First Staphylococcal Cassette Chromosome mec Containing a mecB-Carrying Gene Complex Independent of Transposon Tn6045 in a Macrococcus caseolyticus Isolate from a Canine Infection.

A methicillin-resistant mecB-positive Macrococcus caseolyticus (strain KM45013) was isolated from the nares of a dog with rhinitis. It contained a novel 39-kb transposon-defective complete mecB-carrying staphylococcal cassette chromosome mec element (SCCmecKM45013). SCCmecKM45013 contained 49 coding sequences (CDSs), was integrated at the 3' end of the chromosomal orfX gene, and was delimited at both ends by imperfect direct repeats functioning as integration site sequences (ISSs). SCCmecKM45013 presented two discontinuous regions of homology (SCCmec coverage of 35%) to the chromosomal and transposon Tn6045-associated SCCmec-like element of M. caseolyticus JCSC7096: (i) the mec gene complex (98.8% identity) and (ii) the ccr-carrying segment (91.8% identity). The mec gene complex, located at the right junction of the cassette, also carried the β-lactamase gene blaZm (mecRm-mecIm-mecB-blaZm). SCCmecKM45013 contained two cassette chromosome recombinase genes, ccrAm2 and ccrBm2, which shared 94.3% and 96.6% DNA identity with those of the SCCmec-like element of JCSC7096 but shared less than 52% DNA identity with the staphylococcal ccrAB and ccrC genes. Three distinct extrachromosomal circularized elements (the entire SCCmecKM45013, ΨSCCmecKM45013 lacking the ccr genes, and SCCKM45013 lacking mecB) flanked by one ISS copy, as well as the chromosomal regions remaining after excision, were detected. An unconventional circularized structure carrying the mecB gene complex was associated with two extensive direct repeat regions, which enclosed two open reading frames (ORFs) (ORF46 and ORF51) flanking the chromosomal mecB-carrying gene complex. This study revealed M. caseolyticus as a potential disease-associated bacterium in dogs and also unveiled an SCCmec element carrying mecB not associated with Tn6045 in the genus Macrococcus.

Effect of an intervertebral disk spacer on stiffness after monocortical screw/polymethylmethacrylate fixation in simulated and cadaveric canine cervical vertebral columns.

OBJECTIVE To determine the biomechanical effect of an intervertebral spacer on construct stiffness in a PVC model and cadaveric canine cervical vertebral columns stabilized with monocortical screws/polymethylmethacrylate (PMMA). STUDY DESIGN Biomechanical study. SAMPLE POPULATION PVC pipe; cadaveric canine vertebral columns. METHODS PVC model-PVC pipe was used to create a gap model mimicking vertebral endplate orientation and disk space width of large-breed canine cervical vertebrae; 6 models had a 4-mm gap with no spacer (PVC group 1); 6 had a PVC pipe ring spacer filling the gap (PCV group 2). Animals-large breed cadaveric canine cervical vertebral columns (C2-C7) from skeletally mature dogs without (cadaveric group 1, n = 6, historical data) and with an intervertebral disk spacer (cadaveric group 2, n = 6) were used. All PVC models and cadaver specimens were instrumented with monocortical titanium screws/PMMA. Stiffness of the 2 PVC groups was compared in extension, flexion, and lateral bending using non-destructive 4-point bend testing. Stiffness testing in all 3 directions was performed of the unaltered C4-C5 vertebral motion unit in cadaveric spines and repeated after placement of an intervertebral cortical allograft ring and instrumentation. Data were compared using a linear mixed model approach that also incorporated data from previously tested spines with the same screw/PMMA construct but without disk spacer (cadaveric group 1). RESULTS Addition of a spacer increased construct stiffness in both the PVC model (P < .001) and cadaveric vertebral columns (P < .001) compared to fixation without a spacer. CONCLUSIONS Addition of an intervertebral spacer significantly increased construct stiffness of monocortical screw/PMMA fixation.

Biomechanical comparison between bicortical pin and monocortical screw/polymethylmethacrylate constructs in the cadaveric canine cervical vertebral column.

OBJECTIVE To compare biomechanical stiffness of cadaveric canine cervical spine constructs stabilized with bicortical stainless steel pins and polymethylmethacrylate (PMMA), monocortical stainless steel screws with PMMA, or monocortical titanium screws with PMMA. STUDY DESIGN Biomechanical cadaver study. ANIMALS Eighteen canine cervical vertebral columns (C2-C7) were collected from skeletally mature dogs (weighing 22-32 kg). METHODS Specimens were radiographed and examined by dual energy X-ray absorptiometry. Stiffness of the unaltered C4-C5 intervertebral motion unit was measured in extension, flexion and lateral bending using non-destructive 4-point bend testing. Specimens were then stabilized by (1) bicortical stainless steel pins/PMMA, (2) monocortical stainless steel screws/PMMA, or (3) monocortical titanium screws/PMMA. Mechanical testing was repeated and stiffness data from unaltered specimens and the 3 treatment groups were compared. RESULTS All 3 surgical methods significantly increased stiffness of the C4-C5 motion unit compared with the unaltered specimen (P < .001 for all treatments), but stiffness was not significantly different among the 3 fixation groups (P = .578). CONCLUSIONS In this model, monocortical screw fixation (with stainless steel or titanium screws) was biomechanically equivalent to bicortical fixation.

Improving 4D plan quality for PBS-based liver tumour treatments by combining online image guided beam gating with rescanning.

Pencil beam scanned (PBS) proton therapy has many advantages over conventional radiotherapy, but its effectiveness for treating mobile tumours remains questionable. Gating dose delivery to the breathing pattern is a well-developed method in conventional radiotherapy for mitigating tumour-motion, but its clinical efficiency for PBS proton therapy is not yet well documented. In this study, the dosimetric benefits and the treatment efficiency of beam gating for PBS proton therapy has been comprehensively evaluated. A series of dedicated 4D dose calculations (4DDC) have been performed on 9 different 4DCT(MRI) liver data sets, which give realistic 4DCT extracting motion information from 4DMRI. The value of 4DCT(MRI) is its capability of providing not only patient geometries and deformable breathing characteristics, but also includes variations in the breathing patterns between breathing cycles. In order to monitor target motion and derive a gating signal, we simulate time-resolved beams' eye view (BEV) x-ray images as an online motion surrogate. 4DDCs have been performed using three amplitude-based gating window sizes (10/5/3 mm) with motion surrogates derived from either pre-implanted fiducial markers or the diaphragm. In addition, gating has also been simulated in combination with up to 19 times rescanning using either volumetric or layered approaches. The quality of the resulting 4DDC plans has been quantified in terms of the plan homogeneity index (HI), total treatment time and duty cycle. Results show that neither beam gating nor rescanning alone can fully retrieve the plan homogeneity of the static reference plan. Especially for variable breathing patterns, reductions of the effective duty cycle to as low as 10% have been observed with the smallest gating rescanning window (3 mm), implying that gating on its own for such cases would result in much longer treatment times. In addition, when rescanning is applied on its own, large differences between volumetric and layered rescanning have been observed as a function of increasing number of re-scans. However, once gating and rescanning is combined, HI to within 2% of the static plan could be achieved in the clinical target volume, with only moderately prolonged treatment times, irrespective of the rescanning strategy used. Moreover, these results are independent of the motion surrogate used. In conclusion, our results suggest image guided beam gating, combined with rescanning, is a feasible, effective and efficient motion mitigation approach for PBS-based liver tumour treatments.

Canine rabies in Australia: a review of preparedness and research needs

Australia is unique as a populated continent in that canine rabies is exotic, with only one likely incursion in 1867. This is despite the presence of a widespread free-ranging dog population, which includes the naturalized dingo, feral domestic dogs and dingo-dog cross-breeds. To Australia's immediate north, rabies has recently spread within the Indonesian archipelago, with outbreaks occurring in historically free islands to the east including Bali, Flores, Ambon and the Tanimbar Islands. Australia depends on strict quarantine protocols to prevent importation of a rabid animal, but the risk of illegal animal movements by fishing and recreational vessels circumventing quarantine remains. Predicting where rabies will enter Australia is important, but understanding dog population dynamics and interactions, including contact rates in and around human populations, is essential for rabies preparedness. The interactions among and between Australia's large populations of wild, free-roaming and restrained domestic dogs require quantification for rabies incursions to be detected and controlled. The imminent risk of rabies breaching Australian borders makes the development of disease spread models that will assist in the deployment of cost-effective surveillance, improve preventive strategies and guide disease management protocols vitally important. Here, we critically review Australia's preparedness for rabies, discuss prevailing assumptions and models, identify knowledge deficits in free-roaming dog ecology relating to rabies maintenance and speculate on the likely consequences of endemic rabies for Australia.

Serum and fecal canine α1-proteinase inhibitor concentrations reflect the severity of intestinal crypt abscesses and/or lacteal dilation in dogs.

Gastrointestinal (GI) protein loss, due to lymphangiectasia or chronic inflammation, can be challenging to diagnose. This study evaluated the diagnostic accuracy of serum and fecal canine α1-proteinase inhibitor (cα1PI) concentrations to detect crypt abscesses and/or lacteal dilation in dogs. Serum and fecal cα1PI concentrations were measured in 120 dogs undergoing GI tissue biopsies, and were compared between dogs with and without crypt abscesses/lacteal dilation. Sensitivity and specificity were calculated for dichotomous outcomes. Serial serum cα1PI concentrations were also evaluated in 12 healthy corticosteroid-treated dogs. Serum cα1PI and albumin concentrations were significantly lower in dogs with crypt abscesses and/or lacteal dilation than in those without (both P <0.001), and more severe lesions were associated with lower serum cα1PI concentrations, higher 3 days-mean fecal cα1PI concentrations, and lower serum/fecal cα1PI ratios. Serum and fecal cα1PI, and their ratios, distinguished dogs with moderate or severe GI crypt abscesses/lacteal dilation from dogs with only mild or none such lesions with moderate sensitivity (56-92%) and specificity (67-81%). Serum cα1PI concentrations increased during corticosteroid administration. We conclude that serum and fecal α1PI concentrations reflect the severity of intestinal crypt abscesses/lacteal dilation in dogs. Due to its specificity for the GI tract, measurement of fecal cα1PI appears to be superior to serum cα1PI for diagnosing GI protein loss in dogs. In addition, the serum/fecal cα1PI ratio has an improved accuracy in hypoalbuminemic dogs, but serum cα1PI concentrations should be carefully interpreted in corticosteroid-treated dogs.

Validation of an enzyme-linked immunosorbent assay (ELISA) for the measurement of canine S100A12.

BACKGROUND Canine S100 calcium-binding protein A12 (cS100A12) shows promise as biomarker of inflammation in dogs. A previously developed cS100A12-radioimmunoassay (RIA) requires radioactive tracers and is not sensitive enough for fecal cS100A12 concentrations in 79% of tested healthy dogs. An ELISA assay may be more sensitive than RIA and does not require radioactive tracers. OBJECTIVE The purpose of the study was to establish a sandwich ELISA for serum and fecal cS100A12, and to establish reference intervals (RI) for normal healthy canine serum and feces. METHODS Polyclonal rabbit anti-cS100A12 antibodies were generated and tested by Western blotting and immunohistochemistry. A sandwich ELISA was developed and validated, including accuracy and precision, and agreement with cS100A12-RIA. The RI, stability, and biologic variation in fecal cS100A12, and the effect of corticosteroids on serum cS100A12 were evaluated. RESULTS Lower detection limits were 5 μg/L (serum) and 1 ng/g (fecal), respectively. Intra- and inter-assay coefficients of variation were ≤ 4.4% and ≤ 10.9%, respectively. Observed-to-expected ratios for linearity and spiking recovery were 98.2 ± 9.8% (mean ± SD) and 93.0 ± 6.1%, respectively. There was a significant bias between the ELISA and the RIA. The RI was 49-320 μg/L for serum and 2-484 ng/g for fecal cS100A12. Fecal cS100A12 was stable for 7 days at 23, 4, -20, and -80°C; biologic variation was negligible but variation within one fecal sample was significant. Corticosteroid treatment had no clinically significant effect on serum cS100A12 concentrations. CONCLUSIONS The cS100A12-ELISA is a precise and accurate assay for serum and fecal cS100A12 in dogs.

Sexually transmissible diseases, sexual factors and prostate cancer: A review of literature

A review of literature was carried out regarding sexually related factors, sexually transmissible diseases (STD's) and infections with prostate cancer (PC) development risk. The review of literature, in conjunction with the tabulation of studies, suggested that ejaculation and circumcision may play a protective role in the development of PC and that multiple sex partners and an active sex life may play a causal role in the development of PC which may negate and counteract the protective effects of ejaculation and circumcision. HIV infection may plausibly play a function in deteriorating and compromising immune controls on carcinogenesis. Because of the coexistence of a highly active sexual lifestyle and sexual promiscuity with the growing occurence of STD's, their maybe a correlation with the high incidence of prostate cancer in the United States. Potential multi-institutional studies are warranted to confirm the high incidence of this neoplasm with the increasing cases of STD's and if in fact there is a proportional association to further elucidate the factors responsible for its high incidence.^

Relation between acid back -diffusion and luminal surface hydrophobicity in canine gastric mucosa: Effects of salicylate and prostaglandin

The stomach is thought to be protected from luminal acid by a gastric mucosal barrier that restricts the diffusion of acid into tissue. This study tested the hypothesis that the hydrophobic luminal surface of canine gastric mucosa incubated in Ussing chambers, impedes the back-diffusion of luminal acid into the tissue. Isolated sheets of mucosa were treated with cimetidine to inhibit spontaneous acid secretion, and incubated under conditions that prevented significant secretion of luminal bicarbonate. By measuring acid loss from the luminal compartment using the pH-stat technique, acid back-diffusion was continuously monitored; potential difference (PD) was measured as an index of tissue viability. Tissue luminal surface hydrophobicity was estimated by contact angle analysis at the end of each experiment. Addition of 16,16-dimethyl prostaglandin E$\sb2$ to the nutrient compartment enhanced luminal surface hydrophobicity, but did not reduce acid back-diffusion in tissues that maintained a constant PD. 10 mM salicylate at pH 4.00 in the luminal compartment reduced surface hydrophobicity, but this decrease did not occur if 1 ug/ml prostaglandin was present in the nutrient solution. Despite possessing relatively hydrophilic and relatively hydrophobic surface properties, respectively, acid back-diffusion in the absence of salicylate was not significantly different between these two groups. Neither group maintained a PD after incubation with salicylate. Lastly, radiolabelled salicylate was used to calculate the free (non-salicylate associated) acid loss in tissues incubated with salicylate and/or prostaglandin. No significant correlation was found between free acid back-diffusion and luminal surface hydrophobicity. These data do not support the hypothesis that acid back-diffusion in impeded by the hydrophobic surface presented by isolated canine gastric mucosa. ^

Raft association of SNAP receptors acting in apical trafficking in Madin–Darby canine kidney cells

We have investigated the relationships between the apical sorting mechanism using lipid rafts and the soluble N-ethyl maleimide-sensitive factor attachment protein receptor (SNARE) machinery, which is involved in membrane docking and fusion. We first confirmed that anti-alpha-SNAP antibodies inhibit the apical pathway in Madin– Darby canine kidney (MDCK) cells; in addition, we report that a recombinant SNAP protein stimulates the apical transport whereas a SNAP mutant inhibits this transport step. Based on t-SNARE overexpression experiments and the effect of botulinum neurotoxin E, syntaxin 3 and SNAP-23 have been implicated in apical membrane trafficking. Here, we show in permeabilized MDCK cells that antisyntaxin 3 and anti-SNAP-23 antibodies lower surface delivery of an apical reporter protein. Moreover, using a similar approach, we show that tetanus toxin-insensitive, vesicle-associated membrane protein (TI-VAMP; also called VAMP7), a recently described apical v-SNARE, is involved. Furthermore, we show the presence of syntaxin 3 and TI-VAMP in isolated apical carriers. Polarized apical sorting has been postulated to be mediated by the clustering of apical proteins into dynamic sphingolipid-cholesterol rafts. We provide evidence that syntaxin 3 and TI-VAMP are raft-associated. These data support a raft-based mechanism for the sorting of not only apically destined cargo but also of SNAREs having functions in apical membrane-docking and fusion events.

Na,K-ATPase transport from endoplasmic reticulum to Golgi requires the Golgi spectrin–ankyrin G119 skeleton in Madin Darby canine kidney cells

Spectrin (βIΣ∗) and ankyrin (AnkG119) associate with Golgi membranes and the dynactin complex, but their role in vesicle trafficking remains uncertain. We find that the actin-binding domain and membrane-association domain 1 (MAD1) of βI spectrin together form a constitutive Golgi targeting signal in transfected MDCK cells. Expression of this signal in transfected cells disrupts the endogenous Golgi spectrin skeleton and blocks transport of α- and β-Na,K-ATPase and vesicular stomatitis virus-G protein from the endoplasmic reticulum (ER) but does not disrupt the formation of Golgi stacks, the distribution of β-COP, or the transport and surface display of E-cadherin. The Golgi spectrin skeleton is thus required for the transport of a subset of membrane proteins from the ER to the Golgi. We postulate that together with polyfunctional adapter proteins such as AnkG119, Golgi spectrin forms a docking complex that acts prior to the cis-Golgi, presumably with vesicular–tubular clusters (VTCs or ERGIC), to sequester specific membrane proteins into vesicles transiting between the ER and Golgi, and subsequently (probably involving other isoforms of spectrin and ankyrin) to mediate cargo transport within the Golgi and to other membrane compartments. We hypothesize that this vesicular spectrin–ankyrin adapter-protein trafficking (or tethering) system (SAATS) mediates the capture and transport of many membrane proteins and acts in conjunction with vesicle-targeting molecules to effect the efficient transport of cargo proteins.

Association of Rab25 and Rab11a with the Apical Recycling System of Polarized Madin–Darby Canine Kidney Cells

Recent evidence suggests that apical and basolateral endocytic pathways in epithelia converge in an apically located, pericentriolar endosomal compartment termed the apical recycling endosome. In this compartment, apically and basolaterally internalized membrane constituents are thought to be sorted for recycling back to their site of origin or for transcytosis to the opposite plasma membrane domain. We report here that in the epithelial cell line Madin–Darby Canine Kidney (MDCK), antibodies to Rab11a label an apical pericentriolar endosomal compartment that is dependent on intact microtubules for its integrity. Furthermore, this compartment is accessible to a membrane-bound marker (dimeric immunoglobulin A [IgA]) internalized from either the apical or basolateral pole, functionally defining it as the apical recycling endosome. We have also examined the role of a closely related epithelial-specific Rab, Rab25, in the regulation of membrane recycling and transcytosis in MDCK cells. When cDNA encoding Rab25 was transfected into MDCK cells, the protein colocalized with Rab11a in subapical vesicles. Rab25 transfection also altered the distribution of Rab11a, causing the coalescence of immunoreactivity into multiple denser vesicular structures not associated with the centrosome. Nevertheless, nocodazole still dispersed these vesicles, and dimeric IgA internalized from either the apical or basolateral membrane was detected in endosomes labeled with antibodies to both Rab11a and Rab25. Overexpression of Rab25 decreased the rate of IgA transcytosis and of apical, but not basolateral, recycling of internalized ligand. Conversely, expression of the dominant-negative Rab25T26N did not alter either apical recycling or transcytosis. These results indicate that both Rab11a and Rab25 associate with the apical recycling system of epithelial cells and suggest that Rab25 may selectively regulate the apical recycling and/or transcytotic pathways.