/- 7 h) were also similar. Consistent with the enzyme activity, the levels of catalase protein were higher in Zellweger cells as compared with control cells. On the other hand, there was no difference in the level of catalase mRNA between control and Zellweger cells. Although the rate of synthesis in Zellweger and control cells was initially similar, it was down-regulated to a lower level at approximately 72 h of culture in control fibroblasts as compared with Zellweger cells, which continued to synthesize catalase at the same rate up to 5 days in culture. The presence of similar levels of mRNA in control and Zellweger cells and continued synthesis of catalase in Zellweger cells at a higher level as compared with control cells suggest a loss of regulation at the translational level.

The present study reports the effect of ceramide generated by hydrolysis of membrane sphingomyelin with bacterial sphingomyelinase (SMase) and of cell-permeable ceramide analogues on the expression of manganese superoxide dismutase (MnSOD). Incubation of the rat primary astrocytes with SMase led to a time- and dose-dependent increase in MnSOD activity. The increase in MnSOD activity was accompanied by an increase in MnSOD protein and mRNA. A similar effect on the expression of MnSOD was observed with the addition of cell-permeable ceramide analogues (C2 and C6). On the other hand, C2-dihydroceramide (N-acetylsphinganine), which lacks the functional critical double bond, was ineffective in inducing the expression of MnSOD. Nuclear run-on analysis showed that SMase and ceramide increased the rate of transcription of the MnSOD gene. Besides astrocytes, SMase was also found to induce the expression of MnSOD in rat mesangial cells, C6 glial cells, PC12 cells, and human skin fibroblasts. Markedly higher expression of mRNA, protein, and activity of MnSOD in skin fibroblasts from patients with Farber disease, a human disorder with pathognomonic accumulation of ceramide due to a deficiency of ceramidase, than in normal skin fibroblasts indicate that ceramide may act as a physiological inducer of MnSOD gene expression. However, stimulation of ceramide-mediated DNA fragmentation by antisense knockdown of MnSOD suggests that induction of MnSOD by ceramide is a protective response of the cell.

A polyclonal antibody against a glutathione S:-transferase fusion protein containing the 76 COOH-terminal amino acids of Hex, a divergent homeobox gene, was raised in rabbits. Western blot and immunofluorescence reveal that Hex is a 35-37-kD soluble protein present both in the nucleus and cytoplasm of transfected and nontransfected cultured cells as well as in whole mouse embryo. Confocal microscopy of whole mount immunostained mouse embryos at E7. 5 and E8.5 demonstrates that Hex is differentially localized in the cytoplasm and nucleus of definitive endoderm, developing blood islands, and hepatic diverticulum. In particular, in the region of the foregut that gives rise to the liver, Hex expression is nuclear in the endodermal cells of the hepatic diverticulum, whereas expression is primarily cytoplasmic in cells lateral to the liver-forming region. This suggests that nuclear localization of Hex is involved in early hepatic specification and that compartmentalization of Hex protein plays an important role in its function during mouse development.

Reactive oxygen species (ROS; O2-, H2O2, and OH), normal by-products of cellular metabolic processes, are kept in control by antioxidant enzymes, such as catalase, glutathione peroxidase (GPX) and superoxide dismutases (SODs). To understand the role of antioxidant enzymatic defenses against ROS injury following ischemia-reperfusion, we examined the effect on kidney exposed to varying periods (30, 60 or 90 min) of ischemia followed by different periods of reperfusion. The enzymatic activities and protein levels of catalase, GPX, CuZnSOD and MnSOD were relatively unaffected at 30 min of ischemia followed by 0, 2 or 24 h reperfusion. However, 60 or 90 min of ischemia followed by 0, 2 or 24 h of reperfusion resulted in a decrease in activities and protein levels which paralleled the duration of ischemic injury. MnSOD activity tended to recover towards normal during reperfusion. Examination of the mRNA levels of these antioxidant enzymes demonstrated a severe decrease in mRNA levels of catalase and GPX at a time point of minimal ischemic injury (30 min of ischemia followed by reperfusion) suggesting that loss of mRNA of catalase and GPX may be the first markers of alterations in cellular redox in ischemia-reperfusion injury. Greater loss of mRNA for catalase, GPX and CuZnSOD was observed following longer periods (60 or 90 min) of ischemia. The mRNA for MnSOD was upregulated at all time points of ischemia-reperfusion injury. Actually, the greater decrease in mRNAs for catalase, GPX and CuZnSOD in the acute phase (within 24 h) subsequently showed a further decrease in these enzyme activities in the subacute phase (72 or 120 h after ischemia). These enzyme activities in the 30 min ischemia group, (but not in the 90 min group), already showed tendencies for normalization at 120 h after ischemia. To understand the molecular basis of the loss of mRNA of these antioxidant enzymes during ischemia-reperfusion injury, we examined the rate of transcription by nuclear run-on assays. The similar rates of transcription in control and kidney exposed to ischemia-reperfusion indicates that the loss of mRNA for catalase, GPX and CuZnSOD is possibly due to the increased rate of turnover of their mRNAs. These studies suggest that expression of antioxidant genes during ischemia-reperfusion are not coordinately expressed and that the differential loss of antioxidant enzymes may be the contributing factor(s) towards the heterogeneous renal tissue damage as a result of ischemia-reperfusion induced oxidative stress.

The excessive expression of catalase protein and its activity in cultured skin fibroblast from Zellweger Syndrome (ZS), a disorder of peroxisomal biogenesis, was found to be regulated at the translational level (J. Neurochem. 67: 2373-2378, 1996). Overall there is a considerable increase in the association of catalase mRNA with polysomes in ZS cell lines as compared to control indicating translational upregulation. To investigate the possibility that RNA-protein interactions are involved in the mediation of this increase in translation, the interaction between 3′ untranslated region of human catalase mRNA and human fibroblast cytoplasmic proteins were investigated by RNA gel shift assay technique. Competition experiments demonstrated that all the 600 bases of 3′ UTR (of human catalase gene) was required for efficient binding. Catalase RNA- protein interaction was sensitive to the altered redox state in these in vitro assays and this RNA-protein interaction could be enhanced by the addition of β-mercaptoethanol in cytoplasm from control fibroblast but not in cytoplasm from ZS fibroblast. UV cross linked RNA-protein complexes on SDS polyacrylamide gel electrophoresis revealed the presence of at least four protein bands with approximate molecular masses of 38 kDa, 50 kDa, 66 kDa and 80 kDa. The potential role of these mRNA binding proteins in the regulation of catalase gene expression is discussed.

The effect of endotoxin on antioxidant gene expression and antioxidant enzyme activity in homogenates of the heart, liver, and kidney from Sprague-Dawley rats was compared by quantitation of m-RNA and enzyme activities. Alterations in the message level for Cu-Zn superoxide dismutase (SOD), Mn SOD, and catalase varied with the tissue type, length of exposure to endotoxin, and dose of endotoxin. In general, endotoxin treatment reduced Cu-Zn SOD expression in the heart and liver, but had no noticeable effect in the kidney. Mn SOD message levels were increased in the heart and kidney but decreased in the liver. Catalase expression was reduced in the kidney and increased marginally in the heart and liver. With regard to enzyme activity, endotoxin treatment reduced Cu-Zn SOD activity in the heart, liver, and kidney. Mn SOD activity showed little change in the heart, but increased in the liver and, to a lesser extent, in the kidney. Catalase activity showed little change in the heart and kidney but was decreased at 12 h in the liver. The differing responses of tissues to the oxidant stress of endotoxin exposure should be considered when evaluating the effect of endotoxin on antioxidant enzymes.

Dicer is a ribonuclease whose major role is to generate mature microRNAs, although additional functions have been proposed. Deletion of Dicer leads to embryonic lethality in mice. To study the role of Dicer in adults, we generated mice in which administration of tamoxifen induces deletion of Dicer. Surprisingly, disruption of Dicer in adult mice induced lipid accumulation in the small intestine. To dissect the underlying mechanisms, we carried out miRNA, mRNA, and proteomic profiling of the small intestine. The proteomic analysis was done using mice metabolically labeled with heavy lysine (SILAC mice) for an in vivo readout. We identified 646 proteins, of which 80 were up-regulated2-fold and 75 were down-regulated. Consistent with the accumulation of lipids, Dicer disruption caused a marked decrease of microsomal triglyceride transfer protein, long-chain fatty acyl-CoA ligase 5, fatty acid binding protein, and very-long-chain fatty acyl-CoA dehydrogenase, among others. We validated these results using multiple reaction monitoring (MRM) experiments by targeting proteotypic peptides. Our data reveal a previously unappreciated role of Dicer in lipid metabolism. These studies demonstrate that a systems biology approach by integrating mouse models, metabolic labeling, gene expression profiling, and quantitative proteomics can be a powerful tool for understanding complex biological systems.

Fish ribosomal RNA genes (rDNA) have been compared by restriction endonuclease digestion followed by Southern hybridization using rRNA or cloned rRNA genes as labelled probes. In several species belonging to the orders Cypriniformes and Perciformes, the simple restriction patterns revealed a high degree of size class homogeneity among the rDNA repeats and similar restriction map within a species. Different species have different restriction patterns and fragment lengths arising mostly out of different length of the nontranscribed spacer. Polymorphic restriction sites are present in some species. The species-specific differences in fragment lengths produced in rDNA by some restriction enzymes can thus be used to study interspecific fish hybrids.

The developmental plasticity of adult pancreas is evidenced by the ability to undergo conversion between different epithelial cell types. Specific examples of such conversions include acinar to ductal metaplasia, ductal to islet metaplasia, and generation of ductal structures within islets. Although 90% of human pancreatic cancers are classified as ductal adenocarcinoma, markers of all pancreatic epithelial cell types (acini, ductal, and endocrine) as well as markers of gastric and intestinal lineages can be detected in these tumors. In recent years considerable knowledge has been gained regarding regulation of cellular differentiation and various signaling pathways involved in normal and neoplastic pancreas through studies of pancreatic cancer and immortalized ductal cell lines. However, these studies provide little insight into the context of normal developmental cues, the disruption of which leads to organ pathology. Here we have described a detailed method for preparation, maintenance, and manipulation of adult and embryonic mouse pancreas. These methods may be utilized in studies involving normal epithelial differentiation, contributing to improved understanding of pancreatic development and disease.

Notch signaling regulates cell fate decisions in a variety of adult and embryonic tissues, and represents a characteristic feature of exocrine pancreatic cancer. In developing mouse pancreas, targeted inactivation of Notch pathway components has defined a role for Notch in regulating early endocrine differentiation, but has been less informative with respect to a possible role for Notch in regulating subsequent exocrine differentiation events. Here, we show that activated Notch and Notch target genes actively repress completion of an acinar cell differentiation program in developing mouse and zebrafish pancreas. In developing mouse pancreas, the Notch target gene Hes1 is co-expressed with Ptf1-P48 in exocrine precursor cells, but not in differentiated amylase-positive acinar cells. Using lentiviral delivery systems to induce ectopic Notch pathway activation in explant cultures of E10.5 mouse dorsal pancreatic buds, we found that both Hes1 and Notch1-IC repress acinar cell differentiation, but not Ptf1-P48 expression, in a cell-autonomous manner. Ectopic Notch activation also delays acinar cell differentiation in developing zebrafish pancreas. Further evidence of a role for endogenous Notch in regulating exocrine pancreatic differentiation was provided by examination of zebrafish embryos with homozygous mindbomb mutations, in which Notch signaling is disrupted. mindbomb-deficient embryos display accelerated differentiation of exocrine pancreas relative to wild-type clutchmate controls. A similar phenotype was induced by expression of a dominant-negative Suppressor of Hairless [Su(H)] construct, confirming that Notch actively represses acinar cell differentiation during zebrafish pancreatic development. Using transient transfection assays involving a Ptf1-responsive reporter gene, we further demonstrate that Notch and Notch/Su(H) target genes directly inhibit Ptf1 activity, independent of changes in expression of Ptf1 component proteins. These results define a normal inhibitory role for Notch in the regulation of exocrine pancreatic differentiation.

The role of miRNA processing in the maintenance of adult pancreatic acinar cell identity and during the initiation and progression of pancreatic neoplasia has not been studied in detail. In this work, we deleted Dicer specifically in adult pancreatic acinar cells, with or without simultaneous activation of oncogenic Kras. We found that Dicer is essential for the maintenance of acinar cell identity. Acinar cells lacking Dicer showed increased plasticity, as evidenced by loss of polarity, initiation of epithelial-to-mesenchymal transition (EMT) and acinar-to-ductal metaplasia (ADM). In the context of oncogenic Kras activation, the initiation of ADM and pancreatic intraepithelial neoplasia (PanIN) were both highly sensitive to Dicer gene dosage. Homozygous Dicer deletion accelerated the formation of ADM but not PanIN. In contrast, heterozygous Dicer deletion accelerated PanIN initiation, revealing complex roles for Dicer in the regulation of both normal and neoplastic pancreatic epithelial identity.

A complimentary DNA clone encoding the entire human palmitoyl-CoA ligase has been isolated from a liver cDNA library and sequenced in it's entirety. The predicted product is a 699 amino acid protein. Southern analysis utilizing the human palmitoyl-CoA ligase gene as a probe revealed varying degrees of similarity amongst various mammalian species. The palmitoyl-CoA ligase gene is highly expressed in liver, heart, skeletal muscle and kidney, and to a lesser extent in brain, lung, placenta and pancreas. The expression of palmitoyl-CoA ligase in various tissue parallels the function of this enzyme in the metabolism of fatty acids in these tissues.