Longitudinal metagenomic profiling of bovine milk to assess the impact of intramammary treatment using a third-generation cephalosporin

Antimicrobial usage in food animals has a direct impact on human health, and approximately 80% of the antibiotics prescribed in the dairy industry are used to treat bovine mastitis. Here we provide a longitudinal description of the changes in the microbiome of milk that are associated with mastitis and antimicrobial therapy. Next-generation sequencing, 16 S rRNA gene quantitative real-time PCR, and aerobic culturing were applied to assess the effect of disease and antibiotic therapy on the milk microbiome. Cows diagnosed with clinical mastitis associated with Gram-negative pathogens or negative aerobic culture were randomly allocated into 5 days of Ceftiofur intramammary treatment or remained as untreated controls. Serial milk samples were collected from the affected quarter and the ipsilateral healthy quarter of the same animal. Milk from the mastitic quarter had a higher bacterial load and reduced microbial diversity compared to healthy milk. Resolution of the disease was accompanied by increases in diversity indexes and a decrease in pathogen relative abundance. Escherichia coli-associated mastitic milk samples had a remarkably distinct bacterial profile, dominated by Enterobacteriaceae, when compared to healthy milk. However, no differences were observed in culture-negative mastitis samples when compared to healthy milk. Antimicrobial treatment had no significant effect on clinical cure, bacteriological cure, pathogen clearance rate or bacterial load

A Further Unique Chondroitin Sulfate from the shrimp Litopenaeus vannamei with Antithrombin Activity that Modulates Acute Inflammation

The detailed structure of a further Chondroitin Sulfate from Litopenaeus vannamei shrimp (sCS) is described. The backbone structure was established by 1H/13C NMR, which identified 3-O-sulfated GlcA, 4-O-sulfated GalNAc, 6-O-sulfated GalNAc, and 4,6-di-O-sulfated GalNAc residues. GlcA is linked to GalNAc 4,6 di S and GlcA 3S is linked to GalNAc 4S, GalNAc 4,6 di-S and GalNAc6S residues. The anticoagulant properties of this sCS were evaluated by activated partial thromboplastin time, anti-IIa, anti-Xa and anti-heparin cofactor II-mediated activities, and sCS failed to stabilise antithrombin in a fluoresence shift assay. The anti-inflammatory effect of sCS was explored using a model of acute peritonitis, followed by leukocyte count and measurement of the cytokines, IL-1β, IL-6 and TNF-α. The compound showed low clotting effects, but high anti-IIa activity and HCII-mediated thrombin inhibition. Its anti-inflammatory effect was shown by leukocyte recruitment inhibition and a decrease in pro-inflammatory cytokine levels. Although the biological role of sCS remains unknown, its properties indicate that it is suitable for studies of multi-potent molecules obtained from natural sources

Influence of dietary n-6 and n-3 lipids upon the development of pulmonary granulomas induced by Schistosoma mansoni eggs

Pulmonary granuloma development was studied in mice injected with 2x103 Schistosoma mansoni eggs through the main tail vein. Mice were divided into three groups and fed with a laboratory manipulated diet differing among them only in the source of fatty acids. Control group (CT) was fed with a soya oil diet; n-6 deficient animals (low n-6 group) received a supplemented coconut fat diet; and the third group, high n-3, was fed with a cod liver oil diet. The expected anti-inflammatory actions associated with essential fatty acids deficiency was noticed in our system. When compared with CT, animals fed with the n-6 deficient diet presented significantly reduced pulmonary granulomas 32 days after egg injection. Mice fed with high n-3 diet had pulmonary granulomas reduced as soon as 16 days post egg injection. Mean granuloma measurements for CT at days 8, 16 and 32 were, in 103μ2, respectively 7.52 5.92, 32.70 18.09, 32.22 19.87; for n-6 deficient animals 17.65 18.85, 36.14 29.14, 24.45 14.06; and for high n-3 group were 11.60 12.67, 9.97 12.34, 25.54 16.66

The consequences of the effects of the chemotherapeutic drug (vincristine) in organs and the influence on the bioavailability of two radio-biocomplexes used for bone evaluations in balb/c female mice

The development of animal model to evaluate the toxicological action of compounds used as pharmaceutical drugs is desired. The model described in this work is based on the capability of drugs to alter the bioavailability of radiopharmaceuticals (radiobiocomplexes) labeled with technetium-99 m(99mTc). There are evidences that the bioavailability or the pharmacokinetic of radiobiocomplexes can be modified by some factors, as drugs, due to their toxicological action in specific organs. Vincristine is anatural product that has been utilized in oncology. The vincristine effect on the bioavailability of the radiobiocomplexes 99mTc- ethylenediphosphonic acid (99mTc-MDP) and 99mTc-pyrophosphate (99mTc- PYP) in Balb/c female mice was evaluated. The fragments of kidney were processed to light microscopy and transmission electron microscopy. The aim of this work was to study at structural and ultrastructural levels the alterations caused by vincristine in organs. One hour after the last dose ofvincristine, 99mTc-PYP or 99mTc-MDP was injected, the animals were sacrificed and the percentage of radioactivity (%ATI) was determined in the isolated organs. Concerning 99mTc-PYP, the %ATI (i) decreased in spleen, thymus, lymph nodes (inguinal and mesentheric), kidney, lung, liver, pancreas, stomach, heart and brain and (ii) increased in bone and thyroid. Concerning 99mTc-MDP, the %ATI (iii) decreased in spleen, thymus, lymph nodes (inguinal and mesentheric), kidney, liver, pancreas,stomach, heart, brain, bone, ovary and uterus. In conclusion, the toxic effect of vincristine in determined organs could be responsible for the alteration of the uptake of the studied radiobiocomplexes

Comparative chromosome painting discloses homologous Segments in distantly related mammals

Comparative chromosome painting, termed ZOO-FISH, using DNA libraries from flow sorted human chromosomes 1,16,17 and X, and mouse chromosome 11 discloses the presence of syntenic groups in distantly related mammalian Orders ranging from primates (Homo sapiens), rodents (Mus musculus), even-toed ungulates (Muntiacus muntjak vaginalis and Muntiacus reevesi) and whales (Balaenoptera physalus). These mammalian Orders have evolved separately for 55-80 million years (Myr). We conclude that ZOO-FISH can be used to generate comparative chromosome maps of a large number of mammalian species

2, 3-Di-O-sulfo glucuronic acid: an unmodified and unusual residue in a highly sulfated chondroitin sulfate from Litopenaeus vannamei

The occurrence of a natural and unmodified highly sulfated chondroitin sulfate from Litopenaeus vannamei heads (sCS) is herein reported. Its partial digestion by Chondroitinases AC and ABC together with its electrophoretic migration profile revealed it as a highly sulfated chondroitin sulfate despite its average molecular weight being similar to CSA. Using orthogonal 1D/2D NMR experiments, the anomeric signals (δ 4.62/106.0) corresponding to unusual 2,3-di-O-Sulfo-GlcA (∼36%), U33S (δ 4.42/84.1, ∼63%) and U22S (4.12/80.1, ∼50%) substitutions were confirmed. In addition, non-sulfated GlcA (δ 4.5/106.3) linked to 4-O- (A14S, 36%) or 6-O-Sulfo (A16S, 28%) GalNAc (δ 4.64/103.5) was observed. Although the biological role of sCS in shrimp is unknown, its influence on hemostasis was also demonstrated. The sCS identification brings to light new questions about the hierarchical model of GAGs biosynthesis and contributes to the better understanding of the subtle relationship between GAGs structure and function

Insights into the role of 3-O-sulfotransferase in heparan sulfate biosynthesis

3-O-Sulfotransferase enzyme (sHS) from Litopenaeus vannamei was cloned and its substrate specificity was investigated against a number of GAG structures, including modified heparin polysaccharides and model oligosaccharides. For the heparin polysaccharides, derived from porcine intestinal mucosa heparin, sulfate groups were incorporated into glucosamine residues containing both N-sulfated and N-acetylated substitution within the regions of the predominant repeating disaccharide, either I–ANS or I–ANAc. However, the resulting polysaccharides did not stabilize antithrombin, which is correlated with anticoagulant activity. It was also shown that the enzyme was able to sulfate disaccharides, I2S–ANS and G–ANAc. The results further illustrate that 3-O-sulfation can be induced outside of the classical heparin-binding pentasaccharide sequence, show that 3-O-sulfation of glucosamine is not a sufficient condition for antithrombin stabilization and suggest that the use of this enzyme during HS biosynthesis may not occur as the final enzymatic step

Neuronal circuitry for pain processing in the dorsal horn

Neurons in the spinal dorsal horn process sensory information, which is then transmitted to several brain regions, including those responsible for pain perception. The dorsal horn provides numerous potential targets for the development of novel analgesics and is thought to undergo changes that contribute to the exaggerated pain felt after nerve injury and inflammation. Despite its obvious importance, we still know little about the neuronal circuits that process sensory information, mainly because of the heterogeneity of the various neuronal components that make up these circuits. Recent studies have begun to shed light on the neuronal organization and circuitry of this complex region

A putative relay circuit providing low-threshold mechanoreceptive input to lamina I projection neurons via vertical cells in lamina II of the rat dorsal horn

Background: Lamina I projection neurons respond to painful stimuli, and some are also activated by touch or hair movement. Neuropathic pain resulting from peripheral nerve damage is often associated with tactile allodynia (touch-evoked pain), and this may result from increased responsiveness of lamina I projection neurons to non-noxious mechanical stimuli. It is thought that polysynaptic pathways involving excitatory interneurons can transmit tactile inputs to lamina I projection neurons, but that these are normally suppressed by inhibitory interneurons. Vertical cells in lamina II provide a potential route through which tactile stimuli can activate lamina I projection neurons, since their dendrites extend into the region where tactile afferents terminate, while their axons can innervate the projection cells. The aim of this study was to determine whether vertical cell dendrites were contacted by the central terminals of low-threshold mechanoreceptive primary afferents. Results: We initially demonstrated contacts between dendritic spines of vertical cells that had been recorded in spinal cord slices and axonal boutons containing the vesicular glutamate transporter 1 (VGLUT1), which is expressed by myelinated low-threshold mechanoreceptive afferents. To confirm that the VGLUT1 boutons included primary afferents, we then examined vertical cells recorded in rats that had received injections of cholera toxin B subunit (CTb) into the sciatic nerve. We found that over half of the VGLUT1 boutons contacting the vertical cells were CTb-immunoreactive, indicating that they were of primary afferent origin. Conclusions: These results show that vertical cell dendritic spines are frequently contacted by the central terminals of myelinated low-threshold mechanoreceptive afferents. Since dendritic spines are associated with excitatory synapses, it is likely that most of these contacts were synaptic. Vertical cells in lamina II are therefore a potential route through which tactile afferents can activate lamina I projection neurons, and this pathway could play a role in tactile allodynia

The nuclear receptors of Biomphalaria glabrata and Lottia gigantea: Implications for developing new model organisms

© 2015 Kaur et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are creditedNuclear receptors (NRs) are transcription regulators involved in an array of diverse physiological functions including key roles in endocrine and metabolic function. The aim of this study was to identify nuclear receptors in the fully sequenced genome of the gastropod snail, Biomphalaria glabrata, intermediate host for Schistosoma mansoni and compare these to known vertebrate NRs, with a view to assessing the snail's potential as a invertebrate model organism for endocrine function, both as a prospective new test organism and to elucidate the fundamental genetic and mechanistic causes of disease. For comparative purposes, the genome of a second gastropod, the owl limpet, Lottia gigantea was also investigated for nuclear receptors. Thirty-nine and thirty-three putative NRs were identified from the B. glabrata and L. gigantea genomes respectively, based on the presence of a conserved DNA-binding domain and/or ligand-binding domain. Nuclear receptor transcript expression was confirmed and sequences were subjected to a comparative phylogenetic analysis, which demonstrated that these molluscs have representatives of all the major NR subfamilies (1-6). Many of the identified NRs are conserved between vertebrates and invertebrates, however differences exist, most notably, the absence of receptors of Group 3C, which includes some of the vertebrate endocrine hormone targets. The mollusc genomes also contain NR homologues that are present in insects and nematodes but not in vertebrates, such as Group 1J (HR48/DAF12/HR96). The identification of many shared receptors between humans and molluscs indicates the potential for molluscs as model organisms; however the absence of several steroid hormone receptors indicates snail endocrine systems are fundamentally different.The National Centre for the Replacement, Refinement and Reduction of Animals in Research, Grant Ref:G0900802 to CSJ, LRN, SJ & EJR [www.nc3rs.org.uk]