Effects of dietary chlortetracycline on the antimicrobial resistance of broiler faecal Streptococcaceae

A breeder flock and a control group of progeny birds were fed antimicrobial-free rations; a second group of progeny received rations supplemented with 50 g chlortetracycline (Ctc)/ton. Effects of dietary Ctc on the distribution of species and biotypes of faecal Gram-positive cocci and their relative resistance to 12 antimicrobial agents were studied. Antimicrobial resistance (AMR) pattern diversity and modal AMR patterns were determined for bacterial species common to all three groups. Numerical taxonomic analysis placed 1321 (97%) of 1360 isolates into eight species or biotypes. The largest cluster (n = 659, 48%) was a biotype of Streptococcus faecalis. Three clusters were biotypes of Streptococcus faecium and contained 580 isolates (42%). The isolates were susceptible to ampicillin and almost uniformly resistant to methicillin, neomycin, streptomycin, sulfadiazine and tetracycline. There were 54 and 47 different AMR patterns, including 0 to 11 and 1 to 11 resistance determinants, in isolates from control and Ctc-fed birds, respectively. Modal AMR patterns for Strep. faecalis and one biotype of Strep. faecium were very similar for all three groups of birds. However, modal patterns in a second biotype of Strep. faecium varied considerably for all three groups. Interpretation of AMR pattern diversities were equivocal among biotypes from both progeny groups. The variable distribution of isolates, proportions of resistant strains, modal patterns and diversity indices among the progeny were probably due to their exposure to different environmental sources of bacteria.     

Cadherin-Mediated Cell Adhesion Is Critical for the Closing of the Mouse Optic Fissure

Coloboma is a congenital disease that contributes significantly to childhood blindness. It results from the failure in closing the optic fissure, a transient opening on the ventral side of the developing eye. Although human and mouse genetic studies have identified a number of genes associated with coloboma, the detailed cellular mechanisms underlying the optic fissure closure and coloboma formation remain largely undefined. N-cadherin-mediated cell adhesion has been shown to be important for the optic fissure closure in zebrafish, but it remains to be determined experimentally how cell-cell adhesions are involved in the mammalian optic fissure closing process. α-catenin is required for cell adhesion mediated by all of the classic cadherin molecules, including N-cadherin. In this study, we used the Cre-mediated conditional knockout technique to specifically delete α-catenin from the developing mouse eye to show that it is required for the successful closing of the optic fissure. In α-catenin conditional mutant optic cups, the major cell fates, including the optic fissure margin, neural retina and retinal pigmented epithelium, are specified normally, and the retinal progenitor cells proliferate normally. However, adherens junctions components, including N-cadherin, β-catenin and filamentous actin, fail to accumulate on the apical side of α-catenin mutant retinal progenitor cells, where adherens junctions are normally abundant, and the organization of the neural retina and the optic fissure margin is disrupted. Finally, the α-catenin mutant retina gradually degenerates in the adult mouse eye. Therefore, our results show that α-catenin-mediated cell adhesion and cell organization are important for the fissure closure in mice, and further suggest that genes that regulate cell adhesion may underlie certain coloboma cases in humans.     

Generation and characterization of mouse monoclonal antibodies to the myelin-associated glycoprotein (MAG)

A panel of mouse monoclonal antibodies to rat and human myelin-associated glycoprotein (MAG) was developed. Normal mice were unresponsive to rat MAG, and successful immunization with rat MAG was only achieved in autoimmune NZB mice. By contrast, all strains of mice were responsive to human MAG. The monoclonal antibodies developed differ with respect to immunoglobulin type, their specificity for human and/or rat MAG, and their recognition of protein or carbohydrate epitopes in MAG. In general, the antibodies that react with the protein backbone recognize both rat and human MAG, whereas a large number of the monoclonal antibodies recognize a carbohydrate determinant in human MAG that is not in rat MAG. Immunocytochemical staining of adult human spinal cord with the monoclonal antibodies resulted in periaxonal staining of myelin sheaths, similar to that produced by well-defined, rabbit, polyclonal anti-MAG serum. In addition, the antibodies recognizing, carbohydrate determinants in human MAG strongly stained oligodendrocyte cytoplasm. These monoclonal antibodies will be of value for the further chemical and biological characterization of MAG.     

The carboxylation status of osteocalcin has important consequences for its structure and dynamics

BackgroundThe carboxylation status of Osteocalcin (Ocn) not only influences formation and structure in bones but also has important endocrine functions affecting energy metabolism and expenditure. In this study, the role of γ-carboxylation of the glutamate residues in the structure-dynamics-function relationship in Ocn is investigated.MethodsThree forms of Ocn, differentially carboxylated at the Glu-17, 21 and 24 residues, along with a mutated form of Ocn carrying Glu/Ala mutations, are modeled and simulated using molecular dynamics (MD) simulation in the presence of calcium ions.ResultsCharacterization of the global conformational dynamics of Ocn, described in terms of the orientational variations within its 3-helical domain, highlights large structural variations in the non-carboxylated osteocalcin (nOcn). The bi-carboxylated Ocn (bOcn) and tri-carboxylated (tOcn) species, in contrast, display relatively rigid tertiary structures, with the dynamics of most regions strongly correlated. Radial distribution functions calculated for both bOcn and tOcn show long-range ordering of the calcium ion distribution around the carboxylated glutamate (γGlu) residues, likely playing an important role in promoting stability of these Ocns. Additionally, the same calcium ions are observed to coordinate with neighboring γGlu, better shielding their negative charges and in turn stabilizing these systems more than do the singly coordinating calcium ions observed in the case of nOcn. bOcn is also found to exhibit a more helical C-terminal structure, that has been shown to activate its cellular receptor GPRC6A, highlighting the allosteric role of Ocn carboxylation in modulating the stability and binding potential of the active C-terminal.ConclusionsThe carboxylation status of Ocn as well and its calcium coordination appear to have a direct influence on Ocn structure and dynamics, possibly leading to the known differences in Ocn biological function.General significanceModification of Ocn sequence or its carboxylation state may provide the blueprint for developing high-affinity peptides targeting its cellular receptor GPRC6A, with therapeutic potential for treatment of metabolic disorders.     

Deformability in the cleavage site of primary microRNA is not sensed by the double‐stranded RNA binding domains in the microprocessor component DGCR8

The prevalence of double‐stranded RNA (dsRNA) in eukaryotic cells has only recently been appreciated. Of interest here, RNA silencing begins with dsRNA substrates that are bound by the dsRNA‐binding domains (dsRBDs) of their processing proteins. Specifically, processing of microRNA (miRNA) in the nucleus minimally requires the enzyme Drosha and its dsRBD‐containing cofactor protein, DGCR8. The smallest recombinant construct of DGCR8 that is sufficient for in vitro dsRNA binding, referred to as DGCR8‐Core, consists of its two dsRBDs and a C‐terminal tail. As dsRBDs rarely recognize the nucleotide sequence of dsRNA, it is reasonable to hypothesize that DGCR8 function is dependent on the recognition of specific structural features in the miRNA precursor. Previously, we demonstrated that noncanonical structural elements that promote RNA flexibility within the stem of miRNA precursors are necessary for efficient in vitro cleavage by reconstituted Microprocessor complexes. Here, we combine gel shift assays with in vitro processing assays to demonstrate that neither the N‐terminal dsRBD of DGCR8 in isolation nor the DGCR8‐Core construct is sensitive to the presence of noncanonical structural elements within the stem of miRNA precursors, or to single‐stranded segments flanking the stem. Extending DGCR8‐Core to include an N‐terminal heme‐binding region does not change our conclusions. Thus, our data suggest that although the DGCR8‐Core region is necessary for dsRNA binding and recruitment to the Microprocessor, it is not sufficient to establish the previously observed connection between RNA flexibility and processing efficiency. Proteins 2015; 83:1165–1179. © 2015 Wiley Periodicals, Inc.     

Pharmacokinetics of Oral Dichloroacetate in Dogs

We characterized the pharmacokinetics and dynamics of dichloroacetate (DCA), an investigational drug for mitochondrial diseases, pulmonary arterial hypertension, and cancer. Adult Beagle dogs were orally administered 6.25 mg/kg q12h DCA for 4 weeks. Plasma kinetics was determined after 1, 14, and 28 days. The activity and expression of glutathione transferase zeta 1 (GSTZ1), which biotransforms DCA to glyoxylate, were determined from liver biopsies at baseline and after 27 days. Dogs demonstrate much slower clearance and greater inhibition of DCA metabolism and GSTZ1 activity and expression than rodents and most humans. Indeed, the plasma kinetics of DCA in dogs is similar to humans with GSTZ1 polymorphisms that confer exceptionally slow plasma clearance. Dogs may be a useful model to further investigate the toxicokinetics and therapeutic potential of DCA.     

Piggy-BACing the Human Genome I: Constructing a Porcine BAC Physical Map Through Comparative Genomics

Availability of the human genome sequence and high similarity between humans and pigs at the molecular level provides an opportunity to use a comparative mapping approach to piggy-BAC the human genome. In order to advance the pig genome sequencing initiative, sequence similarity between large-scale porcine BAC-end sequences (BESs) and human genome sequence was used to construct a comparatively-anchored porcine physical map that is a first step towards sequencing the pig genome. A total of 50,300 porcine BAC clones were end-sequenced, yielding 76,906 BESs after trimming with an average read length of 538 bp. To anchor the porcine BACs on the human genome, these BESs were subjected to BLAST analysis using the human draft sequence, revealing 31.5% significant hits (E < e^?5). Both genic and non-genic regions of homology contributed to the alignments between the human and porcine genomes. Porcine BESs with unique homology matches within the human genome provided a source of markers spaced approximately 70 to 300 kb along each human chromosome. In order to evaluate the utility of piggy-BACing human genome sequences, and confirm predictions of orthology, 193 evenly spaced BESs with similarity to HSA3 and HSA21 were selected and then utilized for developing a high-resolution (1.22 Mb) comparative radiation hybrid map of SSC13 that represents a fusion of HSA3 and HSA21. Resulting RH mapping of SSC13 covers 99% and 97% of HSA3 and HSA21, respectively. Seven evolutionary conserved blocks were identified including six on HSA3 and a single syntenic block corresponding to HSA21. The strategy of piggy-BACing the human genome described in this study demonstrates that through a directed, targeted comparative genomics approach construction of a high-resolution anchored physical map of the pig genome can be achieved. This map supports the selection of BACs to construct a minimal tiling path for genome sequencing and targeted gap filling. Moreover, this approach is highly relevant to other genome sequencing projects.     

On the Origin of Large Flexibility of P-glycoprotein in the Inward-facing State

P-glycoprotein (Pgp) is one of the most biomedically relevant transporters in the ATP binding cassette (ABC) superfamily due to its involvement in developing multidrug resistance in cancer cells. Employing molecular dynamics simulations and double electron-electron resonance spectroscopy, we have investigated the structural dynamics of membrane-bound Pgp in the inward-facing state and found that Pgp adopts an unexpectedly wide range of conformations, highlighted by the degree of separation between the two nucleotide-binding domains (NBDs). The distance between the two NBDs in the equilibrium simulations covers a range of at least 20 Å, including, both, more open and more closed NBD configurations than the crystal structure. The double electron-electron resonance measurements on spin-labeled Pgp mutants also show wide distributions covering both longer and shorter distances than those observed in the crystal structure. Based on structural and sequence analyses, we propose that the transmembrane domains of Pgp might be more flexible than other structurally known ABC exporters. The structural flexibility of Pgp demonstrated here is not only in close agreement with, but also helps rationalize, the reported high NBD fluctuations in several ABC exporters and possibly represents a fundamental difference in the transport mechanism between ABC exporters and ABC importers. In addition, during the simulations we have captured partial entrance of a lipid molecule from the bilayer into the lumen of Pgp, reaching the putative drug binding site. The location of the protruding lipid suggests a putative pathway for direct drug recruitment from the membrane.