Importing genetically altered animals: ensuring quality.

The reproducibility of research using laboratory animals requires reliable management of their quality, in particular of their genetics, health and environment, all of which contribute to their phenotypes. The point at which these biological materials are transferred between researchers is particularly sensitive, as it may result in a loss of integrity of the animals and/or their documentation. Here, we describe the various aspects of laboratory animal quality that should be confirmed when sharing rodent research models. We also discuss how repositories of biological materials support the scientific community to ensure the continuity of the quality of laboratory animals. Both the concept of quality and the role of repositories themselves extend to all exchanges of biological materials and all networks that support the sharing of these reagents.

Impact of Fellow Participation During Colonoscopy on Adenoma Detection Rates.

BACKGROUND: An endoscopist's adenoma detection rate (ADR) is inversely related to interval colorectal cancer risk and cancer mortality. Previous studies evaluating the impact of gastroenterology fellow participation in colonoscopy on ADR have generated conflicting results. AIMS: We aimed to determine the impact of fellow participation, duration of fellowship training, and physician sex on ADR and advanced ADR (AADR). METHODS: We retrospectively analyzed average-risk patients undergoing screening colonoscopy at Veterans Affairs New York Harbor Healthcare System Brooklyn Campus and Kings County Hospital Center. Review of colonoscopy and pathology reports were performed to obtain adenoma-specific details, including the presence of advanced adenoma and adenoma location (right vs. left colon). RESULTS: There were 893 colonoscopies performed by attending only and 502 performed with fellow participation. Fellow participation improved overall ADR (44.6% vs. 35.4%, p < 0.001), right-sided ADR (34.1% vs. 25.2%, p < 0.001), and AADR (15.3% vs. 8.3%, p < 0.001); however, these findings were institution-specific. Year of fellowship training did not impact overall ADR or overall AADR, but did significantly improve right-sided AADR (p-value for trend 0.03). Female attending physicians were associated with increased ADR (47.1% vs. 37.0%, p = 0.0037). Fellow sex did not impact ADR. CONCLUSIONS: Fellow participation in colonoscopy improved overall ADR and AADR, and female attending physicians were associated with improved ADR. Year of fellowship training did not impact overall ADR or AADR.

Regioselective Amidomethylation of 4-Chloro-3-fluoropyridine by Metalation and Minisci-Type Reactions.

The synthesis of a series of 2-amidomethylated pyridines (3-8) was investigated, starting from 4-chloro-3-fluoropyridine. Kinetic deprotonation at -75 °C followed by reaction with DMF gave 2-formyl-4-chloro-3-fluoropyridine 10 regioselectively, which was converted to 2-aminomethyl analogue 1 via sulfinamide 2. Alternatively, Minisci-type amidomethylation under Ag+/persulfate or photoredox-mediated conditions using a series of amino acid derivatives gave (3-8, 19, and 34) in 30-74% yield and isomer ratios in the range 6.7:1 to >50:1. The latter methods gave overall yields similar to that of the deprotonation approach, but were shorter and more amenable to scale-up. In particular, N-Boc analogue 8 was obtained in a single step. The amidomethylations of another six 3-fluoropyridines under the photoredox conditions were briefly examined.

Genome Wide Conditional Mouse Knockout Resources.

The International Knockout Mouse Consortium (IKMC) developed high throughput gene trapping and gene targeting pipelines that produced mostly conditional mutations of more than 18,500 genes in C57BL/6N mouse embryonic stem (ES) cells which have been archived and are freely available to the research community as a frozen resource. From this unprecedented resource more than 6,000 mutant mouse strains have been produced by the IKMC and mostly the International Mouse Phenotyping Consortium (IMPC). In addition, a cre-driver resource was established including 250 inducible cre-driver mouse strains in a C57BL/6 background. Complementing the cre-driver resource, a collection of comprising 27 cre-driver rAAVs has also been produced. The resources can be easily accessed at the IKMC/IMPC web portal (www.mousephenotype.org). The IKMC/IMPC resource is a standardized reference library of mouse models with defined genetic backgrounds that enables the analysis of gene-disease associations in mice of different genetic makeup and should therefore have a major impact on biomedical research.

Capsaicin Synthesis Requires in Situ Phenylalanine and Valine Formation in in Vitro Maintained Placentas from Capsicum chinense.

Capsaicinoids (CAP) are nitrogenous metabolites formed from valine (Val) and phenylalanine (Phe) in the placentas of hot Capsicum genotypes. Placentas of Habanero peppers can incorporate inorganic nitrogen into amino acids and have the ability to secure the availability of the required amino acids for CAP biosynthesis. In order to determine the participation of the placental tissue as a supplier of these amino acids, the effects of blocking the synthesis of Val and Phe by using specific enzyme inhibitors were analyzed. Isolated placentas maintained in vitro were used to rule out external sources' participation. Blocking Phe synthesis, through the inhibition of arogenate dehydratase, significantly decreased CAP accumulation suggesting that at least part of Phe required in this process has to be produced in situ. Chlorsulfuron inhibition of acetolactate synthase, involved in Val synthesis, decreased not only Val accumulation but also that of CAP, pointing out that the requirement for this amino acid can also be fulfilled by this tissue. The presented data demonstrates that CAP accumulation in in vitro maintained placentas can be accomplished through the in situ availability of Val and Phe and suggests that the synthesis of the fatty acid chain moiety may be a limiting factor in the biosynthesis of these alkaloids.

Human immunodeficiency virus patients with low CD4 counts are more likely to have precancerous polyps identified during index colonoscopy.

BACKGROUND: Antiretroviral treatment (ART) has improved the life expectancy of patients living with human immunodeficiency virus (HIV). As these patients age, they are at increased risk for developing non-acquired immunodeficiency syndrome defining malignancies (NADMs) such as colon cancers. AIM: To determine which factors are associated with the development of precancerous polyps on screening colonoscopy in patients with HIV and to investigate whether HIV disease status, measured by viral load and CD4 count, might influence precancerous polyp development. METHODS: A retrospective review of records at two urban academic medical centers was performed for HIV patients who had a screening colonoscopy between 2005-2015. Patients with a history of colorectal cancer or polyps, poor bowel preparation, or inflammatory bowel disease were excluded. Demographic data such as sex, age, race, and body mass index (BMI) as well as information regarding the HIV disease status such as CD4 count, viral load, and medication regimen were collected. Well-controlled patients were defined as those that had viral load < 50 copies, and poorly-controlled patients were those with viral load ≥ 50. Patients were also stratified based on their CD4 count, comparing those with a low CD4 count to those with a high CD4 count. Using colonoscopy reports in the medical record, the size, histology, and number of polyps were recorded for each patient. Precancerous polyps included adenomas and proximal serrated polyps. Data was analyzed using Fisher's exact tests and logistic regression through SAS 3.8 software. RESULTS: Two hundred and seven patients met our inclusion criteria. The mean age was 56.13 years, and 58% were males. There were no significant differences in terms of age, race or ethnicity, insurance, and smoking status between patients with CD4 counts above or below 500. BMI was lower in patients with CD4 count < 500 as compared to those with count > 500 (P = 0.0276). In patients with CD4 > 500, 53.85% of patients were female, and 70.87% of patients with CD4 < 500 were male (P = 0.0004). Only 1.92% of patients with CD4 ≥ 500 had precancerous polyps vs 10.68% of patients with CD4 < 500 (P = 0.0102). When controlled for sex, BMI, and ART use, patients with CD4 < 500 were 9.01 times more likely to have precancerous polyps [95% confidence interval (CI): 1.69-47.97; P = 0.0100]. Patients taking non-nucleoside reverse transcriptase inhibitors were also found to be 10.23 times more likely to have precancerous polyps (95%CI: 1.08-97.15; P = 0.0428). There was not a significant difference noted in precancerous polyps between those that had viral loads greater or less than 50 copies. CONCLUSION: Patients with low CD4 counts were more likely to have precancerous polyps on their screening colonoscopy although the etiology for this association is unclear. We also found an increased risk of precancerous polyps in patients taking non-nucleoside reverse transcriptase inhibitors, which is contradictory to prior literature showing ART has decreased the risk of development of NADMs. However, there have not been studies looking at colorectal cancer and ART by drug class, to our knowledge. Further prospective studies are needed to determine the effect of HIV control and therapies on polyp development.

Second generation N-(1,2-diphenylethyl)piperazines as dual serotonin and noradrenaline reuptake inhibitors: improving metabolic stability and reducing ion channel activity.

New N-(1,2-diphenylethyl)piperazines 6 are disclosed as dual serotonin and noradrenaline reuptake inhibitors (SNRI) which may have potential in treating stress urinary incontinence (SUI). In this Letter, we present new data for SNRI PF-526014 (4) including performance in a canine in vivo model of SUI, cardiovascular assessment, pharmacokinetics in dog and determination of the primary routes of metabolism in vitro. Starting from 4, detailed structure activity relationships established that potent dual SNRIs could be achieved by appropriate substitution of the phenyl rings (6: R; R(1)) combined with a preferred stereochemistry. From this set of compounds, piperazine (-)-6a was identified as a potent and selective dual SNRI with improved metabolic stability and reduced ion channel activity when compared to 4. Based on this profile, (-)-6a was selected for further evaluation in a preclinical model of SUI.

Utility of the NeuroTrax computerized battery for cognitive screening in Parkinson's disease: comparison with the MMSE and the MoCA.

To determine the utility of a computerized assessment in Parkinson's disease (PD), we compared the cognitive performance of 50 PD patients on the NeuroTrax computerized battery relative to the mini-mental state examination (MMSE) and the Montreal Cognitive Assessment (MoCA). The results revealed fair agreement between impairment on the NeuroTrax and the MMSE (kappa=.291, p=.031) but only slight agreement between the NeuroTrax and the MoCA (kappa=.138, p = .054) and between the MoCA and the MMSE (kappa = .168, p = .069). The NeuroTrax identified 52% of the sample as average or above, 40% as below average, and 8% as impaired. The MoCA identified 54% of the sample as impaired (28% average or above and 18% below average), while the MMSE identified 66% as average or above (20% below average and 14% impaired). Several stepwise regressions revealed that executive and verbal functions were the best predictors of cognitive functioning on the NeuroTrax, while memory recall, serial sevens, naming, and abstraction were the best predictors on the MoCA. These results suggest that although the NeuroTrax may be useful in identifying executive cognitive deficits in PD, similar to the MMSE the NeuroTrax may lack optimal sensitivity. While the MoCA is sensitive, it may be too stringent in overclassifying PD patients as impaired.

4-Piperidines and 3-pyrrolidines as dual serotonin and noradrenaline reuptake inhibitors: design, synthesis and structure-activity relationships.

Single enantiomer [(aryloxy)(pyridinyl)methyl]piperidine and pyrrolidine derivatives 5-9 are inhibitors of monoamine reuptake. Structure-activity relationships established that monoamine reuptake inhibition are functions of amine, pyridine isomer, aryloxy ring substitution and stereochemistry. Consequently, selective NRIs, selective SRIs, dual SNRIs and triple SNDRIs were all identified. Dual SNRIs 5l-a and 9c were evaluated in additional pharmacology and pharmacokinetic studies as representative examples from this series.

N-[(3S)-Pyrrolidin-3-yl]benzamides as novel dual serotonin and noradrenaline reuptake inhibitors: impact of small structural modifications on P-gp recognition and CNS penetration.

The structure-activity relationship and the synthesis of novel N-[(3S)-pyrrolidin-3-yl]benzamides as dual serotonin and noradrenaline monoamine reuptake inhibitors (SNRI) is described. Preferred compound 9 aka PF-184,298 is a potent SNRI with good selectivity over dopamine reuptake inhibition (DRI), good in vitro metabolic stability, weak CYP inhibition and drug-like physicochemical properties consistent with CNS target space. Evaluation in an in vivo preclinical model of stress urinary incontinence showed 9 significantly increased urethral tone at free plasma concentrations consistent with its in vitro primary pharmacology.

Pyridyl-phenyl ether monoamine reuptake inhibitors: Impact of lipophilicity on dual SNRI pharmacology and off-target promiscuity.

A novel series of pyridyl-phenyl ethers are disclosed, which possess dual 5-HT and NA reuptake pharmacology with good selectivity over dopamine reuptake inhibition. An analysis of the relationship between lipophilicity and pharmacology highlighted that potent dual SNRI activity was only achievable at c log P>3.5. The series was found to possess significant polypharmacology issues, and we concluded that this off-target promiscuity was related to lipophilicity.

N-Benzyl-N-(pyrrolidin-3-yl)carboxamides as a new class of selective dual serotonin/noradrenaline reuptake inhibitors.

The structure-activity relationship and the synthesis of novel N-benzyl-N-(pyrrolidin-3-yl)carboxamides as dual serotonin (5-HT) and noradrenaline (NA) monoamine reuptake inhibitors are described. Compounds such as 18 exhibited dual 5-HT and NA reuptake inhibition, good selectivity over dopamine (DA) reuptake inhibition and drug-like physicochemical properties consistent with CNS target space. Compound 18 was selected for further preclinical evaluation.

Upgrading mouse health and welfare: direct benefits of a large-scale rederivation programme.

We report the outcome of a 30-month programme to rederive 310 specific pathogen-free mouse strains to populate a new individually ventilated cage barrier facility at the Mary Lyon Centre (MLC), Medical Research Council (MRC) Harwell. The mice were rederived in a self-contained quarantine suite and embryo-recipient females were health-screened to assess microbiological status, before moving their offspring into the new facility. The MLC currently houses approximately 49,000 mice in about 9750 cages and we have 30 months of follow-up health screen data. Embryo rederivation and hysterectomy have high safety margins; however, the precaution of performing the programme in isolators facilitated the containment and decontamination of two mouse hepatitis virus (MHV) infection outbreaks. Rederivation of the colony has eliminated endemic MHV, mouse adenovirus type 2 (MAV-2), Theiler's murine encephalomyelitis virus, pinworms, intestinal protozoa, Pasteurella pneumotropica, Helicobacter spp. and mites. The improvements in microbiological status have had notable benefits for mouse health and welfare and the science at MRC Harwell. Previously important clinical entities such as sudden death associated with lactation ileus in C3H/HeH mice, early weight loss associated with inflammatory bowel disease in B6-TgN(HDexon1)61Gpb and B6TgN-(HD82Gln)81Dbo (Huntington) mice and early weight loss in male mice mutagenized with N-ethyl-N-nitrosourea have been markedly reduced or eliminated.

Gastrointestinal Manifestations of Systemic Sclerosis.

Systemic sclerosis (SSc) is a rare autoimmune disease characterized by fibroproliferative alterations of the microvasculature leading to fibrosis and loss of function of the skin and internal organs. Gastrointestinal manifestations of SSc are the most commonly encountered complications of the disease affecting nearly 90% of the SSc population. Among these complications, the esophagus and the anorectum are the most commonly affected. However, this devastating disorder does not spare any part of the gastrointestinal tract (GIT), and includes the oral cavity, esophagus, stomach, small and large bowels as well as the liver and pancreas. In this review, we present the current understanding of the pathophysiologic mechanisms of SSc including vasculopathy, endothelial to mesenchymal transformation as well as the autoimmune pathogenetic pathways. We also discuss the clinical presentation and diagnosis of each part of the GIT affected by SSc. Finally, we highlight the latest developments in the management of this disease, addressing the severe malnutrition that affects this vulnerable patient population and ways to assess and improve the nutritional status of the patients.

Matrix metalloproteinase-3 differences in oral and skin fibroblasts.

While skin wounds heal by scarring, wounds of oral mucosa show privileged healing with minimal scar formation. Our hypothesis was that phenotypic differences between oral and skin fibroblasts underlie these differences in healing. The aims of this study were to compare MMP-3 expression by oral and skin fibroblasts and investigate a role for MMP-3 in mediating collagen gel contraction. Oral fibroblasts induced significantly greater gel contraction than did paired skin cells. Inhibition of MMP activity significantly inhibited gel contraction by both cell types. Specific inhibition of MMP-3 activity reduced gel contraction by oral, but not skin, fibroblasts. Oral fibroblasts produced significantly higher levels of MMP-3 than did skin fibroblasts at all levels studied. TGF-beta1 and -beta3 isoforms stimulated MMP-3 expression at mRNA, protein, and activity levels by both fibroblast populations. Results suggest that increased MMP-3 production by oral fibroblasts may underlie the differences in wound-healing outcome seen in skin and oral mucosa.

Antibiotic loaded microspheres as antimicrobial delivery systems for medical applications.

In this paper, we present the preparation and antibiotic loading of polymeric microspheres, composed of copolymers derived from fatty acid/amino acid components, as new polymeric platforms for antibiotic delivery systems. New polymeric materials were used to prepare microspheres with and without immobilized model antibiotics (streptomycin, chloramphenicol and amphotericin B) by a W/O/W double-emulsion/solvent evaporation method, in which chloroform and poly(vinyl alcohol) are used as the solvent and emulsifier, respectively. The antimicrobial activity of the microspheres was tested against Gram-positive S. aureus, Gram-negative E. coli bacteria, and C. albicans, as a representative of a fungus. The new polymeric microspheres are particularly effective carriers for streptomycin, exhibiting antibacterial activity against all tested microorganisms.

Effects of time of progesterone supplementation on embryo development and interferon-tau production in the cow.

We have investigated the effects of the timing of progesterone supplementation on early embryo development in mature, non-lactating Holstein-Friesian cows. Animals were inseminated 72 h (day 1) and 96 h following prostaglandin injection and were either left as untreated controls (n=6) or received progesterone supplementation from either days 5 to 9 (early; n=6) or from days 12 to 16 (late; n=6). Daily plasma samples were collected until day 16, when cows were slaughtered and reproductive tracts recovered and flushed to collect embryos and to measure interferon-tau activity. Both early and later progesterone supplementation resulted in marked increases in plasma progesterone (P<0.01). Early, but not late, progesterone supplementation resulted in a fourfold increase in trophoblast length (P<0.01) and a sixfold increase in uterine concentration of interferon-tau (P<0.05). The results demonstrate that progesterone supplementation during the postovulatory rise, but not later in the luteal phase, increases embryo development and interferon-tau production.

New injectable elastomeric biomaterials for hernia repair and their biocompatibility.

Complications associated with implantation of polymeric hernia meshes remain a difficult surgical challenge. We report here on our work, developing for the first time, an injectable viscous material that can be converted to a solid and elastic implant in vivo, thus successfully closing herniated tissue. In this study, long-chain fatty acids were used for the preparation of telechelic macromonomers end-capped with methacrylic functionalities to provide UV curable systems possessing high biocompatibility, good mechanical strength and flexibility. Two different systems, comprising urethane and ester bonds, were synthesized from non-toxic raw materials and then subjected to UV curing after injection of viscous material into the cavity at the abdominal wall during hernioplasty in a rabbit hernia model. No additional fixation or sutures were required. The control group of animals was treated with commercially available polypropylene hernia mesh. The observation period lasted for 28 days. We show here that artificially fabricated defect was healed and no reherniation was observed in the case of the fatty acid derived materials. Importantly, the number of inflammatory cells found in the surrounding tissue was comparable to these found around the standard polypropylene mesh. No inflammatory cells were detected in connective tissues and no sign of necrosis has been observed. Collectively, our results demonstrated that new injectable and photocurable systems can be used for minimally invasive surgical protocols in repair of small hernia defects.

Passing on the medicinal chemistry baton: training undergraduates to be industry-ready through research projects between the University of Nottingham and GlaxoSmithKline.

In this article we describe a radically different industry-academia collaboration between the School of Chemistry, University of Nottingham, and GlaxoSmithKline (GSK), aiming to train students in research and give them an insight into medicinal chemistry as practiced in industry. The project concerns the discovery of potent and selective αvß6 integrin antagonists to treat idiopathic pulmonary fibrosis; the synthetic chemistry is performed by a group of ten final-year undergraduates and the biological and physicochemical screening data are generated by GSK. The project planning, organisation and operation are discussed, together with some of the challenges and rewards of working with undergraduates.

Influence of ceria nanoparticles on chemical structure and properties of segmented polyesters.

In this work, we present new nanocomposite materials derived from segmented copolyesters, comprising ethylene terephthalate (PET) segments and dimerized linoleic acid (DLA), and nanometric cerium oxide particles (CeO2). Nanoparticles were incorporated in situ during polycondensation in various concentrations, from 0.1 up to 0.6 wt.%. It was found that preparation of nanocomposites in situ, during polycondensation, had no significant influence on changes in segmental composition as determined from (1)H and (13)C, as well as 2D NMR. Thermal analysis and calculated degree of crystallinity showed that increasing concentration of ceria nanoparticles lead to an increase in mass content of PET crystallites in hard segments. The XRD investigations also showed an increased intensity of characteristic signals with increasing ceria concentration. Simultaneously, the incorporation of CeO2 led to an increase in tensile strength and elongation at break, indicating a reinforcing and plasticizing effect of ceria nanoparticles. However, the modulus at 10% strain decreased with increasing amount of nanoparticles. The in vitro culture of human cardiac progenitor cells (hCPCs) on the new materials indicated a homogenous cell displacement across the samples after 5 days with no signs of cytotoxicity, indicating good biocompatibility in vitro of CeO2-based nanocomposites and a potential for biomedical applications.