Effects of preservation method on canine (Canis lupus familiaris) fecal microbiota.

Studies involving gut microbiome analysis play an increasing role in the evaluation of health and disease in humans and animals alike. Fecal sampling methods for DNA preservation in laboratory, clinical, and field settings can greatly influence inferences of microbial composition and diversity, but are often inconsistent and under-investigated between studies. Many laboratories have utilized either temperature control or preservation buffers for optimization of DNA preservation, but few studies have evaluated the effects of combining both methods to preserve fecal microbiota. To determine the optimal method for fecal DNA preservation, we collected fecal samples from one canine donor and stored aliquots in RNAlater, 70% ethanol, 50:50 glycerol:PBS, or without buffer at 25 °C, 4 °C, and -80 °C. Fecal DNA was extracted, quantified, and 16S rRNA gene analysis performed on Days 0, 7, 14, and 56 to evaluate changes in DNA concentration, purity, and bacterial diversity and composition over time. We detected overall effects on bacterial community of storage buffer (F-value = 6.87, DF = 3, P < 0.001), storage temperature (F-value=1.77, DF = 3, P = 0.037), and duration of sample storage (F-value = 3.68, DF = 3, P < 0.001). Changes in bacterial composition were observed in samples stored in -80 °C without buffer, a commonly used method for fecal DNA storage, suggesting that simply freezing samples may be suboptimal for bacterial analysis. Fecal preservation with 70% ethanol and RNAlater closely resembled that of fresh samples, though RNAlater yielded significantly lower DNA concentrations (DF = 8.57, P < 0.001). Although bacterial composition varied with temperature and buffer storage, 70% ethanol was the best method for preserving bacterial DNA in canine feces, yielding the highest DNA concentration and minimal changes in bacterial diversity and composition. The differences observed between samples highlight the need to consider optimized post-collection methods in microbiome research

A duchenne muscular dystrophy gene hot spot mutation in dystrophin-deficient Cavalier King Charles Spaniels is amenable to exon 51 skipping

BACKGROUND Duchenne muscular dystrophy (DMD), which afflicts 1 in 3500 boys, is one of the most common genetic disorders of children. This fatal degenerative condition is caused by an absence or deficiency of dystrophin in striated muscle. Most affected patients have inherited or spontaneous deletions in the dystrophin gene that disrupt the reading frame resulting in unstable truncated products. For these patients, restoration of the reading frame via antisense oligonucleotide-mediated exon skipping is a promising therapeutic approach. The major DMD deletion "hot spot" is found between exons 45 and 53, and skipping exon 51 in particular is predicted to ameliorate the dystrophic phenotype in the greatest number of patients. Currently the mdx mouse is the most widely used animal model of DMD, although its mild phenotype limits its suitability in clinical trials. The Golden Retriever muscular dystrophy (GRMD) model has a severe phenotype, but due to its large size, is expensive to use. Both these models have mutations in regions of the dystrophin gene distant from the commonly mutated DMD "hot spot". METHODOLOGY/PRINCIPAL FINDINGS Here we describe the severe phenotype, histopathological findings, and molecular analysis of Cavalier King Charles Spaniels with dystrophin-deficient muscular dystrophy (CKCS-MD). The dogs harbour a missense mutation in the 5' donor splice site of exon 50 that results in deletion of exon 50 in mRNA transcripts and a predicted premature truncation of the translated protein. Antisense oligonucleotide-mediated skipping of exon 51 in cultured myoblasts from an affected dog restored the reading frame and protein expression. CONCLUSIONS/SIGNIFICANCE Given the small size of the breed, the amiable temperament and the nature of the mutation, we propose that CKCS-MD is a valuable new model for clinical trials of antisense oligonucleotide-induced exon skipping and other therapeutic approaches for DMD

Commentary on key aspects of fecal microbiota transplantation in small animal practice

The gastrointestinal tract of dogs, cats, and other mammals including humans harbors millions of beneficial microorganisms that regulate and maintain health. Fecal microbiota transplantation (FMT) is a procedure involving the administration of a fecal infusion from a healthy individual (donor) to a patient with disease to help improve health. Despite the effectiveness of FMT to treat intestinal disorders in humans, in particular recurrent Clostridium difficile infection, there is a paucity of scientific data regarding the application of FMT in veterinary patients. Here, we outline key aspects of FMT in small animal practice

The Fecal Microbiome in Cats with Diarrhea

Recent studies have revealed that microbes play an important role in the pathogenesis of gastrointestinal (GI) diseases in various animal species, but only limited data is available about the microbiome in cats with GI disease. The aim of this study was to evaluate the fecal microbiome in cats with diarrhea. Fecal samples were obtained from healthy cats (n = 21) and cats with acute (n = 19) or chronic diarrhea (n = 29) and analyzed by sequencing of 16S rRNA genes, and PICRUSt was used to predict the functional gene content of the microbiome. Linear discriminant analysis (LDA) effect size (LEfSe) revealed significant differences in bacterial groups between healthy cats and cats with diarrhea. The order Burkholderiales, the families Enterobacteriaceae, and the genera Streptococcus and Collinsella were significantly increased in diarrheic cats. In contrast the order Campylobacterales, the family Bacteroidaceae, and the genera Megamonas, Helicobacter, and Roseburia were significantly increased in healthy cats. Phylum Bacteroidetes was significantly decreased in cats with chronic diarrhea (>21 days duration), while the class Erysipelotrichi and the genus Lactobacillus were significantly decreased in cats with acute diarrhea. The observed changes in bacterial groups were accompanied by significant differences in functional gene contents: metabolism of fatty acids, biosynthesis of glycosphingolipids, metabolism of biotin, metabolism of tryptophan, and ascorbate and aldarate metabolism, were all significantly (p<0.001) altered in cats with diarrhea. In conclusion, significant differences in the fecal microbiomes between healthy cats and cats with diarrhea were identified. This dysbiosis was accompanied by changes in bacterial functional gene categories. Future studies are warranted to evaluate if these microbial changes correlate with changes in fecal concentrations of microbial metabolites in cats with diarrhea for the identification of potential diagnostic or therapeutic targets.The open access fee for this work was funded through the Texas A&M University Open Access to Knowledge (OAK) Fund

Toward a Deeper Understanding of the Meaning of Marriage Among Black Men

Black men benefit from healthy, satisfying marriages in domains of physical, psychological, and financial well-being. Yet marriage among Black men has declined and remains elusive for many. One gap in the research concerns the positive meaning that Black men find in their marriages. Prior research has failed to collect in-depth accounts of Black men’s experiences of marriage. The purpose of this qualitative study is to explore the meaning of marriage among 52 Black men, using interview data. Findings highlight four themes in the meaning of marriage—secure emotional support, lifelong commitment, enhanced life success, and secure attachment. Two themes emerged from the data related to important influences on the construction of meaning relative to marriage—faith, and the dynamics of give and take. Responses among the men concerning the change in marriage over time related to transitions in American marriages and a deepened respect for marriage. Implications are discussed

Percutaneous coronary intervention in asians- are there differences in clinical outcome?

<p>Abstract</p> <p>Background</p> <p>Ethnic differences in clinical outcome after percutaneous coronary intervention (PCI) have been reported. Data within different Asian subpopulations is scarce. We aim to explore the differences in clinical profile and outcome between Chinese, Malay and Indian Asian patients who undergo PCI for coronary artery disease (CAD).</p> <p>Methods</p> <p>A prospective registry of consecutive patients undergoing PCI from January 2002 to December 2007 at a tertiary care center was analyzed. Primary endpoint was major adverse cardiovascular events (MACE) of myocardial infarction (MI), repeat revascularization and all-cause death at six months.</p> <p>Results</p> <p>7889 patients underwent PCI; 7544 (96%) patients completed follow-up and were included in the analysis (79% males with mean age of 59 years ± 11). There were 5130 (68%) Chinese, 1056 (14%) Malays and 1001 (13.3%) Indian patients. The remaining 357 (4.7%) patients from other minority ethnic groups were excluded from the analysis. The primary end-point occurred in 684 (9.1%) patients at six months. Indians had the highest rates of six month MACE compared to Chinese and Malays (Indians 12% vs. Chinese 8.2% vs. Malays 10.7%; OR 1.55 95%CI 1.24-1.93, p < 0.001). This was contributed by increased rates of MI (Indians 1.9% vs. Chinese 0.9% vs. Malays 1.3%; OR 4.49 95%CI 1.91-10.56 p = 0.001), repeat revascularization (Indians 6.5% vs. Chinese 4.1% vs. Malays 5.1%; OR 1.64 95%CI 1.22-2.21 p = 0.0012) and death (Indians 11.4% vs. Chinese 7.6% vs. Malays 9.9%; OR 1.65 95%CI 1.23-2.20 p = 0.001) amongst Indian patients.</p> <p>Conclusion</p> <p>These data indicate that ethnic variations in clinical outcome exist following PCI. In particular, Indian patients have higher six month event rates compared to Chinese and Malays. Future studies are warranted to elucidate the underlying mechanisms behind these variations.</p

Expression and Activity of a Novel Cathelicidin from Domestic Cats

Cathelicidins are small cationic antimicrobial peptides found in many species including primates, mammals, marsupials, birds and even more primitive vertebrates, such as the hagfish. Some animals encode multiple cathelicidins in their genome, whereas others have only one. This report identifies and characterizes feline cathelicidin (feCath) as the sole cathelicidin in domestic cats (Felis catus). Expression of feCath is predominantly found in the bone marrow, with lower levels of expression in the gastrointestinal tract and skin. By immunocytochemistry, feCath localizes to the cytoplasm of neutrophils in feline peripheral blood. Structurally, the mature feCath sequence is most similar to a subgroup of cathelicidins that form linear α-helices. feCath possesses antimicrobial activity against E. coli D31, Salmonella enterica serovar Typhimurium (IR715), Listeria monocytogenes and Staphylococcus pseudintermedius (clinical isolate) similar to that of the human ortholog, LL-37. In contrast, feCath lacks the DNA binding activity seen with LL-37. Given its similarity in sequence, structure, tissue expression, and antimicrobial activity, the cathelicidin encoded by cats, feCath, belongs to the subgroup of linear cathelicidins found not only in humans, but also non-human primates, dogs, mice, and rats

The Role of Practitioner Resilience and Mindfulness in Effective Practice: A Practice-Based Feasibility Study.

A growing body of literature attests to the existence of therapist effects with little explanation of this phenomenon. This study therefore investigated the role of resilience and mindfulness as factors related to practitioner wellbeing and associated effective practice. Data comprised practitioners (n = 37) and their patient outcome data (n = 4980) conducted within a stepped care model of service delivery. Analyses employed benchmarking and multilevel modeling to identify more and less effective practitioners via yoking of therapist factors and nested patient outcomes. A therapist effect of 6.7 % was identified based on patient depression (PHQ-9) outcome scores. More effective practitioners compared to less effective practitioners displayed significantly higher levels of mindfulness as well as resilience and mindfulness combined. Implications for policy, research and practice are discussed

Bryostatin Modulates Latent HIV-1 Infection via PKC and AMPK Signaling but Inhibits Acute Infection in a Receptor Independent Manner

HIV's ability to establish long-lived latent infection is mainly due to transcriptional silencing in resting memory T lymphocytes and other non dividing cells including monocytes. Despite an undetectable viral load in patients treated with potent antiretrovirals, current therapy is unable to purge the virus from these latent reservoirs. In order to broaden the inhibitory range and effectiveness of current antiretrovirals, the potential of bryostatin was investigated as an HIV inhibitor and latent activator. Bryostatin revealed antiviral activity against R5- and X4-tropic viruses in receptor independent and partly via transient decrease in CD4/CXCR4 expression. Further, bryostatin at low nanomolar concentrations robustly reactivated latent viral infection in monocytic and lymphocytic cells via activation of Protein Kinase C (PKC) -α and -δ, because PKC inhibitors rottlerin and GF109203X abrogated the bryostatin effect. Bryostatin specifically modulated novel PKC (nPKC) involving stress induced AMP Kinase (AMPK) inasmuch as an inhibitor of AMPK, compound C partially ablated the viral reactivation effect. Above all, bryostatin was non-toxic in vitro and was unable to provoke T-cell activation. The dual role of bryostatin on HIV life cycle may be a beneficial adjunct to the treatment of HIV especially by purging latent virus from different cellular reservoirs such as brain and lymphoid organs

An Abundant Dysfunctional Apolipoprotein A1 in Human Atheroma

Recent studies have indicated that high-density lipoproteins (HDLs) and their major structural protein, apolipoprotein A1 (apoA1), recovered from human atheroma are dysfunctional and are extensively oxidized by myeloperoxidase (MPO). In vitro oxidation of either apoA1 or HDL particles by MPO impairs their cholesterol acceptor function. Here, using phage display affinity maturation, we developed a high-affinity monoclonal antibody that specifically recognizes both apoA1 and HDL that have been modified by the MPO-H2O2-Cl− system. An oxindolyl alanine (2-OH-Trp) moiety at Trp72 of apoA1 is the immunogenic epitope. Mutagenesis studies confirmed a critical role for apoA1 Trp72 in MPO-mediated inhibition of the ATP-binding cassette transporter A1 (ABCA1)-dependent cholesterol acceptor activity of apoA1 in vitro and in vivo. ApoA1 containing a 2-OH-Trp72 group (oxTrp72-apoA1) is in low abundance within the circulation but accounts for 20% of the apoA1 in atherosclerosis-laden arteries. OxTrp72-apoA1 recovered from human atheroma or plasma is lipid poor, virtually devoid of cholesterol acceptor activity and demonstrated both a potent proinflammatory activity on endothelial cells and an impaired HDL biogenesis activity in vivo. Elevated oxTrp72-apoA1 levels in subjects presenting to a cardiology clinic (n = 627) were associated with increased cardiovascular disease risk. Circulating oxTrp72-apoA1 levels may serve as a way to monitor a proatherogenic process in the artery wall