Small RNAs contribute to citrus Huanglongbing tolerance by manipulating methyl salicylate signaling and exogenous methyl salicylate primes citrus groves from emerging infection.

Citrus production is severely threatened by the devastating Huanglongbing (HLB) disease globally. By studying and analyzing the defensive behaviors of an HLB-tolerant citrus cultivar 'Shatangju', we discovered that citrus can sense Candidatus Liberibacter asiaticus (CLas) infection and induce immune responses against HLB, which can be further strengthened by both endogenously produced and exogenously applied methyl salicylate (MeSA). This immune circuit is turned on by an miR2977-SAMT (encoding a citrus Salicylate [SA] O-methyltransferase) cascade, by which CLas infection leads to more in planta MeSA production and aerial emission. We provided both transgenic and multi-year trail evidences that MeSA is an effective community immune signal. Ambient MeSA accumulation and foliage application can effectively induce defense gene expression and significantly boost citrus performance. We also found that miRNAs are battle fields between citrus and CLas, and about 30% of the differential gene expression upon CLas infection are regulated by miRNAs. Furthermore, CLas hijacks host key processes by manipulating key citrus miRNAs, and citrus employs miRNAs that coordinately regulate defense-related genes. Based on our results, we proposed that miRNAs and associated components are key targets for engineering or breeding resistant citrus varieties. We anticipate that MeSA-based management, either induced expression or external application, would be a promising tool for HLB control.

RLL5, an F-box-containing protein, involved in preventing transgene silencing and in maintaining global DNA methylation in Arabidopsis.

RNA-directed DNA methylation (RdDM) and ROS1-dependent active DNA demethylation pathways are antagonistic processes that dynamically regulate site-specific methylation. In this study, we obtained a mutant with reduced luciferase (LUC) luminescence by genetic screening, which was named rll5-1 (for reduced LUC luminescence 5-1). The rll5-1 mutant showed narrower, frizzled and curly leaves, and the low-LUC-luminescence phenotype in the rll5-1 mutant can be largely restored by DNA methylation inhibitor 5-Aza-2'-deoxycytidine. Map-based cloning coupled with genome resequencing data revealed that a nucleotide substitution of G to A was found at the 124th bp of ORF of At4G10190, leading to an aspartate-to-asparagine change at position 42 in such a protein. Bisulfite sequencing data indicated that DNA methylation of 3' region of the double 35S promoter that drives the LUC expression was appreciably increased. Further analysis revealed that there were 4747 hypo-DMRs and 936 hyper-DMRs found in the rll5-1 genome, and the hypo-DMRs was predominantly distributed on TEs, which appeared to stem from the downregulation of a few RdDM pathway genes and DNA methyltransferase genes. Closer inspection demonstrated that there were 1229 hypo-DMRs commonly shared among rll5-1, nrpd1-3 and nrpe1-11, and a total of 1349 hypo-DMRs were common to rll5-1 and cmt2 mutants. Thus, these studies demonstrate the roles of RLL5 in preventing transgene silencing and in maintaining genome-wide DNA methylation in a direct/indirect or locus-specific manner.

Roles of MEM1 in safeguarding Arabidopsis genome against DNA damage, inhibiting ATM/SOG1-mediated DNA damage response, and antagonizing global DNA hypermethylation.

Arabidopsis methylation elevated mutant 1 (mem1) mutants have elevated levels of global DNA methylation. In this study, such mutant alleles showed increased sensitivity to methyl methanesulfonate (MMS). In mem1 mutants, an assortment of genes engaged in DNA damage response (DDR), especially DNA-repair-associated genes, were largely upregulated without MMS treatment, suggestive of activation of the DDR pathway in them. Following MMS treatment, expression levels of multiple DNA-repair-associated genes in mem1 mutants were generally lower than in Col-0 plants, which accounted for the MMS-sensitive phenotype of the mem1 mutants. A group of DNA methylation pathway genes were upregulated in mem1 mutants under non-MMS-treated conditions, causing elevated global DNA methylation, especially in RNA-directed DNA methylation (RdDM)-targeted regions. Moreover, MEM1 seemed to help ATAXIA-TELANGIECTASIA MUTATED (ATM) and/or SUPPRESSOR OF GAMMA RESPONSE 1 (SOG1) to fully activate/suppress transcription of a subset of genes regulated simultaneously by MEM1 and ATM and/or SOG1, because expression of such genes decreased/increased consistently in mem1 and atm and/or sog1 mutants, but the decreases/increases in the mem1 mutants were not as dramatic as in the atm and/or sog1 mutants. Thus, our studies reveals roles of MEM1 in safeguarding genome, and interrelationships among DNA damage, activation of DDR, DNA methylation/demethylation, and DNA repair.

Roles of IDM3 and SDJ1/2/3 in Establishment and/or Maintenance of DNA Methylation in Arabidopsis.

Previous studies had demonstrated that in Arabidopsis, IDM3 is involved in ROS1-mediated DNA demethylation pathway, and SUVH-SDJ complex functions as a DNA methylation reader complex for enhancing gene transcription, which presumably recruits ROS1 to the promoters of target genes for DNA demethylation. Here, our analyses, however, showed that the IDM3 and SDJ1/2/3, the components of the SUVH-SDJ complex, are implicated in establishing and/or maintaining DNA methylation as well through DDR (DRD1-DMS3-RDM1) complex. idm3-3 or sdj1/2/3 mutations led to genome-wide DNA hypomethylation, and both mutants shared a large number of common hypo-DMRs (Differentially Methylated Regions) with rdm1-4 and dms3-4, suggesting that IDM3 and SDJ1/2/3 help establish and/or maintain DNA methylation, mediated by RdDM pathway, at a subset of genomic regions largely through DDR complex. IDM3 is able to strongly interact with RDM1 and DMS3, but weakly with SDJ1 and SDJ3; SDJ1 and SDJ3 is capable of interacting separately with RDM1 and DMS3. Furthermore, comparisons of DNA methylation features in idm3-3 and sdj1/2/3 indicated that idm3-3 and sdj1/2/3 mutations make differential impacts on DNA methylation levels and patterns on a genome-wide scale, indicating that they are targeted to quite distinct genomic regions for aiding in DNA methylation. Further analyses on ChIP-seq data demonstrated that RDM1, DMS3 and NRPE1 are enriched in IDM3- and SDJ1/2/3-targted regions. Altogether, our results provide clear demonstration that IDM3 and SDJ1/2/3 play a part in establishing and/or maintaining DNA methylation of a group of genomic regions, through the DDR complex.

Increased Drought Resistance 1 Mutation Increases Drought Tolerance of Upland Rice by Altering Physiological and Morphological Traits and Limiting ROS Levels.

To discover new mutants conferring enhanced tolerance to drought stress, we screened a mutagenized upland rice (Oryza sativa) population (cv. IAPAR9) and identified a mutant, named idr1-1 (increased drought resistance 1-1), with obviously increased drought tolerance under upland field conditions. The idr1-1 mutant possessed a significantly enhanced ability to tolerate high-drought stresses. Map-based cloning revealed that the gene LOC_Os05g26890, residing in the mapping region of IDR1 locus, carried a single-base deletion in the idr1-1 mutant. IDR1 encodes the Gα subunit of the heterotrimeric G protein (also known as RGA1), and this protein was localized in nucleus and to plasma membrane or cell periphery. Further investigations indicated that the significantly increased drought tolerance in idr1-1 mutants stemmed from a range of physiological and morphological changes, including greater leaf potentials, increased proline contents, heightened leaf thickness and upregulation of antioxidant-synthesizing and drought-induced genes, under drought-stressed conditions. Especially, reactive oxygen species (ROS) production might be remarkably impaired, while ROS-scavenging ability appeared to be markedly enhanced due to significantly elevated expression of ROS-scavenging enzyme genes in idr1-1 mutants under drought-stressed conditions. In addition, idr1-1 mutants showed reduced expression of OsBRD1. Altogether, these results suggest that mutation of IDR1 leads to alterations in multiple layers of regulations, which ultimately leads to changes in the physiological and morphological traits and limiting of ROS levels, and thereby confers obviously increased drought tolerance to the idr1-1 mutant.

Involvement of MEM1 in DNA demethylation in Arabidopsis.

KEY MESSAGE: MEM1 participates in ROS1-mediated DNA demethylation pathway, and acts functionally as ROS3 to counteract the effects of RdDM pathway.mem1mutation leads to large numbers of hyper-DMRs inArabidopsisgenome. In higher plants, DNA methylation performs important functions in silencing transcribed genes and transposable elements (TEs). Active DNA demethylation mediated by REPRESSOR OF SILENCING 1 (ROS1) is able to antagonize the action of DNA methylation caused by RNA-directed DNA methylation (RdDM) pathway, which plays critical roles in keeping DNA methylation at a proper level. In this study, a new mutant named mem1 (for methylation elevated mutant 1) was isolated from a genetic screen of T-DNA insertional mutant population for lines with elevated DNA methylation at a particular locus through Chop-PCR method. MEM1 possesses a Zf-C3HC domain, and is localized in nucleus as well as highly expressed in cotyledons. Whole-genome bisulfite sequencing data showed that knockout mutation of MEM1 leads to 4519 CG, 1793 CHG and 12739 CHH hyper-DMRs (for differentially methylated regions). Further analysis indicated that there are 2751, 2216 and 2042 overlapped CG hyper-DMRs between mem1-1and three mutants, i.e. ros1-4, rdd and ros3-2, respectively; 797, 2514, and 6766 overlapped CHH hyper-DMRs were observed between mem1-1 and three such mutants, respectively; mem1 nrpd1-3 and mem1 rdm1 double mutants showed nearly complete or partial loss of hypermethylation at 4 tested loci, suggesting that MEM1 performs similar functions as DNA glycosylase/lyases in counteracting excessive DNA methylation, and MEM1 plays important roles as REPRESSOR OF SILENCING 3 (ROS3) in erasing CHH methylation caused by the RdDM pathway. Together, these data demonstrate the involvement of MEM1 in ROS1-mediated DNA demethylation pathway and functional connections between MEM1 and ROS3.

Characterization and fine mapping of Arabidopsis RLL3, a locus required for DNA demethylation pathway.

DNA methylation pattern is found to be established by the combined actions of DNA methylation and demethylation. Compared to the DNA methylation pathway, DNA demethylation pathway, however, remains largely unknown. To better understand the DNA demethylation pathway, we performed genetic screening for Arabidopsis mutants with increased genomic DNA methylation levels through a 2 × 35S:LUC (LUC, luciferase) reporter system. A mutant with reduced LUC luminescence was identified by such a system, therefore named rll3-1 (for reduced LUC luminescence 3-1). The rll3-1 mutant exhibited pleiotropic developmental defects, such as delayed bolting as well as flowering, more branches, etc. By map-based cloning approach, rll3 locus that contains a single nuclear recessive mutation as revealed by the genetic analysis was mapped to a region between molecular markers CL102_B1 M1 and CL102_B3M1, which are located in bacterial artificial chromosome (BAC) clones F9P14 and F12K11, respectively, on chromosome 1. Chop-PCR analysis indicated that a total of seven tested loci displayed elevated DNA methylation levels. Whole-genome bisulfite sequencing further revealed 1536 loci exhibiting increased DNA methylation levels relative to Col-LUC control, among which there are 507 such loci overlapping between the rll3-1 and ros1-7 mutants, suggestive of a functional association between RLL3 and REPRESSOR OF SILENCING 1 (ROS1). Further investigations demonstrated that the expression levels of a few genes (like ROS1, IDM1, etc.), which are involved in DNA demethylation pathway, remained unchanged in the rll3-1 mutant, indicating that the increased DNA methylation levels in rll3-1 mutant are not attributable to downregulation of such genes. Taken together, our studies provide a demonstration of the involvement of RLL3 in the DNA demethylation pathway.

Quantitative Proteomic Analysis Provides Insights into Rice Defense Mechanisms against Magnaporthe oryzae.

Blast disease is one of the major rice diseases, and causes nearly 30% annual yield loss worldwide. Resistance genes that have been cloned, however, are effective only against specific strains. In cultivation practice, broad-spectrum resistance to various strains is highly valuable, and requires researchers to investigate the basal defense responses that are effective for diverse types of pathogens. In this study, we took a quantitative proteomic approach and identified 634 rice proteins responsive to infections by both Magnaporthe oryzae strains Guy11 and JS153. These two strains have distinct pathogenesis mechanisms. Therefore, the common responding proteins represent conserved basal defense to a broad spectrum of blast pathogens. Gene ontology analysis indicates that the “responding to stimulus" biological process is explicitly enriched, among which the proteins responding to oxidative stress and biotic stress are the most prominent. These analyses led to the discoveries of OsPRX59 and OsPRX62 that are robust callose inducers, and OsHSP81 that is capable of inducing both ROS production and callose deposition. The identified rice proteins and biological processes may represent a conserved rice innate immune machinery that is of great value for breeding broad-spectrum resistant rice in the future.

Sanger Sequencing for BRCA1 c.68_69del, BRCA1 c.5266dup and BRCA2 c.5946del Mutation Screen on Pap Smear Cytology Samples.

Three sets of polymerase chain reaction (PCR) primers were designed for heminested PCR amplification of the target DNA fragments in the human genome which include the site of BRCA1 c.68_69del, BRCA1 c.5266dup and BRCA2 c.5946del respectively, to prepare the templates for direct Sanger sequencing screen of these three founder mutations. With a robust PCR mixture, crude proteinase K digestate of the fixed cervicovaginal cells in the liquid-based Papanicolaou (Pap) cytology specimens can be used as the sample for target DNA amplification without pre-PCR DNA extraction, purification and quantitation. The post-PCR products can be used directly as the sequencing templates without further purification or quantitation. By simplifying the frontend procedures for template preparation, the cost for screening these three founder mutations can be reduced to about US $200 per test when performed in conjunction with human papillomavirus (HPV) assays now routinely ordered for cervical cancer prevention. With this projected price structure, selective patients in a high-risk population can be tested and each provided with a set of DNA sequencing electropherograms to document the absence or presence of these founder mutations in her genome to help assess inherited susceptibility to breast and ovarian cancer in this era of precision molecular personalized medicine.

Human cytomegalovirus infection of M1 and M2 macrophages triggers inflammation and autologous T-cell proliferation.

Macrophages (MΦ) are first targets during human cytomegalovirus (HCMV) infection and are thought to be crucial for viral persistence and dissemination. However, since MΦ are also a first line of defense and key modulators of the immune response, these cells are at the crossroad between protection and viral pathogenesis. To date, the MΦ-specific contribution to the immune response against HCMV is still poorly understood. In view of the opposite roles of M1 and M2 MΦ during initiation and resolution of the immune response, we characterized the effects of HCMV infection on classically activated M1 MΦ and alternatively activated M2 MΦ. Although HCMV susceptibility was higher in M2 MΦ, HCMV established a productive and persistent infection in both types of MΦ. Upon HCMV encounter, both types of MΦ acquired similar features of classical activation and secreted high levels of proinflammatory cytokines and chemokines. As a functional consequence, conditioned media obtained from HCMV-infected M1 and M2 MΦ potently activated freshly isolated monocytes. Finally, compared to HCMV-infected monocyte-derived dendritic cells, infected M1 and M2 MΦ were more efficient in stimulating proliferation of autologous T cells from HCMV-seropositive donors at early times (24 h) postinfection, while the MΦ immunostimulatory properties were reduced, but not abrogated, at later times (72 h postinfection). In summary, our findings indicate that MΦ preserve proper antigen presentation capacity upon HCMV infection while enhancing inflammation, thus suggesting that MΦ play a role in the maintenance of the large HCMV-specific T-cell repertoire in seropositive individuals.

A novel low temperature PCR assured high-fidelity DNA amplification.

As previously reported, a novel low temperature (LoTemp) polymerase chain reaction (PCR) catalyzed by a moderately heat-resistant (MHR) DNA polymerase with a chemical-assisted denaturation temperature set at 85 °C instead of the conventional 94-96 °C can achieve high-fidelity DNA amplification of a target DNA, even after up to 120 PCR thermal cycles. Furthermore, such accurate amplification is not achievable with conventional PCR. Now, using a well-recognized L1 gene segment of the human papillomavirus (HPV) type 52 (HPV-52) as the template for experiments, we demonstrate that the LoTemp high-fidelity DNA amplification is attributed to an unusually high processivity and stability of the MHR DNA polymerase whose high fidelity in template-directed DNA synthesis is independent of non-existent 3'-5' exonuclease activity. Further studies and understanding of the characteristics of the LoTemp PCR technology may facilitate implementation of DNA sequencing-based diagnostics at the point of care in community hospital laboratories.

Discharge of iodine-containing contrast media into the environment - problem analysis and implementation of measures to reduce discharge by means of separation toilets - experience from a pilot project. / Herausforderung Eintrag jodhaltiger Röntgenkontrastmitteln in die Umwelt ­ Problemanalyse sowie Umsetzung erster Maßnahmen zur Reduktion des Eintrags mittels Trenntoiletten ­ Erfahrungen aus einem Pilotprojekt.

PURPOSE: Environmental aspects and sustainability are becoming increasingly important. In addition to energy consumption, the consumption and environmental discharge of contrast agents pose a particular challenge. Because of their desired stability, X-ray contrast agents (XCAs) are deposited in surface water at a rate of up to 400 tons per year. MATERIALS AND METHODS: In a pilot project, a set of measures (installation of specific separation toilets, the establishment of feedback systems, interviews, questionnaires, and observation) was implemented to sensitize patients and staff to the problem of XCAs during outpatient CT examinations and a retention and recovery system for XCAs was evaluated. RESULTS: In the initial baseline phase, a separation toilet with an additional collection system and a feedback/button system was installed. The built-in feedback system indicated that the separation toilets were used by approx. 16 % of patients without measures. In two subsequent intervention phases, accompanying measures significantly (p < 0.01) increased the use of these separation toilets to 21 % and 25 %, respectively. The measures to reduce the discharge of XCAs were positively assessed by both staff and patients. CONCLUSION: Measures to reduce the discharge of XCAs into the environment have a high acceptance among staff and patients. The subsequent installation of separation toilets is one possibility to achieve on-site retention of XCAs. However, this measure is likely to be of high value only if patients stay on site for a correspondingly long time, as is the case in cardiology, for example. KEY POINTS: · The input of X-ray contrast agents into the environment is relevant in light of the quantity. · Measures to reduce the discharge of X-ray contrast agents into the environment have been investigated in pilot projects. · The (subsequent) installation of separation toilets is possible and allows retention of X-ray contrast agents. · This measure is considered useful by patients and staff. · The financing of these measures needs to be clarified. CITATION FORMAT: · Beer M, Schuler J, Kraus E et al. Discharge of iodine-containing contrast media into the environment - problem analysis and implementation of measures to reduce discharge by means of separation toilets - experience from a pilot project. Fortschr Röntgenstr 2023; 195: 1122 - 1127.

Thromboembolism after Astra Zeneca COVID-19 vaccine: Not always PF4- antibody mediated.

Cases of thromboembolic events in 2021 flared up the discussion about the safety of Astra Zeneca's AZD1222 vaccine. We hereby report three cases of pulmonary embolism (PE), one case of extended portal vein thrombosis, and one case of combined portal vein thrombosis and PE within 2 weeks after vaccination with the Astra Zeneca AZD1222 vaccine in a 60-year-old, a 50-year old, a 33-year-old, a 30-year old, and a 40-year-old male in that year. All patients were healthy before. In three patients, we observed thrombocytopenia and to some extent unusually low antibody levels for the Spike Protein (S-protein), while the other two had normal thrombocyte counts. Only one patient had anti-platelet factor 4 (PF4)-antibodies detectable as it has been described in the "heparin-induced thrombocytopenia (HIT)-like" disease of "vaccine-induced prothrombotic immune thrombocytopenia" (VIPIT) and we therefore assume that heterogeneous mechanisms led to PE. Therefore, we advise to collect and report more cases, in order to determine the age-related risks of vaccination balanced against the benefits of immunity to SARS-COV-2 for the AZD1222 vaccine in order to gain knowledge for the next pandemic.

Interactions of pancreatic cancer and stellate cells are mediated by FGFR1-III isoform expression.

BACKGROUND/AIMS: Human pancreatic cancer is characterised by an extensive desmoplastic reaction. Activation of pancreatic stellate cells (PSCs) and their interactions with pancreatic cancer cells seem to be of essential importance in this process. Expression of fibroblast growth factor (FGF) receptor (FGFR) 1 splice variants may be of special interest in this communication as they are known to modulate the malignant phenotype of pancreatic cancer. The aim of the present study was to characterize interactions between PSCs and pancreatic cancer cells focusing on the Ig-domain III variants of fibroblast growth factor (FGF) receptor (FGFR) 1. METHODOLOGY: Expression of FGF ligands and FGFR1-III isoforms was determined by immunoblotting and specific RT-PCR analysis, respectively. RESULTS: PSCs and COLO-357, MIA PaCa-2, and PANC-1 pancreatic cancer cells expressed and secreted FGF2 and FGF5. Both FGFR1-III isoforms were coexpressed in PSCs and cancer cells. Conditioned medium of COLO-357 cells induced expression of both FGFR1- III isoforms in PSCs. In contrast, conditioned medium of PSCs induced FGFR1-IIIc, but reduced FGFR1- IIIb expression in the cancer cells. Neutralizing the effects of FGFs by heparin-sepharose precipitation abolished these effects completely. FGF2 and other growth factors secreted by PSCs resulted in upregulation of FGFR1-IIIc and downregulation of FGFR1-IIIb in pancreatic cancer cells. CONCLUSIONS: We identified in this study a mechanism based on tumor-stroma interactions involving PSCs that can contribute to enhance the malignant phenotype of human pancreatic cancer.

Identification of c-FLIP(L) and c-FLIP(S) as critical regulators of death receptor-induced apoptosis in pancreatic cancer cells.

BACKGROUND: Evasion of apoptosis is a hallmark of pancreatic cancer. However, the underlying mechanisms are still only partly understood and may involve antiapoptotic proteins such as c-FLIP. Here, the role of c-FLIP in the regulation of death receptor-mediated apoptosis in pancreatic cancer was investigated. METHODS: Expression of c-FLIP(L) and c-FLIP(S) was analysed in primary pancreatic carcinoma samples, pancreatic carcinoma cell lines and primary tumour cells together with its function as a regulator of death receptor-induced apoptosis by knockdown and overexpression studies and through modulation by chemotherapeutics. RESULTS: c-FLIP is expressed in pancreatic intraepithelial neoplasm (PanIN) lesions and in pancreatic ductal adenocarcinomas, whereas normal pancreatic ducts were consistently negative for c-FLIP. Simultaneous downregulation of c-FLIP(L) and c-FLIP(S) as well as individual knockdown of either isoform by RNA interference significantly enhances TRAIL (tumour necrosis factor-related apoptosis-inducing ligand)- and CD95-induced caspase activation and caspase-dependent apoptosis. Also, pretreatment with chemotherapeutic drugs--that is, 5-fluorouracil (5-FU), cisplatin or gemcitabine--downregulates c-FLIP and renders cells sensitive to death receptor-triggered apoptosis. Similarly, primary cultured pancreatic cancer cells are primed for TRAIL-induced apoptosis by pre-exposure to 5-FU or cisplatin. Mechanistic studies revealed that 5-FU-mediated suppression of c-FLIP results in increased TRAIL-induced recruitment and activation of caspase-8 at the death-inducing signalling complex (DISC), leading to caspase-3 activation and caspase-dependent cell death. Overexpression of c-FLIP(L) rescues cells from 5-FU- or cisplatin-mediated sensitisation for TRAIL-induced apoptosis, indicating that c-FLIP suppression is a key event in this chemotherapy-mediated sensitisation to TRAIL. Further, concomitant neutralisation of c-FLIP and XIAP acts in concert to potentiate TRAIL-induced apoptosis. CONCLUSIONS: Both the long and the short isoform of the antiapoptotic protein c-FLIP are critical regulators of death receptor-induced apoptosis in pancreatic carcinoma cells and are suppressed by chemotherapeutics. Targeting either c-FLIP(L) or c-FLIP(S) is sufficient to promote death receptor-induced apoptosis in pancreatic carcinoma cells. These findings have important implications for the design of TRAIL-based combination protocols in pancreatic cancer.

c-Jun N-terminal kinase 2 suppresses pancreatic cancer growth and invasion and is opposed by c-Jun N-terminal kinase 1.

The c-Jun N-terminal protein kinases (JNKs) JNK1 and JNK2 can act as either tumor suppressors or pro-oncogenic kinases in human cancers. The isoform-specific roles for JNK1 and JNK2 in human pancreatic cancer are still unclear, the question which should be addressed in this project. Human pancreatic cancer cell lines MIA PaCa-2 and PANC-1 clones were established either expressing either JNK1 or -2 shRNA in a stable manner. Basal anchorage-dependent and -independent cell growth, single-cell movement, and invasion using the Boyden chamber assay were analyzed. Xenograft growth was assessed using an orthotopic mouse model. All seven tested pancreatic cancer cell lines expressed JNKs as did human pancreatic cancer samples determined by immunohistochemistry. Pharmacological, unspecific JNK inhibition (SP600125) reduced cell growth of all cell lines but PANC-1. Especially inhibition of JNK2 resulted in overall increased oncogenic potential with increased proliferation and invasion, associated with alterations in cytoskeleton structure. Specific inhibition of JNK1 revealed opposing functions. Overall, JNK1 and JNK2 can exert different functions in human pancreatic cancer and act as counter players for tumor invasion. Specifically modulating the activity of JNKs may be of potential therapeutic interest in the future.

PSP/reg inhibits cultured pancreatic stellate cell and regulates MMP/ TIMP ratio.

BACKGROUND: Pancreatic stellate cells (PSC) play a central role in fibrogenesis associated with acute and chronic pancreatitis. Pancreatic stone protein/regenerating protein (PSP/reg) belongs to a family of secretory stress proteins (SSP) that are constitutively synthesized by pancreatic acinar cells and upregulated dramatically during acute and chronic pancreatitis. Assuming a protective role of this stress protein, we investigated its effects on human PSC. MATERIAL AND METHODS: Pancreatic stellate cells were obtained by outgrowth from fibrotic human pancreas tissue. PSP/reg was expressed in the yeast Pichia pastoris and purified from medium supernatants. PSP/reg was added at concentrations of 100 ng/mL to cultured PSC. Cell proliferation was determined by bromodeoxyuridine incorporation. PSC migration was assessed by a wound healing assay. Extracellular matrix (collagen type I and fibronectin), matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs) were demonstrated on protein level. RESULTS: Pancreatic stone protein/regenerating protein inhibited PSC proliferation and migration. Soluble collagen I and fibronectin were reduced after the addition of PSP/reg. PSP/reg slightly decreased the synthesis of MMP-1 and MMP-2 and strongly decreased TIMP-1 and TIMP-2 concentrations in PSC supernatants. CONCLUSIONS: Our work describes a novel aspect that in vitro PSP/reg reduces PSC activity (proliferation and migration) and stimulates fibrolysis by increasing MMP/TIMP ratio. The findings suggest that PSP/reg might have a protective function in the repair phase of acute and chronic pancreatitis by promoting resolution of fibrosis. We highlight PSP/reg as an antifibrogenic protein in pancreatic injury.

Opposing Oncogenic Functions of p38 Mitogen-activated Protein Kinase Alpha and Beta in Human Pancreatic Cancer Cells.

BACKGROUND/AIM: The p38 family of mitogen-activated protein kinases (MAPK) includes four isoforms: p38α, -ß, -γ and -δ. The aim of this study was to elucidate possible functions of p38α and p38ß in human pancreatic cancer. MATERIALS AND METHODS: Isoform expression was determined in seven human pancreatic cancer cell lines. After shRNA based selective knockdown of p38α and p38ß, in vitro growth and migration as well as in vivo tumorigenicity were assessed. RESULTS: All pancreatic cancer cells expressed p38 isoforms. Knockdown of p38α and p38ß inhibited in vitro growth. Migration was markedly reduced in p38α shRNA expressing clones, but not altered by p38ß knockdown. While in vivo inhibition of p38ß decreased tumor formation and growth, the knockdown of p38α significantly enhanced tumorigenicity. CONCLUSION: p38 MAPKs may exert isoform specific functions in pancreatic cancer. Selective targeting may contribute to individualized treatment of pancreatic cancer in the future.

Stimulation of stellate cells by injured acinar cells: a model of acute pancreatitis induced by alcohol and fat (VLDL).

Mechanisms leading to acute pancreatitis after a fat-enriched meal combined with excess alcohol are incompletely understood. We have studied the effects of alcohol and fat (VLDL) on pancreatic acinar cell (PAC) function, oxidative stress, and repair mechanisms by pancreatic stellate cells (PSC) leading to fibrogenesis. To do so, PAC (rat) were isolated and cultured up to 24 h. Ethanol and/or VLDL were added to PAC. We measured PAC function (amylase, lipase), injury (lactic dehydrogenase), apoptosis (TUNEL, Apo2.7, annexin V binding), oxidative stress, and lipid peroxidation (conjugated dienes, malondialdehyde, chemoluminescence); we also measured PSC proliferation (bromodeoxyuridine incorporation), matrix synthesis (immunofluorescence of collagens and fibronectin, fibronectin immunoassay), and fatty acids in PAC supernatants (gas chromatography). Within 6 h, cultured PAC degraded and hydrolyzed VLDL completely. VLDL alone (50 microg/ml) and in combination with alcohol (0.2, 0.5, and 1% vol/vol) induced PAC injury (LDL, amylase, and lipase release) within 2 h through generation of oxidative stress. Depending on the dose of VLDL and alcohol, apoptosis and/or necrosis were induced. Antioxidants (Trolox, Probucol) reduced the cytotoxic effect of alcohol and VLDL. Supernatants of alcohol/VLDL-treated PAC stimulated stellate cell proliferation and extracellular matrix synthesis. We concluded that, in the presence of lipoproteins, alcohol induces acinar cell injury. Our results provide a biochemical pathway for the clinical observation that a fat-enriched meal combined with excess alcohol consumption can induce acinar cell injury (acute pancreatitis) followed by repair mechanisms (proliferation and increased matrix synthesis in PSC).