Untargeted GC-MS metabolomics to identify and classify bioactive compounds in Combretum platypetalum subsp. oatesii (Rolfe) Exell (Combretaceae).

INTRODUCTION: Combretum platypetalum is used in traditional African healing practices against different infections. Unfortunately, no scientific knowledge of its phytochemical composition exists, except for the isolation of two compounds from the leaves. Scientific study has been limited to the leaves only, despite the applications of stems and roots in traditional medicine practice and natural product drug discovery programs. OBJECTIVE: Omics was applied to identify and classify different volatile and semivolatile bioactive compounds in the leaf, stem, and root parts of C. platypetalum. The thermal stability of the plant constituents at 60-65°C extraction temperature by Soxhlet and maceration at room temperature on the type, class, and concentration of compounds in the leaf was further investigated. METHOD: A GC-MS untargeted metabolomics approach, automated deconvolution by the Automated Mass Spectral Deconvolution and Identification System (AMDIS) for GC-MS data, preprocessing by Metab R, and multivariate statistical data analysis were employed in this study. RESULTS: A total of 97 phytoconstituents, including 17 bioactive compounds belonging to the terpenoids, flavonoids, long-chain fatty acids, and other unclassified structural arrangements distributed across C. platypetalum, were identified for the first time. A correlation (r = 0.782; P = 0.000) between Soxhlet and maceration extraction methods relative to resolved chromatographic peak areas of metabolites was established. CONCLUSION: Findings corroborate the reported bio-investigation of its leaf extracts, its traditional uses, and previous findings from the Combretum genus. The results substantiate the possible applications of C. platypetalum in natural product drug discovery and provide a guide for future investigations.

Choose wisely: Network, ontology and annotation resources for the analysis of Staphylococcus aureus omics data.

Staphylococcus aureus (S. aureus) is a prominent human and livestock pathogen investigated widely using omic technologies. Critically, due to availability, low visibility or scattered resources, robust network and statistical contextualisation of the resulting data is generally under-represented. Here, we present novel meta-analyses of freely-accessible molecular network and gene ontology annotation information resources for S. aureus omics data interpretation. Furthermore, through the application of the gene ontology annotation resources we demonstrate their value and ability (or lack-there-of) to summarise and statistically interpret the emergent properties of gene expression and protein abundance changes using publically available data. This analysis provides simple metrics for network selection and demonstrates the availability and impact that gene ontology annotation selection can have on the contextualisation of bacterial omics data.

The highly abundant urinary metabolite urobilin interferes with the bicinchoninic acid assay.

Estimation of total protein concentration is an essential step in any protein- or peptide-centric analysis pipeline. This study demonstrates that urobilin, a breakdown product of heme and a major constituent of urine, interferes considerably with the bicinchoninic acid (BCA) assay. This interference is probably due to the propensity of urobilin to reduce cupric ions (Cu(2+)) to cuprous ions (Cu(1+)), thus mimicking the reduction of copper by proteins, which the assay was designed to do. In addition, it is demonstrated that the Bradford assay is more resistant to the influence of urobilin and other small molecules. As such, urobilin has a strong confounding effect on the estimate of total protein concentrations obtained by BCA assay and thus this assay should not be used for urinary protein quantification. It is recommended that the Bradford assay be used instead.

Optical coherence tomography.

Optical coherence tomography (OCT) is a non-contact method for imaging the topological and internal microstructure of samples in three dimensions. OCT can be configured as a conventional microscope, as an ophthalmic scanner, or using endoscopes and small diameter catheters for accessing internal biological organs. In this Primer, we describe the principles underpinning the different instrument configurations that are tailored to distinct imaging applications and explain the origin of signal, based on light scattering and propagation. Although OCT has been used for imaging inanimate objects, we focus our discussion on biological and medical imaging. We examine the signal processing methods and algorithms that make OCT exquisitely sensitive to reflections as weak as just a few photons and that reveal functional information in addition to structure. Image processing, display and interpretation, which are all critical for effective biomedical imaging, are discussed in the context of specific applications. Finally, we consider image artifacts and limitations that commonly arise and reflect on future advances and opportunities.

Ultrathin side-viewing needle probe for optical coherence tomography.

We present the smallest reported side-viewing needle probe for optical coherence tomography (OCT). Design, fabrication, optical characterization, and initial application of a 30-gauge (outer diameter 0.31 mm) needle probe are demonstrated. Extreme miniaturization is achieved by using a simple all-fiber probe design incorporating an angle-polished and reflection-coated fiber-tip beam deflector. When inserted into biological tissue, aqueous interstitial fluids reduce the probe's inherent astigmatism ratio to 1.8, resulting in a working distance of 300 µm and a depth-of-field of 550 µm with beam diameters below 30 µm. The needle probe was interfaced with an 840 nm spectral-domain OCT system and the measured sensitivity was shown to be only 7 dB lower than that of a comparable galvo-scanning sample arm configuration. 3D OCT images of lamb lungs were acquired over a depth range of ~600 µm, showing individual alveoli and bronchioles.

A provider-based water planning and management model--WaterSim 4.0--for the Phoenix Metropolitan Area.

Uncertainty in future water supplies for the Phoenix Metropolitan Area (Phoenix) are exacerbated by the near certainty of increased, future water demands; water demand may increase eightfold or more by 2030 for some communities. We developed a provider-based water management and planning model for Phoenix termed WaterSim 4.0. The model combines a FORTRAN library with Microsoft C# to simulate the spatial and temporal dynamics of current and projected future water supply and demand as influenced by population demographics, climatic uncertainty, and groundwater availability. This paper describes model development and rationale. Water providers receive surface water, groundwater, or both depending on their portfolio. Runoff from two riverine systems supplies surface water to Phoenix while three alluvial layers that underlie the area provide groundwater. Water demand was estimated using two approaches. One approach used residential density, population projections, water duties, and acreage. A second approach used per capita water consumption and separate population growth estimates. Simulated estimates of initial groundwater for each provider were obtained as outputs from the Arizona Department of Water Resources (ADWR) Salt River Valley groundwater flow model (GFM). We compared simulated estimates of water storage with empirical estimates for modeled reservoirs as a test of model performance. In simulations we modified runoff by 80%-110% of the historical estimates, in 5% intervals, to examine provider-specific responses to altered surface water availability for 33 large water providers over a 25-year period (2010-2035). Two metrics were used to differentiate their response: (1) we examined groundwater reliance (GWR; that proportion of a providers' portfolio dependent upon groundwater) from the runoff sensitivity analysis, and (2) we used 100% of the historical runoff simulations to examine the cumulative groundwater withdrawals for each provider. Four groups of water providers were identified, and discussed. Water portfolios most reliant on Colorado River water may be most sensitive to potential reductions in surface water supplies. Groundwater depletions were greatest for communities who were either 100% dependent upon groundwater (urban periphery), or nearly so, coupled with high water demand projections. On-going model development includes linking WaterSim 4.0 to the GFM in order to more precisely model provider-specific estimates of groundwater, and provider-based policy options that will enable "what-if" scenarios to examine policy trade-offs and long-term sustainability of water portfolios.

Measuring airway dimensions during bronchoscopy using anatomical optical coherence tomography.

Airway dimensions are difficult to quantify bronchoscopically because of optical distortion and a limited ability to gauge depth. Anatomical optical coherence tomography (aOCT), a novel imaging technique, may overcome these limitations. This study evaluated the accuracy of aOCT against existing techniques in phantom, excised pig and in vivo human airways. Three comparative studies were performed: 1) micrometer-derived area measurements in 10 plastic tubes were compared with aOCT-derived area; 2) aOCT-derived airway compliance curves from excised pig airways were compared with curves derived using an endoscopic technique; and 3) airway dimensions from the trachea to subsegmental bronchi were measured using aOCT in four anaesthetised patients during bronchoscopy and compared with computed tomography (CT) measurements. Measurements in plastic tubes revealed aOCT to be accurate and reliable. In pig airways, aOCT-derived compliance measurements compared closely with endoscopic data. In human airways, dimensions measured with aOCT and CT correlated closely. Bland-Altman plots showed that aOCT diameter and area measurements were higher than CT measurements by 7.6% and 15.1%, respectively. Airway measurements using aOCT are accurate, reliable and compare favourably with existing imaging techniques. Using aOCT with conventional bronchoscopy allows real-time measurement of airway dimensions and could be useful clinically in settings where knowledge of airway calibre is required.

Quantifying tracheobronchial tree dimensions: methods, limitations and emerging techniques.

The ability to measure airway dimensions is important for clinicians, interventional bronchoscopists and researchers in order to accurately quantify structural abnormalities and track their changes over time or in response to treatment. Most quantitative airway measurements are based on X-ray computed tomography and, more recently, on multidetector computed tomography. Quantitative bronchoscopic techniques have also been developed, although these are less widely employed. Emerging techniques, including magnetic resonance imaging, endoscopic optical coherence tomography, endobronchial ultrasound and confocal endomicroscopy, provide new research tools with potential clinical applications. An understanding of issues related to the acquisition, processing and analysis of images, and how such issues impact on imaging the tracheobronchial tree, is essential in order to assess measurement accuracy and to make effective use of the newer methods. This article contributes to this understanding by providing a comprehensive review of current and emerging techniques for quantifying airway dimensions.

A Four-Biomarker Blood Signature Discriminates Systemic Inflammation Due to Viral Infection Versus Other Etiologies.

The innate immune system of humans and other mammals responds to pathogen-associated molecular patterns (PAMPs) that are conserved across broad classes of infectious agents such as bacteria and viruses. We hypothesized that a blood-based transcriptional signature could be discovered indicating a host systemic response to viral infection. Previous work identified host transcriptional signatures to individual viruses including influenza, respiratory syncytial virus and dengue, but the generality of these signatures across all viral infection types has not been established. Based on 44 publicly available datasets and two clinical studies of our own design, we discovered and validated a four-gene expression signature in whole blood, indicative of a general host systemic response to many types of viral infection. The signature's genes are: Interferon Stimulated Gene 15 (ISG15), Interleukin 16 (IL16), 2',5'-Oligoadenylate Synthetase Like (OASL), and Adhesion G Protein Coupled Receptor E5 (ADGRE5). In each of 13 validation datasets encompassing human, macaque, chimpanzee, pig, mouse, rat and all seven Baltimore virus classification groups, the signature provides statistically significant (p < 0.05) discrimination between viral and non-viral conditions. The signature may have clinical utility for differentiating host systemic inflammation (SI) due to viral versus bacterial or non-infectious causes.

The effect of levamisole on cell-mediated immunity and suppressor cell function.

The immunopotentiating drug, levamisole, was found to augment human lymphocyte responses to allogeneic cells and plant mitogens in vitro. The effect was critically dose dependent and, at high doses, suppression rather than augmentation of the immune response was observed. Our hypothesis that augmentation of immune responses by the drug is due to the selective impairment of immunoregulatory suppressor activity was tested in a model using human splenic and thymic suppressor cells. Contrary to expectation, the drug was found to be capable of augmenting suppressor activity rather than abolishing it. It is concluded that levamisole is a nonspecific stimulator of lymphocyte function, irrespective of the role played by these cells in the human response.

Dose dependence of immunopotentiation and tumor regression induced by levamisole.

Breast cancer was induced in female Sprague-Dawley rats by 7,12-dimethylbenz(a)anthracene. Once tumors had become established, they were treated with varying doses of the immunopotentiating drug, levamisole. Tumor growth was measured in the various dosage groups, and at 6 months after tumor induction the animals were sacrificed. Their immunological competence at this time was measured by the mitogen responses of splenic lymphocytes. Untreated animals with breast cancer were found to be immunosuppressed compared to normal animals. The drug levamisole resulted in immunopotentiation, but at high doses it was immunosuppressive. Tumor regression was observed at doses that resulted in immunopotentiation, but not at high doses. There was a significant correlation between immune competence and tumor regression. It is concluded that levamisole can cause regression of breast cancer in the rat but that this effect is critically dependent on the dose of the drug; these observations confirm previous studies carried out on human cells in vitro. It is recommended that high doses of the drug be avoided in human clinical trials and that the patients who receive this drug should have their immune responses carefully monitored.

Mobile Health Technology Can Objectively Capture Physical Activity (PA) Targets Among African-American Women Within Resource-Limited Communities-the Washington, D.C. Cardiovascular Health and Needs Assessment.

BACKGROUND: Little is understood about using mobile health (mHealth) technology to improve cardiovascular (CV) health among African-American women in resource-limited communities. METHODS: We conducted the Washington, D.C. CV Health and Needs Assessment in predominantly African-American churches in city wards 5, 7, and 8 with the lowest socioeconomic status based on community-based participatory research (CBPR) principles. The assessment measured CV health factors: body mass index (BMI), fasting blood glucose and cholesterol, blood pressure, fruit/vegetable (F/V) intake, physical activity (PA), and smoking. Participants were trained to use a PA monitoring wristband to measure 30 days of PA, wirelessly upload the PA data to hubs at the participating churches, and access their data from a church/home computer. CV health factors were compared across weight classes. RESULTS: Among females (N = 78; 99 % African-American; mean age = 59 years), 90 % had a BMI categorized as overweight/obese. Across weight classes, PA decreased and self-reported sedentary time (ST) increased (p ≤ 0.05). Diastolic blood pressure and glucose increased across weight classes (p ≤ 0.05); however, cholesterol, glucose, and BP were near intermediate CV health goals. CONCLUSIONS: Decreased PA and increased ST are potential community intervention targets for overweight and obese African-American women in resource-limited Washington D.C. areas. mHealth technology can assist in adapting CBPR intervention resources to improve PA for African-American women in resource-limited communities.

Contrast and depth enhancement in two-photon microscopy of human skin ex vivo by use of optical clearing agents.

We investigate the application of hyperosmotic optical clearing agents to improve the image contrast and penetration depth in two-photon microscopy of human dermis ex vivo. We show that the agents glycerol, propylene glycol, and glucose all convey significant improvements and we provide results on their dynamic behaviour and the reversibility of the effect. At suitable concentrations, such agents have the potential to be compatible with living tissue and may possibly enhance in-vivo deep-tissue imaging.

Use of web-based data collection to evaluate analgesic administration and the decision for surgery in horses with colic.

REASONS FOR PERFORMING STUDY: There is no current veterinary information with regard to data entry on a website. We therefore completed a prospective study on use of analgesics in relation to the need for abdominal surgery using a web-based survey instrument. OBJECTIVES: To establish an internet-based data entry system that could be used to record prospective data on horses with colic. HYPOTHESIS: Failure of horses to respond to the initial administration of an analgesic is an indicator of the need for abdominal surgery. METHODS: A survey was developed to determine if the response to administration of an analgesic during a veterinarian's first examination of a horse with colic was related to the need for surgery. Veterinarians were contacted via AAEP and ECN listservs requesting submission of cases using a log-in system at a data entry website. The survey was completed by submission of a data entry page. RESULTS: Twenty-seven veterinarians submitted details of 119 cases of colic, 28 of which required surgery. The need for surgery was significantly associated with moderate or severe pain observed during the first examination compared to mild or no pain, and with constant pain or return of pain after administration of one or more analgesics and the need to administer a second analgesic treatment. Abnormal findings on rectal examination were not associated with the need for surgery, whereas total absence or decrease of intestinal sounds was significantly related to the odds that surgery was needed. The web-based data collection was successful in collecting all data points on the survey for each case. CONCLUSIONS: This study reinforced veterinarians' use of response to therapy as a way to determine the need for abdominal surgery. POTENTIAL RELEVANCE: When a specific diagnosis has not been made, lack of response or resumption of colic after analgesic administration should be considered as an indicator of the need for surgery. Web-based collection of data allows veterinary practitioners to participate in clinical research by providing prospective data.

Decreased reactivity to sweetness following chronic exposure to mild unpredictable stress or acute administration of pimozide.

Consumption of a palatable wet mash was examined in rats subjected chronically (4-10 weeks) to unpredictable mild stress. Intake of mash containing 0, 10%, or 20% additional sucrose was normal in stressed animals. In control animals, the addition of 30% or 40% sucrose caused a decrease in the quantity of mash consumed, but increased the rate of eating. Both the increase in eating rate and the decrease in intake, at high sucrose concentration, were markedly attenuated in stressed animals (which therefore had higher intakes of very sweet mash and lower rates of eating, relative to control animals). Like chronic mild stress, the dopamine receptor antagonist pimozide (0.2 mg/kg) also increased the intake of a wet mash with 30% added sucrose, while decreasing the rate of consumption. Stressed animals were relatively insensitive to pimozide, though there were significant additive effects on duration of eating (increased) and on postprandial resting (suppressed). The failure of stressed animals to adapt their intake to increases in sweetness, and the similarities between the effects of chronic mild stress and acute pimozide, are compatible with the hypothesis that animals exposed to chronic mild stress are anhedonic.

Dopaminergic mechanism of imipramine action in an animal model of depression.

Rats, subjected chronically (10-12 weeks) to a variety of mild, unpredictable stressors, showed a decrease in their consumption of weak sucrose solutions; normal behavior was restored by chronic (5-9 weeks) treatment with the tricyclic antidepressant imipramine. Acute administration of the dopamine receptor antagonist pimozide or the specific dopamine D2 receptor antagonist raclopride had no effect in nonstressed animals and in vehicle-treated stressed animals, but both drugs selectively reversed the improvement of performance in imipramine-treated stressed animals. The 5HT antagonist metergoline increased sucrose consumption in all groups. The data suggest that the mechanism of action of imipramine in this model is an increase in functional activity at dopamine (DA) synapses.

Exercise-induced changes in plasma vitamin B-6 concentrations do not vary with exercise intensity.

This study examined the effect of high vs moderate exercise intensity on changes in concentration of plasma pyridoxal 5'-phosphate (PLP), pyridoxal (PL), and 4-pyridoxic acid (4-PA) in human subjects. Eight physically active subjects were tested twice at 60% and 85% maximum oxygen consumption (VO2max) for 30 and 20 min, respectively, on a bicycle ergometer. Blood samples were obtained before and during exercise. Data were adjusted for changes in plasma volume calculated from changes in hematocrit. PLP concentrations significantly increased during exercise (P < 0.0001), with 79% of the rise in PLP concentration occurring within 5 min. 4-PA concentration increased steadily through exercise and was 23% higher at 20 min than at 0 min. Exercise intensity had no effect on the magnitude or rate of either increase for either PLP or 4-PA. PL concentration did not vary with exercise duration, but was significantly higher at 60% compared with 85% VO2max (P = 0.001). No significant differences were observed in glucose concentration. The data do not support hypotheses in the literature that PLP concentration rises during exercise to support exercise-induced shifts in substrate utilization.

The human splenic suppressor cell.

Studies of human spleen cell suspensions show that they contain a population of cells which can inhibit the mixed lymphocyte reaction. The cells appear to be able to suppress the patient's own response to an antigenic challenge in slightly greater degree than their ability to suppress a nonspecific mixed lymphocyte culture. The suppressive effect is dependent on cell dose and is linearly related to the log of cell concentration. At low dose these cells have no suppressive effect and, in fact, behave as stimulators. Exposure of these cells to an environment containing immunosuppressive drug abrogates their suppressor activity. Manipulation of these cells may prove to be of value in the control of graft rejection.

Influence of maternal hyperthyroidism in the rat on the expression of neuronal and astrocytic cytoskeletal proteins in fetal brain.

Maternal hypothyroidism during pregnancy impairs brain function in human and rat offspring, but little is known regarding the influence of maternal hyperthyroidism on neurodevelopment. We have previously shown that the expression of neuronal and glial differentiation markers in fetal brain is compromised in hypothyroid rat dam pregnancies and have now therefore extended this investigation to hyperthyroid rat dams. Study groups comprised partially thyroidectomised dams, implanted with osmotic pumps infusing either vehicle (TX dams) or a supraphysiological dose of thyroxine (T4) (HYPER dams), and euthyroid dams infused with vehicle (N dams). Cytoskeletal protein abundance was determined in fetal brain at 21 days of gestation by immunoblot analysis. Relative to N dams, circulating total T4 levels were reduced to around one-third in TX dams but were doubled in HYPER dams. Fetal brain weight was increased in HYPER dams, whereas litter size and fetal body weight were reduced in TX dams. Glial fibrillary acidic protein expression was similar in HYPER and TX dams, being reduced in both cases relative to N dams. alpha-Internexin (INX) abundance was reduced in HYPER dams and increased in TX dams, whereas neurofilament 68 (NF68) exhibited increased abundance in HYPER dams. Furthermore, INX was inversely related to - and NF68 directly related to - maternal serum total T4 levels, independently of fetal brain weight. In conclusion, maternal hyperthyroidism compromises the expression of neuronal cytoskeletal proteins in late fetal brain, suggestive of a pattern of accelerated neuronal differentiation.

Maternal thyroid status regulates the expression of neuronal and astrocytic cytoskeletal proteins in the fetal brain.

Maternal thyroid hormone (TH) crosses the placenta and is postulated to regulate fetal brain development. However, TH-dependent stages of fetal brain development remain to be characterised. We have therefore compared the levels of several neuronal and glial cytoskeletal proteins in fetal brains from normal (N) and partially thyroidectomised (TX) rat dams by immunoblotting. Pregnancies were studied both before and after the onset of fetal TH secretion, which occurs at 17.5 days gestation (dg) in the rat. Maternal hypothyroidism disrupted fetal growth, so that fetal body and brain weights were reduced near term. Vimentin expression was unaffected, however, indicating normal acquisition of neuronal and glial precursor cells. Fetal brain levels of glial fibrillary acidic protein (GFAP) were reduced at 21 dg, suggesting delayed astrocytic differentiation, although regression analysis demonstrated appropriate GFAP levels for brain weight. Levels of alpha-internexin, the earliest neurofilament protein expressed in fetal brain were reduced at 16 dg in TX dams, but increased at 21 dg. The ontogeny of neurofilament-L was also perturbed in these pregnancies, with deficient levels apparent at both 16 and 21 dg. These effects on neuronal cytoskeletal proteins were unrelated to fetal brain growth retardation. These findings confirm that maternal hypothyroidism disrupts early fetal brain development. Early disturbances in neuronal differentiation are not corrected by the onset of fetal TH secretion. Such disturbances may contribute to the neurological damage observed in children born to hypothyroxinaemic mothers.