RESUMO
Fluorescent lipid probes such as 1-palmitoyl-2-(6-[7-nitro-2-1,3-benzoxadiazol-4-yl]amino-hexanoyl)-sn-glycero-3-phosphocholine (C6 NBD-PC) have been used extensively to study the kinetics of lipid flip-flop. However, the efficacy of these probes as reliable reporters of native lipid translocation has never been tested. In this study, sum-frequency vibrational spectroscopy (SFVS) was used to measure the kinetics of C6 NBD-PC lipid flip-flop and the flip-flop of native lipids in planar supported lipid bilayers. C6 NBD-PC was investigated at concentrations of 1 and 3 mol. % in both chain-matched 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and chain-mismatched 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) to assess the ability of C6 NBD-PC to mimic the behavior of the surrounding matrix lipids. It was observed that C6 NBD-PC exhibited faster flip-flop kinetics compared to the native lipids in both DPPC and DSPC matrices, with notably accelerated rates in the chain-mismatched DSPC system. SFVS was also used to measure the acyl chain orientation and gauche content of C6 NBD-PC in both DPPC and DSPC membranes. In the DSPC matrix (chain mismatched), C6 NBD-PC was more disordered in terms of both gauche content and acyl tilt, whereas it maintained an orientation similar to that of the native lipids in the DPPC matrix (chain matched). In addition, the flip-flop kinetics of C6 NBD-PC were also measured using second-harmonic generation (SHG) spectroscopy, by probing the motion of the NBD chromophore directly. The flip-flop kinetics measured by SHG were consistent with those obtained from SFVS. This study also marks the first instance of phospholipid flip-flop kinetics being measured via SHG. The results of this study clearly demonstrate that C6 NBD-PC does not adequately mimic the behavior of native lipids within a membrane. These findings also highlight the significant impact of the lipid matrix on the flip-flop behavior of the fluorescently labeled lipid, C6 NBD-PC.
Assuntos
Bicamadas Lipídicas , Bicamadas Lipídicas/química , Fosfatidilcolinas/química , 1,2-Dipalmitoilfosfatidilcolina/química , 1,2-Dipalmitoilfosfatidilcolina/análogos & derivados , Cinética , Corantes Fluorescentes/química , Vibração , Análise Espectral/métodosRESUMO
Planar supported lipid bilayers (PSLBs) are an ideal model for the study of lipid membrane structures and dynamics when using sum-frequency vibrational spectroscopy (SFVS). In this paper, we describe the construction of asymmetric PSLBs and the basic SFVS theory needed to understand and make measurements on these membranes. Several examples are presented, including the determination of phospholipid orientation and measuring phospholipid transmembrane translocation (flip-flop).
Assuntos
Bicamadas Lipídicas , Análise Espectral , Bicamadas Lipídicas/química , Análise Espectral/métodos , Vibração , Fosfolipídeos/química , Lipídeos de Membrana/químicaRESUMO
Demand for nonhuman primates in research has increased over the past several years, while nonhuman primate supply remains a challenge in the United States. Global nonhuman primate supply issues make it increasingly important to maximize domestic colony production. To explore how housing conditions across primate breeding colonies impact infant survival and animal production more broadly, we collected medical records from 7959 rhesus macaques (Macaca mulatta) and 492 pigtail macaques (Macaca nemestrina) across seven breeding facilities and used generalized mixed-effect modeling to determine prenatal and infant survival odds by housing type and group size. Infant survival odds for each housing type and group size varied for prenatal, neonatal, early infant, and late infant age groups. Odds of prenatal survival were lowest in paired indoor housing and small and medium outdoor groups. No housing type performed better than large outdoor groups for neonatal survival. Odds of early infant survival was greatest in indoor and mixed indoor/outdoor housing compared to large outdoor enclosures. Large outdoor housing was associated with higher survival odds for late infant survival compared to small and medium outdoor housing. These results may influence housing choices at macaque breeding facilities hoping to maximize infant success, although there are relative care costs, the promotion of species-typical behaviors, and infrastructure factors to also consider. Our study used an interinstitutional collaboration that allowed for the analysis of more infant macaque medical records than ever before and used the broad variations across the seven national primate research centers to make the results applicable to many other facilities housing macaques.
Assuntos
Cruzamento , Abrigo para Animais , Humanos , Gravidez , Feminino , Animais , Macaca mulatta , Macaca nemestrinaRESUMO
We report a rare case of functional insulinomas in a 16.7-year-old female Rhesus macaque (Macaca mulatta) that was presented with neuroglycopenic signs to the breeding colony hospital at the Tulane National Primate Research Center. At initial and follow-up examinations, the animal was consistently hypoglycaemic and was clinically maintained with additional fruits, other high-sugar food items and dextrose supplementation. Occasional episodes of seizure and collapse resolved quickly on administration of high-sugar food items. At necropsy, the uncinate process of the pancreas had a 2.2 cm diameter, red, round, firm neoplastic mass, and another neoplasm was identified on histological examination of the head of pancreas. Histologically, neoplastic cells exhibited neuroendocrine packeting, resembled pancreatic islet cells and immunolabelled for chromogranin A, synaptophysin and insulin but not for somatostatin, gastrin or pancreatic polypeptide. A few cells immunolabelled for glucagon. The clinical signs and gross and histological findings were consistent with functional insulinomas.
Assuntos
Insulinoma , Insulinas , Neoplasias Pancreáticas , Animais , Cromogranina A , Feminino , Gastrinas , Glucagon , Glucose , Hipoglicemiantes , Insulinoma/diagnóstico , Insulinoma/patologia , Insulinoma/veterinária , Macaca mulatta , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/veterinária , Polipeptídeo Pancreático , Somatostatina , Açúcares , SinaptofisinaRESUMO
Background: Clinical measurements commonly used to evaluate overall health of laboratory animals including complete blood count, serum chemistry, weight, and immunophenotyping, differ with respect to age, development, and environment. This report provides comprehensive clinical and immunological reference ranges for pediatric rhesus macaques over the first year of life. Methods: We collected and analyzed blood samples from 151 healthy rhesus macaques, aged 0-55 weeks, and compared mother-reared infants to two categories of nursery-reared infants; those on an active research protocol and those under derivation for the expanded specific-pathogen-free breeding colony. Hematology was performed on EDTA-anticoagulated blood using a Sysmex XT2000i, and serum clinical chemistry was performed using the Beckman AU480 chemistry analyzer. Immunophenotyping of whole blood was performed with immunofluorescence staining and subsequent flow cytometric analysis on a BD LSRFortessa. Plasma cytokine analysis was performed using a Millipore multiplex Luminex assay. Results: For hematological and chemistry measurements, pediatric reference ranges deviate largely from adults. Comparison of mother-reared and nursery-reared animals revealed that large differences depend on rearing conditions and diet. Significant differences found between two nursery-reared cohorts (research and colony animals) indicate large influences of experimental factors and anesthetic events on these parameters. Immune cells and cytokine responses presented with distinct patterns for infants depending on age, birth location, and rearing conditions. Conclusions: Our results illustrate how the immune system changed over time and that there was variability among pediatric age groups. Reference ranges of results reported here will support interpretations for how infection and treatment may skew common immune correlates used for assessment of pathology or protection in research studies as well as help veterinarians in the clinical care of infant non-human primates. We highlighted the importance of using age-specific reference comparisons for pediatric studies and reiterated the utility of rhesus macaques as a model for human studies. Given the rapid transformation that occurs in multiple tissue compartments after birth and cumulative exposures to antigens as individuals grow, a better understanding of immunological development and how this relates to timing of infection or vaccination will support optimal experimental designs for developing vaccines and treatment interventions.
RESUMO
Idiopathic chronic enterocolitis (ICE) is one of the most commonly encountered and difficult to manage diseases of captive rhesus macaques (Macaca mulatta). The etiology is not well understood, but perturbations in gut microbial communities have been implicated. Here we evaluated the effects of a 14-day course of vancomycin, neomycin, and fluconazole on animals affected with ICE, comparing treated, untreated, and healthy animals. We performed microbiome analysis on duodenal and colonic mucosal samples and feces in order to probe bacterial and/or fungal taxa potentially associated with ICE. All treated animals showed a significant and long-lasting improvement in stool consistency over time when compared to untreated and healthy controls. Microbiome analysis revealed trends associating bacterial community composition with ICE, particularly lineages of the Lactobacillaceae family. Sequencing of DNA from macaque food biscuits revealed that fungal sequences recovered from stool were dominated by yeast-derived food additives; in contrast, bacteria in stool appeared to be authentic gut residents. In conclusion, while validation in larger cohorts is needed, the treatment described here was associated with significantly improved clinical signs; results suggested possible correlates of microbiome structure with disease, though no strong associations were detected between single microbes and ICE.