H2A.Z overexpression suppresses senescence and chemosensitivity in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most intractable and devastating malignant tumors. Epigenetic modifications such as DNA methylation and histone modification regulate tumor initiation and progression. However, the contribution of histone variants in PDAC is unknown. Here, we demonstrated that the histone variant H2A.Z is highly expressed in PDAC cell lines and PDAC patients and that its overexpression correlates with poor prognosis. Moreover, all three H2A.Z isoforms (H2A.Z.1, H2A.Z.2.1, and H2A.Z.2.2) are highly expressed in PDAC cell lines and PDAC patients. Knockdown of these H2A.Z isoforms in PDAC cell lines induces a senescent phenotype, cell cycle arrest in phase G2/M, increased expression of cyclin-dependent kinase inhibitor CDKN2A/p16, SA-ß-galactosidase activity and interleukin 8 production. Transcriptome analysis of H2A.Z-depleted PDAC cells showed altered gene expression in fatty acid biosynthesis pathways and those that regulate cell cycle and DNA damage repair. Importantly, depletion of H2A.Z isoforms reduces the tumor size in a mouse xenograft model in vivo and sensitizes PDAC cells to gemcitabine. Overexpression of H2A.Z.1 and H2A.Z.2.1 more than H2A.Z.2.2 partially restores the oncogenic phenotype. Therefore, our data suggest that overexpression of H2A.Z isoforms enables cells to overcome the oncoprotective barrier associated with senescence, favoring PDAC tumor grow and chemoresistance. These results make H2A.Z a potential candidate as a diagnostic biomarker and therapeutic target for PDAC.

Curcumin induces p53-independent inactivation of Nrf2 during oxidative stress-induced apoptosis.

The transcription factor Nrf2 (nuclear factor erythroid 2-related factor 2) is a master regulator of a battery of antioxidant and detoxificant genes with cytoprotective function. Since Nrf2 inactivation is necessary for the complete execution of apoptosis in the presence of extensive cellular damage caused by oxidative stress, constant activation of Nrf2 may protect tumoral cells from apoptosis. The tumor suppressor gene p53 has been suggested to participate in apoptosis-related repression of Nrf2. Thus, we studied the inactivation of Nrf2 during oxidant-induced apoptosis in a p53 dysfunctional cellular model. Using curcumin dose-response assay and time-response assay in an immortalized lymphoblastoid cell line (control line 45), we observed a time-dependent increase in apoptotic markers such as deoxyribonucleic acid (DNA) fragmentation, phosphatidylserine exposure, and caspase-3, caspase-9 and poly (ADP-ribose) polymerases (PARP) cleavage. Interestingly, at early times of exposure to a proapoptotic dose of curcumin (15 µM), we observed nuclear accumulation of Nrf2 and the expression of Nrf2 target genes, whereas at late exposure times we found a reduction of total and nuclear protein levels of Nrf2 as well as downregulation of Nrf2 target genes in the absence of p53 activation. These data suggest that apoptosis-related inactivation of Nrf2 could occur in a p53 dysfunctional background, opening the possible occurrence of p53-independent mechanism to explain Nrf2 inactivation during apoptosis.

mTORC1 Prevents Epithelial Damage During Inflammation and Inhibits Colitis-Associated Colorectal Cancer Development.

Epithelial cells lining the intestinal mucosa constitute a selective-semipermeable barrier acting as first line of defense in the organism. The number of those cells remains constant during physiological conditions, but disruption of epithelial cell homeostasis has been observed in several pathologies. During colitis, epithelial cell proliferation decreases and cell death augments. The mechanism responsible for these changes remains unknown. Here, we show that the pro-inflammatory cytokine IFNγ contributes to the inhibition of epithelial cell proliferation in intestinal epithelial cells (IECs) by inducing the activation of mTORC1. Activation of mTORC1 in response to IFNγ was detected in IECs present along the crypt axis and in colonic macrophages. mTORC1 inhibition enhances cell proliferation, increases DNA damage in IEC. In macrophages, mTORC1 inhibition strongly reduces the expression of pro-inflammatory markers. As a consequence, mTORC1 inhibition exacerbated disease activity, increased mucosal damage, enhanced ulceration, augmented cell infiltration, decreased survival and stimulated tumor formation in a model of colorectal cancer CRC associated to colitis. Thus, our findings suggest that mTORC1 signaling downstream of IFNγ prevents epithelial DNA damage and cancer development during colitis.

14-3-3 Proteins regulate Akt Thr308 phosphorylation in intestinal epithelial cells.

Akt activation has been associated with proliferation, differentiation, survival and death of epithelial cells. Phosphorylation of Thr308 of Akt by phosphoinositide-dependent kinase 1 (PDK1) is critical for optimal stimulation of its kinase activity. However, the mechanism(s) regulating this process remain elusive. Here, we report that 14-3-3 proteins control Akt Thr308 phosphorylation during intestinal inflammation. Mechanistically, we found that IFNγ and TNFα treatment induce degradation of the PDK1 inhibitor, 14-3-3η, in intestinal epithelial cells. This mechanism requires association of 14-3-3ζ with raptor in a process that triggers autophagy and leads to 14-3-3η degradation. Notably, inhibition of 14-3-3 function by the chemical inhibitor BV02 induces uncontrolled Akt activation, nuclear Akt accumulation and ultimately intestinal epithelial cell death. Our results suggest that 14-3-3 proteins control Akt activation and regulate its biological functions, thereby providing a new mechanistic link between cell survival and apoptosis of intestinal epithelial cells during inflammation.

A prime-boost vaccination of mice with attenuated Salmonella expressing a 30-mer peptide from the Trichinella spiralis gp43 antigen.

Protection against Trichinella infections has been achieved using various parasite antigens and adjuvants. Recently, we reported that immunization of mice with an attenuated Salmonella strain displaying a 30-mer peptide (residues 210-239) from the Trichinella spiralis gp43 antigen using the ShdA autotransporter induced partial protection against T. spiralis infection. To improve the efficacy of vaccination, we used the MisL autotransporter system to display the Ts30mer peptide on the surface of Salmonella enterica ser. Typhimurium in combination with a prime-boost vaccination strategy. This vector and immunization regimen induced superior protection against T. spiralis when compared to our previously reported approach. Data presented herein showed a significant reduction in adult worm and muscle larvae burdens, high IgG titers, and increased production of intestinal mucus with entrapped adult worms. This prime-boost vaccination scheme is a suitable strategy to elicit enhanced protective immunity against T. spiralis.

A novel identification scheme for genus Mycobacterium, M. tuberculosis complex, and seven mycobacteria species of human clinical impact.

Recently, the incidence of human mycobacterial infections due to species other than M. tuberculosis has increased worldwide. Since disease control depends on appropriate antimicrobial therapy, the precise identification of these species of clinical importance has become a major public health concern. Identification of mycobacteria has been hampered because of the lack of specific, rapid, and inexpensive methods. Therefore, we aimed at designing and validating a bacterial lysate-based polymerase chain reaction identification scheme. This scheme can classify clinical isolates into: (1) the genus Mycobacterium, (2) the M. tuberculosis complex, (3) the nontuberculous mycobacteria, and (4) the species M. avium, M. intracellulare, M. abscessus, M. chelonae, M. fortuitum and M. bovis of clinical importance, and M. gordonae, the most commonly encountered nonpathogenic species in clinical laboratories. By using M. fortuitum and M. avium lysates as models, the method sensitivity was determined to be 372 pg of DNA. In a blind parallel comparison between our approach and conventional biochemical tests, both assays correctly categorized 75 patient's mycobacterial isolates. However, our approach only required 4-9 h for categorization compared with at least 15 days by conventional tests. Furthermore, our methodology could also detect M. fortuitum and M. avium from liquid cultures, after only 2 and 6 days, respectively, of incubation. Our new identification scheme is therefore sensitive, specific, rapid, and economic. Additionally, it can help to provide proper treatment to patients, to control these diseases, and to improve our knowledge of the epidemiology of mycobacteriosis, all urgently needed, particularly in developing countries.

Production and characterization of a monoclonal antibody specific for NS3 protease and the ATPase region of Dengue-2 virus.

Dengue is considered a reemerging disease of worldwide distribution. The Dengue virus non-structural protein 3 (NS3) is known to possess ATPase, helicase, and protease activities that are a constitutive part of the replication complex of Dengue virus. In this report, we discuss the cloning, expressing, and purifying of the Dengue-2 NS3 protein, to immunize mice and then generate monoclonal antibodies (MAbs). Our results show the production of MAbs specific to NS3 protein of Dengue-2 virus, which by immunofluorescence recognize the native protein in experimentally infected endothelial cells (HMEC). Likewise, C6/36-infected lisates were used in Western blots, and observed the specific characteristic band that defines the NS3 protein. We conclude that these antibodies may be a useful tool, not only to study the replicative process of Dengue virus, but also to generate specific diagnostic tools for Dengue infection.

Actin induction during PMA and cAMP-dependent signal pathway activation in Entamoeba histolytica trophozoites.

Activation of PKC or cAMP-dependent signalling pathways in Entamoeba histolytica triggers the phosphorylation of proteins involved in actin rearrangements necessary for adhesion and locomotion. Analogous motifs to SRE and CRE sequences--known to respond to PMA and cAMP--were identified within the 5' regulatory region (5'RR) of one of the parasite actin genes. These sequences could be involved in the actin transcriptional upregulation reported during signalling. To test this hypothesis, a plasmid containing the 5'RR of the actin gene fused to the bacterial neomycin gene (neo) was used for stable transfection. Expression of neo and endogenous actin was measured after stimulation of transfected amoebae by PMA and dcAMP. It was found that both compounds induced neo and actin expression and showed a co-operative effect in the induction of neo. Induction by PMA or dcAMP failed if the directing amoebic 5'RR lacked SRE and CRE motifs. Transfection of amoebae with plasmid constructs, containing either progressive deletions of the actin 5'RR or site-directed mutations of the SRE and CRE-like motifs, corroborated that these sequences and a co-ordinated participation of PKC- and PKA-activated transcription factors are responsible for the increments in neo and actin mRNAs. In vivo, these PMA and cAMP-response elements could play an important role in regulating actin expression and organization in signalling processes activated during tissue invasion.

Molecular characterization of two genes encoding betaine aldehyde dehydrogenase from amaranth. Expression in leaves under short-term exposure to osmotic stress or abscisic acid.

A genomic clone (ahybadh4) and a cDNA (ahybadh17) both encoding betaine aldehyde dehydrogenase (BADH; EC 1.2.1.8) were isolated from the plant Amaranthus hypochondriacus L. The ahybadh4 gene extends 9 kilobases (kb) containing 15 exons with an open reading frame (ORF) of 501 amino acids (aa), a 1.3kb 5' untranslated region (UTR) and a 3' UTR of 0.3kb. The ahybadh17 cDNA encodes a BADH isoform of 500aa which contains 10aa substitutions with respect to AHYBADH4. Both encoded proteins share 98% identity at the amino acid level. Comparison of amaranth BADHs with other reported sequences showed high similarity. Analysis of ahybadh17 expression in amaranth leaves showed that mRNA and BADH protein are present in non-treated amaranth leaves and both transiently increased under short-term exposure to abscisic acid (ABA) and osmotic stress treatments.

Agrobacterium-mediated transformation of Amaranthus hypochondriacus: light- and tissue-specific expression of a pea chlorophyll a/b-binding protein promoter.

Mature embryos of Amaranthus hypochondriacus (amaranth) were used to develop an in vitro culture system for plant regeneration and genetic transformation. Plants were regenerated from embryo-derived callus cultivated on Murashige and Skoog medium supplemented with 10 µM 2,4-dichlorophenoxyacetic acid or 3,6-dichloro-2-methoxybenzoic acid and 10% coconut liquid endosperm. Transgenic plants were obtained by inoculation of mature embryo explants with a disarmed Agrobacterium strain containing the plasmid pGV2260(pEsc4), which carried the genes encoding neomycin phosphotransferase type II and ß-glucuronidase. The presence of transgenes in the genome of transformed amaranth plants and their progeny was demonstrated by Southern blot hybridization. Tissue specific and light-inducible expression directed by a pea chlorophyll a/b-binding protein promoter was observed in transgenic amaranth plants and their progeny.