High diversity among environmental Escherichia coli isolates from a bovine feedlot

Approximately 280 Escherichia coli isolates were isolated from a bovine feedlot at the University of Connecticut campus via enrichment in lauryl tryptose broth and random selection from MacConkey plates. The E. coli subspecies diversity was estimated by employing whole-cell BOX-PCR genomic fingerprints. A total of 89 distinct operational taxonomic units (OTUs) were identified by employing a criterion of 85% fingerprint similarity as a surrogate for an OTU, while the Chao1 index estimated the E. coli population richness at 128 OTUs. One genotype (at a similarity level of 60%) dominated the population at 66% regardless of sampling depth or location, while no significant vertical distribution pattern was observed in terms of genotype, mobility, antibiotic resistance profile, or biofilm-forming ability. Motility, measured by a soft agar assay, had a very broad range among the E. coli population and was positively correlated with biofilm-forming ability in minimal medium (Spearman's rank correlation coefficient r = 0.619, P < 10(-4)) but not in Luria broth. Only an estimated 48% of the population possessed gene agn43, which encodes Ag43, a phase-variable outer membrane protein that has been implicated in biofilm formation in minimal medium. We observed significantly more biofilm formation in both minimal medium and Luria broth for agn43(+) strains, with a larger effect in minimal medium. This study represents an exhaustive inventory of extant E. coli population diversity at a bovine feedlot and reveals significant subspecies heterogeneity in interfacial behavior.     

Differential Migration and Activation Profile of Monocytes after Trophoblast Interaction

Macrophages at the maternal-placental interface coordinate opposite demands under the control of trophoblast cells such as the response against pathogens on one hand, and apoptotic cell clearance and wound healing with the production of suppressor cytokines. Here, we investigated whether trophoblast cells induce maternal monocyte activation towards an alternative activated macrophage profile and whether bacterial or viral stimuli modulate their migratory properties. We used an in vitro model of the maternal-placental interface represented by co-cultures of CD14+ cells isolated from fertile women with first trimester trophoblast cell line (Swan-71 cells) in the presence or absence of pathogen associated molecular pattern (PAMP) stimuli lipopolysaccharide (LPS), peptidoglycan (PGN) or poly [I:C]). Maternal CD14+ cells showed increased CD16 and CD39 expression, both markers associated to an alternative activation profile, with no changes in CD80 expression after trophoblast cell interaction. These changes were accompanied by increased IL-10 and decreased IL-12 production by CD14+ cells. After stimulation with LPS, PGN or poly [I:C], monocytes co-cultured with trophoblast cells had lower production of TNF-α and IL-1β compared with non co-cultured monocytes. Interestingly, monocyte migration towards trophoblast cells was prevented in the presence of LPS or PGN but not after 24h of stimulation with poly [I:C]. LPS or PGN also decreased CCR5, CXCL-8 and CCL5 expression. Finally, trophoblast cells co-cultured with monocytes in the presence of pathological stimuli failed to increase chemokine expression, indicating a bidirectional effect. In conclusion, trophoblast might ‘instruct’ maternal monocytes to express an alternative activation profile and restrain their early recruitment under pathological threats as one of the first strategies to avoid potential tissue damage at the maternal-placental interface.     

ER–plasma membrane contact sites contribute to autophagosome biogenesis by regulation of local PI3P synthesis

The double‐membrane‐bound autophagosome is formed by the closure of a structure called the phagophore, origin of which is still unclear. The endoplasmic reticulum (ER) is clearly implicated in autophagosome biogenesis due to the presence of the omegasome subdomain positive for DFCP1, a phosphatidyl‐inositol‐3‐phosphate (PI3P) binding protein. Contribution of other membrane sources, like the plasma membrane (PM), is still difficult to integrate in a global picture. Here we show that ER–plasma membrane contact sites are mobilized for autophagosome biogenesis, by direct implication of the tethering extended synaptotagmins (E‐Syts) proteins. Imaging data revealed that early autophagic markers are recruited to E‐Syt‐containing domains during autophagy and that inhibition of E‐Syts expression leads to a reduction in autophagosome biogenesis. Furthermore, we demonstrate that E‐Syts are essential for autophagy‐associated PI3P synthesis at the cortical ER membrane via the recruitment of VMP1, the stabilizing ER partner of the PI3KC3 complex. These results highlight the contribution of ER–plasma membrane tethers to autophagosome biogenesis regulation and support the importance of membrane contact sites in autophagy.     

Ultrasonographic determination of ovarian follicular. Development in superovulated heifers pretreated with FSH-P at the beginning of the estrous cycle

On Day 3 of the estrous cycle (estrus = Day 0), dairy heifers were given either 10 mg i.m. FSH-P (FSH-P primed; n = 9) or a saline vehicle (saline primed; n = 9). On Day 10, all heifers were superovulated with FSH-P (total = 27.7 mg i.m.) in declining doses over 5 d. Heifers were inseminated artificially at estrus. From Day 2 until estrus, the number and size of follicles >2 mm were monitored daily by ultrasonography. The mean (+/- SEM) number of corpora lutea (CL) (6.2 +/- 1.5 vs 10.7 +/- 0.9; P<0.05) and the mean number of recovered embryos and unfertilized ova (3.6 +/- 1.7 vs 8.4 +/- 2.2; P<0.05) were lower in FSH-P-primed than in saline-primed heifers. Prior to initiation of superovulation, follicles >10 mm appeared on Days 6 to 7 in saline-primed heifers but only on Days 8 to 10 in FSH-P-primed heifers (P<0.05). Also, until Day 10, the mean number of follicles 4 to 6 mm and 7 to 10 mm was higher (P<0.05) in FSH-P-primed than in saline-primed heifers. After initiation of the superovulatory treatment (Day 10 to estrus), saline-primed heifers had a greater and faster increase in the mean number of follicles >10 mm (P<0.02) than FSH-P-primed heifers did. Depletion in the number of follicles 2 to 3 mm (P<0.001) between Day 10 and estrus and in the number of follicles 4 to 6 mm (P<0.05) between Day 12 and estrus occurred in both groups of heifers. Decreased superovulatory response and embryo recovery in FSH-P-primed heifers may have been due to the presence of large follicles (>10 mm) prior to the initiation of the superovulatory treatment which reduced the ability of small follicles to grow into larger size classes during superovulatory treatment.     

Immunohistochemical identification and comparison of glial cell lineage in foetal, neonatal, adult and neoplastic human adrenal medulla

The differentiation of glial cells in developing, neonatal, adult and neoplastic human adrenal medulla has been studied immunohistochemically. From 8 to 28 weeks' gestational age, S-100 protein and its β-subunit revealed two different glial cell populations in adrenal glands, namely Schwann-like and sustentacular cells. Schwann-like cells were spindle-shaped cells forming a continuous layer around groups of sympathetic neuroblasts, often in contact with Schwann cells of nerve fibres entering neuroblastic groups. Sustentacular cells were round or oval cells with dendritic cytoplasmic processes; they were not associated with nerve fibres and mingled both with sympathetic neuroblasts and differentiating chromaffin cells. The developmental fate of Schwann-like cells was different from that of sustentacular cells. Schwann-like cells disappeared from the 28th week of gestational age, in association with the disappearance of sympathetic neuroblastic groups, and they were rarely found in neonatal and adult adrenal medulla. In contrast, sustentacular cells persisted between medullary chromaffin cells, and their number and dendritic cytoplasmic processes progressively increased from foetus to adult. In eight cases of primitive adrenal neuroblastic tumours of neonatal age (five undifferentiated neuroblastomas and three ganglioneuroblastomas), Schwann-like cells were found at the periphery of tumoral nests with a lobular growth pattern, while rare sustentacular cells were associated with neuroblasts. In two cases of adult phaeochromocytomas, only sustentacular cells were detected between chromaffin tumoral cells. Our findings suggest that the glial cell types and their distribution in primitive adrenal medulla tumours closely resemble those observed during development in the groups of adrenal sympathetic neuroblasts and in the clusters of chromaffin cells     

Site directed mutagenesis of subunit 8 of yeast mitochondrial ATP synthase: Functional and import properties of a series of C-terminally truncated forms

The function of the positively charged C-terminal region of mitochondrially encoded subunit 8 of yeast mitochondrial ATP synthase was investigated using derivatives truncated at each of the 3 positively charged residues (Arg³⁷, Arg⁴² and Lys⁴⁷). Each construct, allotopically expressed in the nucleus, was tested for its ability to import and assemble functionally into ATP synthase in yeast cells unable to synthesize mitochondrial subunit 8. The efficiency of import of each construct into isolated wild-type yeast mitochondria was also determined. One construct truncated at the penultimate residue of subunit 8 (Lys⁴⁷) functions in vivo and shows efficient import in vitro. Thus subunit 8 can function with only two positively charged residues. The remainder of the subunit 8 variants failed to rescue in vivo. Since they all show greatly reduced or undetectable import in vitro, presumably because of the increased hydrophobic character of the subunit 8 moiety in the chimaeric precursors, the status of these variants as regards assembly and function is not clear.     

The potential role of spherocrystals in the detoxification of essential trace metals following exposure to Cu and Zn in the fighting conch <Emphasis Type="Italic">Strombus</Emphasis> (<Emphasis Type="Italic">Lobatus</Emphasis>) <Emphasis Type="Italic">pugilis</Emphasis>

Crypt cells—one of the three cell types composing Strombidae digestive tubules—are characterized by the presence of numerous metal-containing phosphate granules termed spherocrystals. We explored the bioaccumulation and detoxification of metals in Strombidae by exposing wild fighting conch Strombus pugilis for 9 days to waterborne CuSO₄ and ZnSO₄. The total amount of Cu and Zn was determined in the digestive gland and in the rest of the body by Inductively Coupled Plasma (ICP) analyses. The digestive gland spherocrystal metal content was investigated based on the semi-quantitative energy dispersive X-ray (EDX) elemental analysis. ICP analyses of unexposed individuals revealed that 87.0?±?5.9% of the Zn is contained in the digestive gland, where its concentration is 36 times higher than in the rest of the body. Regarding Cu, 25.8?±?16.4% of the metal was located in the digestive gland of the control individuals, increasing to 61.5?±?16.4% in exposed individuals. Both Cu and Zn concentrations in the digestive gland increased after exposures, pointing to a potential role of this organ in the detoxification of these metals. EDX analysis of spherocrystals revealed the presence of Ca, Cl, Fe, K, Mg, P, and Zn in unexposed individuals. No difference was found in the relative proportion of Zn in spherocrystals of exposed versus control individuals. Contrastingly, copper was never detected in the spherocrystals from controls and Zn-exposed individuals, but the relative proportion of Cu in spherocrystals of Cu-exposed individuals varied from 0.3 to 5.7%. Our results show the direct role of spherocrystals in Cu detoxification.     

<Emphasis Type="Italic">Paenibacillus</Emphasis> sp. A59 GH10 and GH11 Extracellular Endoxylanases: Application in Biomass Bioconversion

The cost-efficient degradation of xylan to fermentable sugars is of particular interest in second generation bioethanol production, feed, food, and pulp and paper industries. Multiple potentially secreted enzymes involved in polysaccharide deconstruction are encoded in the genome of Paenibacillus sp. A59, a xylanolytic soil bacterium, such as three endoxylanases, seven GH43 β-xylosidases, and two GH30 glucuronoxylanases. In secretome analysis of xylan cultures, ten glycoside hydrolases were identified, including the three predicted endoxylanases, confirming their active role. The two uni-modular xylanases, a 32-KDa GH10 and a 20-KDa GH11, were recombinantly expressed and their activity on xylan was confirmed (106 and 85 IU/mg, respectively), with differences in their activity pattern. Both endoxylanases released mainly xylobiose (X2) and xylotriose (X3) from xylan and pre-treated biomasses (wheat straw, barley straw, and sweet corn cob), although only rGH10XynA released xylose (X1). rGH10XynA presented optimal conditions at pH 6, with thermal stability at 45–50 °C, while rGH11XynB showed activity in a wider range of pH, from 5 to 9, and was thermostable only at 45 °C. Moreover, GH11XynB presented sigmoidal kinetics on xylan, indicating possible cooperative binding, which was further supported by the structural model. This study provides a detailed analysis of the complete set of carbohydrate-active enzymes encoded in Paenibacillus sp. A59 genome and those effectively implicated in hemicellulose hydrolysis, contributing to understanding the mechanisms necessary for the bioconversion of this polysaccharide. Moreover, the two main free secreted xylanases, rGH10XynA and rGH11XynB, were fully characterized, supporting their potential application in industrial bioprocesses on lignocellulosic biomass.