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The objective evaluates the anatomy of elephant grass under different defoliation frequencies and levels of insertion on the tiller. The experiment was set up as a completely randomized design with...

Background: Reports in a northwestern Mexico state linked arsenic (As) in drinking water to DNA damage in people from indigenous communities. However, this correlation remains under discussion due to unknown variables related to nutrition, customs, and the potential presence of other metal(oid)s. Methods: To determine this association, we sampled water from three Yaqui towns (C&oacute, corit, V&iacute, cam, and P&oacute, tam), and analyzed the metals by ICP-OES. We exposed four separate groups, with five male CD-1 mice each, to provide further insight into the potential effects of untreated drinking water. Results: The maximum concentrations of each metal(oid) in &micro, g&middot, L&minus, 1 were Sr(819) &gt, Zn(135) &gt, As(75) &gt, Ba(57) &gt, Mo(56) &gt, Cu(17) &gt, Al(14) &gt, Mn(12) &gt, Se(19). Histological studies revealed brain cells with angulation, satellitosis, and reactive gliosis with significant statistical correlation with Mn and As. Furthermore, the liver cells presented hepatocellular degeneration. Despite the early response, there is no occurrence of both statistical and significative changes in hematological parameters. Conclusions: The obtained results provide experimental insights to understand the potential effects of untreated water with low As and Mn contents in murine models. This fact is noteworthy because of the development of histological changes on both the brain and liver at subchronic exposure.

Norovirus (NoV) is the leading cause of epidemic and sporadic gastroenteritis worldwide; a high number of those cases are attributed to the consumption of contaminated food. Crop producers have used several strategies to inactivate the virus present in these products and thus stop the NoV transmission chain. Physical methods such as gamma radiation show excellent results in the inactivation of bacteria, but its effect on NoV has been little studied. This study aimed to evaluate the effect of gamma radiation for NoV inactivation, and over the surface topographic characteristics of strawberry cells, as a prototype of soft fruit. A 10% suspension of GII norovirus-positive stool samples were treated with either 200 mg/L of sodium hypochlorite (NaClO) or gamma-irradiated at doses of 5, 10, 15 and 20 kilograys (kGy). Viral inactivation was determined by measuring the integrity of viral capsid using RNase A alone or in combination with proteinase K followed by RT-qPCR. The effect over cellular surface topology characteristics of the fruit was measured by atomic force microscopy (AFM) and confocal microscopy. High doses of radiation (20 kGy) were necessary to detect a significant (p 0.05) decrease of up to 1.26 log

Hepatocellular carcinoma (HCC) ranks fifth in occurrence and second in mortality of all cancers. The development of effective therapies for HCC is urgently needed. Anticancer drugs targeted to the liver-specific asialoglycoprotein receptors (ASGPRs) are viewed as a promising potential treatment for HCC. ASGPRs facilitate the recognition and endocytosis of molecules, and possibly vehicles with galactose end groups, by the liver. In this study, bovine serum albumin (BSA) was conjugated with lactose using a thermal treatment. The formation of lactosylated BSA (BSA-Lac) was confirmed by a change of the chemical structure, increased molecular mass, and Ricinus communis lectin recognition. Subsequently, the low-crosslinking BSA-Lac nanoparticles (LC BSA-Lac NPs) and high-crosslinking BSA-Lac nanoparticles (HC BSA-Lac NPs) were synthesized. These nanoparticles presented spherical shapes with a size distribution of 560 &plusmn, 18.0 nm and 539 &plusmn, 9.0 nm, as well as an estimated surface charge of &minus, 26 &plusmn, 0.15 mV and &minus, 24 &plusmn, 0.45 mV, respectively. Both BSA-Lac NPs were selectively recognized by ASGPRs as shown by biorecognition, competition, and inhibition assays using an in vitro model of HCC. This justifies pursuing the strategy of using BSA-Lac NPs as potential drug nanovehicles with selective direction toward hepatocellular carcinoma.

The targeted drug delivery has been studied as one of the main methods in medicine to ensure successful treatments of diseases. Pharmaceutical sciences are using micro or nano carriers to obtain a controlled delivery of drugs, able to selectively interact with pathogens, cells or tissues. In this work, we modified bovine serum albumin (BSA) with lactose, obtaining a neoglycan (BSA-Lac). Subsequently, we synthesized glyconanoparticles (NPBSA-Lac) with the premise that it would be recognized by microbial galactose specific lectins. NPBSA-Lac were tested for bio-recognition with adhesins of E. coli K88 and Ricinus communis agglutinin I (RCA). Glycation of BSA with lactose was analyzed by electrophoresis, infrared spectroscopy and fluorescence. Approximately 41 lactoses per BSA molecule were estimated. Nanoparticles were obtained using water in oil emulsion method and spheroid morphology with a range size of 300-500 nm was observed. Specific recognition of NPBSA-Lac by RCA and E. coli K88 was displayed by aggregation of nanoparticles analyzed by dynamic light scattering and atomic force microscopy. The results indicate that the lactosylated nanovectors could be targeted at the E. coli K88 adhesin and potentially could be used as a transporter for an antibacterial drug.

The objective of this study was to evaluate the effect of cutting age on the production, chemical composition, degradation kinetics and anatomy of Leucena (Leucaena leucocephala). The experimental design was a completely randomized design with a factorial arrangement 2x4 (two types of tissues and four cutting ages) for the production data of dry matter and 3x4 (three degrees of degrees of digestion of tissues and four cutting ages). Observed effect (P < 0.05) for the total production of DM of Leucena in function of different ages. The tissue types grain and non-grain showed maximum production at 70 days of age, with production of 2333,00 and 716.60 kg DM ha-1, respectively. The parameters of degradation of DM evaluated decreased significantly with the increase in the maturity of the plant, in the same way the chemical composition presented behavior inherent to the advance of age. The effective degradability DM also decreased with the increase in the rate of passage (2, 5, and 8% h-1). The highest rate of degradation (c) was obtained for 30 days. With the advance in plant maturity increases the proportion of vascular tissue lignificad influencing parameters of ruminal degradation of Leucena. The ages assessed influenced the chemical composition of the Leucena (P < 0.05), where the levels of dry matter, crude protein, acid detergent fiber and lignin and ash showed increasing linear behavior. The cutting age of 70 days offers an optimal point regarding the proportion of anatomical tissues correlated with the degradation and chemical composition of the Leucena.

The compounds 2,9,25,32-tetraoxo-4,7,27,30-tetrakis(carboxymethyl)-1,4,7,10,24,27,30,33-octaaza-17,40-dioxa[10.1.10.1]paracyclophane and 2,9,25,32-tetraoxo-4,7,27,30-tetrakis(carboxymethyl)-1,4,7,10,24,27,30,33-octaaza[10.1.10.1]paracyclophane binuclear copper complexes (Cu2PO and Cu2PC, respectively) were studied by determining their antioxidant capacity using the TROLOX equivalent antioxidant capacity (TEAC) assay, and their cytotoxicity on cultured cells, as well as the superoxide dismutase (SOD)-like activity. Cu2PO had an antioxidant capacity (0.1 g eq TROLOX mol−1) within the order of magnitude of ascorbic acid, and both, Cu2PO and Cu2PC were nontoxic to cultured peripheral mononuclear blood cells. The SOD-like activity was evaluated using the nitroblue tetrazolium assay, and both compounds presented an excellent activity: for Cu2PO, the IC50 was 52 nM and for Cu2PC an IC50 of 0.5 μM was obtained comparable to CuZn SOD IC50 17 nM (Fernandes et al., J Inorg Biochem 2007;101:849–858). These results suggest that synthetic binuclear macrocycles are good candidates to be used as synthetic bioactive molecules with applications in biomedicine.

This work presents the fabrication and characterization of electro-sprayed core-shell particles that were composed of maize bran arabinoxylans (MBAX) with insulin in the core, and maize wastewater arabinoxylans (MWAX) with Bifidobacterium in the shell. Two concentrations of MBAX (3 and 6% w/v) and MWAX (6 and 10% w/v) were evaluated. The particles fabricated with MBAX at 6% (w/v) in the core and MWAX at 10% (w/v) in the shell were more stable, presented spherical shape and no aggregation being therefore selected to be loaded with insulin and probiotics. These particles presented a size of 2.9 mm. Scanning electron microscopy analysis of the particle cross section revealed the presence of both, a smooth (shell) and a porous (core) microstructure. Confocal laser scanning microscopy confirmed the core-shell structure of the particles and the viability of the probiotic entrapped. Gastrointestinal simulation strongly suggests that these particles are not degraded in the stomach and small intestine and that 76% of the carried insulin is released in colon. These results indicate that insulin and Bifidobacterium encapsulation by tetraaxial electro spraying can be a feasible and adequate technique to produce arabinoxylan capsules containing both insulin and probiotics. © 2018 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2018, 135, 46411.

The aim of this study was to characterize the immune responses of DCs after infection with four different EU strains of PRRSV and whether they show any ability to immunomodulate T cells activation. Our results show that all EU strains can efficiently infect and replicate in DCs. Nevertheless, SLA-II levels remained unaltered in DC infected by all EU PRRSV strains, whereas SLA-I expression was only reduced when strain 2992 was used. IL-10 production was induced by three EU PRRSV strains, being strain 2992 the highest inducer. However, no induction of Treg cells, measured by CD25 and Foxp3 expression on lymphocytes co-cultured with infected DCs, was found. TGF-β induction was not detected in DC infected with any EU strain tested. In conclusion, DCs infected with EU PRRSV strains exhibited an unbalanced ability to stimulate T cell response and was strain dependent. However, Treg cells were not induced, at least in vitro.

Antigen-presenting cells (APCs) are strategically placed in all anatomic sites with high antigen exposure such as the respiratory system. The aim of this study was to evaluate phenotypic and functional properties of APCs from the lung (L-Cs), mediastinal lymph node (LN-Cs) and bronchoalveolar lavage cells (BAL-Cs). The APCs were first analyzed based on forward scatter and side scatter profiles and the selection of MHC-II(high)CD172a(+) cells (referred to as APCs); then the expression of CD1a, CD163, CD206, CD16 and CD11R3 was evaluated in the APCs. The results showed that CD1a, CD163 and CD206 were differentially expressed among L-Cs, LN-Cs and BAL-Cs, suggesting the phenotype MHC-II(high)CD172a(+)CD1a(low/-)CD163(low)CD206(-) for L-Cs and MHC-II(high)CD172a(+)CD1a(+)CD163(low/-)CD206(+) for LN-Cs. BAL-Cs were MHC-II(high)CD172a(+)CD1a(-)CD163(high)CD206(+/-). The functional characteristics of L-Cs and LN-Cs were different from those of BAL-Cs, confirming that L-Cs and LN-Cs resemble specialized APCs. In conclusion, we present the characterization of APCs from L-Cs, LN-Cs and BAL-Cs of the porcine respiratory system.

Capping of RNA molecules with m7G is an essential modification which plays several roles in RNA metabolism and processing. In the nucleus it directs pre-mRNAs to the processing pathway and mRNAs and U small nuclear RNAs (U snRNAs) to the export pathway. In the cytoplasm, it regulates both mRNA translation initiation and mRNA turnover. In most eukaryotic organisms m7G capping is restricted to nascent RNA polymerase II (PolII) transcripts, namely, pre- mRNAs and precursors to U snRNAs. In contrast, in trypanosomatid protozoa mRNA capping occurs by a fundamentally different mechanism. It is well established that the m7G cap of mature mRNA is acquired posttranscriptionally by an RNA processing reaction, namely, trans splicing. In this process, the capped spliced leader (SL) sequence is transferred from the SL RNA to the 5′ ends of all trypanosome mRNAs (14, 23, 36). Thus, trans splicing can also be considered a trans-capping reaction. The cap structure of the Trypanosoma brucei and Crithidia fasciculata SL RNA is quite elaborate in that the m7G moiety is linked via a 5′-5′ triphosphate bridge to four methylated nucleotides, resulting in an unusual cap 4 structure (2) which is essential for utilization of the SL RNA in trans splicing (18, 43). At present the identity of the RNA polymerase transcribing the SL RNA genes is not defined, because the standard classification based on transcription inhibitors gave conflicting results as to whether PolII or PolIII is the responsible polymerase (8, 25, 26, 42). In addition to the SL RNA, a subset of trypanosome PolIII transcripts, namely, the U2, U3, and U4 snRNAs, is also initially m7G capped (7, 22, 24, 39). Despite the uncertainty about the polymerase transcribing the SL RNA genes, the fact that some PolIII transcripts are capped suggests that the mechanism of transcript selection by the capping enzyme is different in trypanosomes from that in other eukaryotes. m7G capping is mediated by the stepwise action of three enzymatic activities (for a review, see reference 33). First, the γ-phosphate of a primary transcript is removed by RNA 5′-triphosphatase followed by GTP:RNA guanylyltransferase, or capping enzyme, which caps the RNA by the addition of GMP. Finally, the newly attached guanine residue is methylated at the N-7 position by the action of RNA (guanine-7-)methyltransferase. So far, only the reaction mechanism of guanylyltransferase has been examined in some detail. This enzyme catalyzes two sequential nucleotidyl transfer reactions, with a covalent enzyme-guanylate intermediate (19, 30). In this reaction, nucleophilic attack on the α-phosphate of GTP by guanylyltransferase results in the release of pyrophosphate and the formation of a covalent adduct in which GMP is linked to the guanylyltransferase through a phosphoamide bond to the ɛ-amino group of the catalytic lysine residue (30, 34, 38). To complete the reaction, GMP is transferred to the 5′ end of substrate RNA to yield an inverted 5′-5′ triphosphate bond. The vaccinia virus capping enzyme is a multifunctional protein that carries out all three steps of the capping reaction (21, 40, 44). The protein is organized as a heterodimer, with subunits of 95 and 33 kDa (4, 10, 16, 29, 32). The RNA 5′-triphosphatase and guanylyltransferase have been mapped to the amino-terminal 60-kDa domain of the large subunit, while full (guanine-7-)methyltransferase activity requires both subunits. The subunit structure of the vaccinia virus capping enzyme is only partially maintained in cellular counterparts. In particular, cellular capping enzymes are distinct from their viral counterparts in that there is so far no example of a physical association between the capping and methyltransferase functions. In Saccharomyces cerevisiae, the purified capping enzyme consists of two monofunctional polypeptides: a 52-kDa guanylyltransferase and an 80-kDa triphosphatase (12, 13, 28). The guanylyltransferase is the product of the CEG1 gene, which is essential for cell viability (28). As in higher eukaryotes, the yeast (guanine-7-)methyltransferase is purified as a separate entity and is encoded by the ABD1 gene (15). The monofunctional domain structure of the guanylyltransferase is also present in other fungi, such as Schizosaccharomyces pombe (31) and Candida albicans (49), and in Chlorella virus PBCV-1 (11). In higher eukaryotes, biochemical fractionation has shown that the guanylyltransferase from rat liver copurifies with an RNA triphosphatase activity but that the (guanine-7-)methyltransferase readily separates in early chromatography steps and purifies as an unassociated enzyme (48). Recent cloning of the Caenorhabditis elegans (37, 46) and mammalian (17, 50) capping enzymes showed that these enzymes consist of a single bifunctional polypeptide with two domains: a carboxy-terminal guanylyltransferase domain and an amino-terminal domain with RNA triphosphatase activity. Even though the guanylyltransferase domain is strictly conserved with respect to amino acids that are essential for in vivo function, the RNA triphosphatase domain does not resemble the vaccinia virus triphosphatase domain but rather has significant sequence and mechanistic similarities to the family of protein tyrosine phosphatases. Thus, it would appear from the above examples that unicellular and multicellular organisms differ with respect to the physical organization of enzymatic activities of the capping machinery; whereas unicellular organisms have a monofunctional guanylyltransferase, multicellular organisms have a bifunctional triphosphatase-guanylyltransferase. As a first step toward understanding the process of RNA capping in trypanosomes, we have purified the capping enzyme from C. fasciculata and cloned the corresponding genes from C. fasciculata and T. brucei. Comparison of the predicted amino acid sequences of the trypanosome proteins with those of the available eukaryotic and viral capping enzymes revealed several unique structural features. In particular, the trypanosome capping enzymes are remarkable in that they include a novel amino-terminal domain that displays no resemblance to any other domain associated with capping enzymes.

Swine influenza virus (SwIV) is considered a zoonosis and the fact that swine may act as an intermediate reservoir for avian influenza virus, potentially infectious for humans, highlights its relevance and the need to understand the interaction of different influenza viruses with the porcine immune system. Thus, in vitro porcine bone marrow-derived dendritic cell (poBMDCs) were infected with a circulating SwIV A/Swine/Spain/SF32071/2007(H3N2), 2009 human pandemic influenza virus A/Catalonia/63/2009(H1N1), low pathogenic avian influenza virus (LPAIV) A/Anas plathyrhynchos/Spain/1877/2009(aH7N2) or high pathogenic avian influenza virus (HPAIV) A/Chicken/Italy/5093/1999(aH7N1). Swine influenza virus H3N2 infection induced an increase of SLA-I and CD80/86 at 16 and 24 h post infection (hpi), whereas the other viruses did not. All viruses induced gene expression of NF-κB, TGF-β, IFN-β and IL-10 at the mRNA level in swine poBMDCs to different extents and in a time-dependent manner. All viruses induced the secretion of IL-12 mostly at 24 hpi whereas IL-18 was detected at all tested times. Only swH3N2 induced IFN-α in a time-dependent manner. Swine H3N2, aH7N2 and aH7N1 induced secretion of TNF-α also in a time-dependent manner. Inhibition of NF-κB resulted in a decrease of IFN-α and IL-12 secretion by swH3N2-infected poBMDC at 24 hpi, suggesting a role of this transcription factor in the synthesis of these cytokines. Altogether, these data might help in understanding the relationship between influenza viruses and porcine dendritic cells in the innate immune response in swine controlled through soluble mediators and transcription factors., This work was partly funded by the Project No. CSD 2006-00007, AGL2006-13809-C03-01, AGL2009-12945-C02-01 and AGL2010-22200-C02-01 by the Spanish Government. PhD studies of Mrs. Tufária Mussá and Masssimiliano Baratelli are funded by doctoral grants from the AECID and MICIN respectively.

The aim of this study was to analyze the regulatory T cells (Tregs) induced by the porcine reproductive and respiratory syndrome virus (PRRSV) in pigs. Serum, blood, tonsil, and mediastinal lymph nodes' samples were obtained at different time post-infection (dpi). The frequencies of CD4(+)CD8(-)CD25(+)Foxp3(+), CD4(+)CD8(+)CD25(+)Foxp3(+), or CD4(-)CD8(+)CD25(+)Foxp3(+) phenotypes were determined in PBMC and lymph node cells, and cells producing IL-10 or TGF-β were analyzed. PRRSV increased the number of CD4(+)CD8(+)CD25(+)Foxp3(+) cells at 14 dpi, whereas CD4(+)CD8(-)CD25(+)Foxp3(+) remained constant until 28 dpi. Positive correlation exists between viremia and induced regulatory cells. CD4(+)CD8(+)CD25(+)Foxp3(+)-induced Treg cells were consistently observed in lymphoid tissues. Analysis of IL-10- and TGF-β-producing cell demonstrated that in response to PRRSV, CD4(+)CD8(-)Foxp3(low) and CD4(+)CD8(+)Foxp3(high) cells increase moderately the proportion of IL-10(+) cells. TGF-β was only observed in the CD4(+)CD8(+)Foxp3(high) population after PRRSV stimulation. In conclusion, PRRSV infection increases the frequency of Tregs with the phenotype CD4(+)CD8(+)CD25(+)Foxp3(high) and produces TGF-β.

Porcine reproductive and respiratory syndrome virus (PRRSV) infects mature dendritic cells (mDCs) derived from porcine monocytes and matured with lipopolysaccharide. The infection of mDCs induced apoptosis, reduced the expression of CD80/86 and major histocompatibility complex class II molecules, and increased the expression of interleukin-10, thus suggesting that such mDC modulation results in the impairment of T-cell activation.

To further our understanding of the structural and functional organization of the Trypanosoma brucei genome, we have searched for and analyzed sites in the genome where Pol II transcription units meet Pol III genes. Physical and transcriptional maps of cosmid clones spanning the Pol III-transcribed U2 small nuclear RNA (snRNA) and U3 snRNA/7SL RNA gene loci demonstrated that single-copy Pol II genes are closely associated with Pol III-transcribed genes, being separated from each other by 0.6-3 kb. At the U3/7SL transcriptional domain, two Pol II transcription units converged from either side of the chromosome towards the Pol III genes, suggesting that at least for the chromosome containing the U3 snRNA and 7SL RNA genes, there exist two distinct initiation sites for Pol II. Furthermore, in all cases the Pol III genes hallmark the end of Pol II transcription units, suggesting perhaps a functional role for this genetic arrangement. Lastly, we asked whether the environment within a Pol III transcriptional domain allowed expression of pre-mRNA. To test this we inserted a CAT gene cassette, seemingly promoterless but endowed with pre-mRNA processing signals, in the chromosome between the U3 snRNA and 7SL RNA genes. Interestingly, abundant CAT mRNA was produced suggesting that the Pol III genes in the immediate vicinity did not prevent access of presumably Pol II to the CAT gene cassette. We propose that either CAT mRNA is synthesized by Pol II run-through transcription or by Pol II initiationupstream from the CAT gene.

Delayed development of virus-specific immune response has been observed in pigs infected with the porcine reproductive and respiratory syndrome virus (PRRSV). Several studies support the hypothesis that the PRRSV is capable of modulating porcine immune system, but the mechanisms involved are yet to be defined. In this study, we evaluated the induction of T regulatory cells by PRRSV-infected dendritic cells (DCs). Our results showed that PRRSV-infected DCs significantly increased Foxp3(+)CD25(+) T cells, an effect that was reversible by IFN-alpha treatment, and this outcome was reproducible using two distinct PRRSV strains. Analysis of the expressed cytokines suggested that the induction of Foxp3(+)CD25(+) T cells is dependent on TGF-beta but not IL-10. In addition, a significant up-regulation of Foxp3 mRNA, but not TBX21 or GATA3, was detected. Importantly, our results showed that the induced Foxp3(+)CD25(+) T cells were able to suppress the proliferation of PHA-stimulated PBMCs. The T cells induced by the PRRSV-infected DCs fit the Foxp3(+)CD25(+) T helper 3 (Th3) regulatory cell phenotype described in the literature. The induction of this cell phenotype depended, at least in part, on PRRSV viability because IFN-alpha treatment or virus inactivation reversed these effects. In conclusion, this data supports the hypothesis that the PRRSV succeeds to establish and replicate in porcine cells early post-infection, in part, by inducing Th3 regulatory cells as a mechanism of modulating the porcine immune system.