Secondary Metabolism Inducing Treatments During In Vitro Development of Turmeric (Curcuma longa L.) Rhizomes

Turmeric (Curcuma longa L.) plants that were grown in vitro for 17 or 22 weeks as a fed-batch culture in 2.5 L vessels yielded 39 to 43 g and 62 to 70 g of fresh rhizomes per vessel, respectively (95 % confidence interval). The MS liquid medium was maintained at 6 % sucrose through media addition twice during the experiment. Various methods were employed in attempts to increase secondary metabolism. Antioxidant and total phenolics assays were employed to characterize phytochemical activity. A first experiment exposed four clones to phenylalanine and/or methyl jasmonate (MeJa) from week 12 to 17 in culture. In a second experiment, a clone was given short-term exposure (1.5 weeks) to either proline, a natural proline-rich fish extract, MeJa, or chitosan beginning during the twentieth week of culture. This experiment also included a nitrogen stress treatment (weeks 16–22). The 5-week phenylalanine and MeJa treatments lowered biomass accumulation and antioxidant capacity of the tissue. The magnitude of antioxidant depression was dependent on genotype and, within each genotype, the degree of depression was similar for phenylalanine and MeJa, alone and in combination. In the second experiment, only the low-nitrogen treatment yielded an increase in phenolic content to 4.7 % of dry weight compared to untreated microrhizomes (4.1 % of dry weight). Nitrogen-stressed plants also had less leaf growth, but rhizome mass was unaffected and averaged 63 g FW per vessel. None of the short-term treatments had a significant effect on biomass, antioxidant capacity, or phenolic content. None of the treatments significantly affected radical scavenging, although the low-nitrogen treatment might have improved this activity (p = .1207). Results indicated that plants grown in a high-nitrogen MS media were not responsive to elicitation

The WIC Advisor: A Case Study in Medical Expert System Development

This project provides a good case study of expert system development with untrained experts over a short period of time. We describe the development of a working medical screening and diagnosis expert system for use at the Women, Infants and Children (WIC) clinics in Madison County, Illinois. The system was designed and implemented over the period of four months. A large number of knowledge acquisition techniques were employed, some of them customized in ways that greatly increased their effectiveness. This paper explores the development of THE WIC Advisor, from problem definition through expert system delivery. The knowledge acquisition methods used in creating The WIC Advisor comprise a beneficial case study of several traditional techniques. Protocol analysis, question/answer listing, knowledge acquisition room selection, prototyping, focused interviewing, multiple expert selection, direct questioning, audio-tape transcription, diving the domain, role-playing and teach back were employed [4,5]. Important factors in the success of this expert system were the selection of a limited diagnostic domain, the choice of multiple experts who worked well together, and our continuing efforts to make the experts feel comfortable with the technology and the process. The major benefits of the system include assisting clients between clinic visits, cataloging basic medical data, and providing consistent and verifiable informatio

Association of pol Diversity with Antiretroviral Treatment Outcomes among HIV-Infected African Children

Background: In HIV-infected children, viral diversity tends to increase with age in the absence of antiretroviral treatment (ART). We measured HIV diversity in African children (ages 6–36 months) enrolled in a randomized clinical trial comparing two ART regimens (Cohort I of the P1060 trial). Children in this cohort were exposed to single dose nevirapine (sdNVP) at birth. Methods: HIV diversity was measured retrospectively using a high resolution melting (HRM) diversity assay. Samples were obtained from 139 children at the enrollment visit prior to ART initiation. Six regions of the HIV genome were analyzed: two in gag, one in pol, and three in env. A single numeric HRM score that reflects HIV diversity was generated for each region; composite HRM scores were also calculated (mean and median for all six regions). Results: In multivariable median regression models using backwards selection that started with demographic and clinical variables, older age was associated with higher HRM scores (higher HIV diversity) in pol (P = 0.005) and with higher mean (P = 0.014) and median (P<0.001) HRM scores. In multivariable models adjusted for age, pre-treatment HIV viral load, pre-treatment CD4%, and randomized treatment regimen, higher HRM scores in pol were associated with shorter time to virologic suppression (P = 0.016) and longer time to study endpoints (virologic failure [VF], VF/death, and VF/off study treatment; P<0.001 for all measures). Conclusions: In this cohort of sdNVP-exposed, ART-naïve African children, higher levels of HIV diversity in the HIV pol region prior to ART initiation were associated with better treatment outcomes

Correction: Association of pol Diversity with Antiretroviral Atment Outcomes among HIV-Infected African Children

Background: In HIV-infected children, viral diversity tends to increase with age in the absence of antiretroviral treatment (ART). We measured HIV diversity in African children (ages 6–36 months) enrolled in a randomized clinical trial comparing two ART regimens (Cohort I of the P1060 trial). Children in this cohort were exposed to single dose nevirapine (sdNVP) at birth. Methods: HIV diversity was measured retrospectively using a high resolution melting (HRM) diversity assay. Samples were obtained from 139 children at the enrollment visit prior to ART initiation. Six regions of the HIV genome were analyzed: two in gag , one in pol , and three in env . A single numeric HRM score that reflects HIV diversity was generated for each region; composite HRM scores were also calculated (mean and median for all six regions). Results: In multivariable median regression models using backwards selection that started with demographic and clinical variables, older age was associated with higher HRM scores (higher HIV diversity) in pol (P = 0.005) and with higher mean (P = 0.014) and median (P , 0.001) HRM scores. In multivariable models adjusted for age, pre-treatment HIV viral load, pre- treatment CD4%, and randomized treatment regimen, higher HRM scores in pol were associated with shorter time to virologic suppression (P = 0.016) and longer time to study endpoints (virologic failure [VF], VF/death, and VF/off study treatment; P , 0.001 for all measures). Conclusions:In this cohort of sdNVP-exposed, ART-naı ̈ ve African children, higher levels of HIV diversity in the HIV pol region prior to ART initiation were associated with better treatment outcome

Astrobiological Considerations for the Selection of the Geological Filters on the ExoMars PanCam Instrument

The Panoramic Camera (PanCam) instrument will provide visible–near IR multispectral imaging of the ExoMars rover's surroundings to identify regions of interest within the nearby terrain. This multispectral capability is dependant upon the 12 preselected “geological” filters that are integrated into two wide-angle cameras. First devised by the Imager for Mars Pathfinder team to detect iron oxides, this baseline filter set has remained largely unchanged for subsequent missions (Mars Exploration Rovers, Beagle 2, Phoenix) despite the advancing knowledge of the mineralogical diversity on Mars. Therefore, the geological filters for the ExoMars PanCam will be redesigned to accommodate the astrobiology focus of ExoMars, where hydrated mineral terrains (evidence of past liquid water) will be priority targets. Here, we conduct an initial investigation into new filter wavelengths for the ExoMars PanCam and present results from tests performed on Mars analog rocks. Two new filter sets were devised: one with filters spaced every 50 nm (“F1-12”) and another that utilizes a novel filter selection method based upon hydrated mineral reflectance spectra (“F2-12”). These new filter sets, along with the Beagle 2 filter set (currently the baseline for the ExoMars PanCam), were tested on their ability to identify hydrated minerals and biosignatures present in Mars analog rocks. The filter sets, with varying degrees of ability, detected the spectral features of minerals jarosite, opaline silica, alunite, nontronite, and siderite present in these rock samples. None of the filter sets, however, were able to detect fossilized biomat structures and small (<2 mm) mineralogical heterogeneities present in silica sinters. Both new filter sets outperformed the Beagle 2 filters, with F2-12 detecting the most spectral features produced by hydrated minerals and providing the best discrimination between samples. Future work involving more extensive testing on Mars analog samples that exhibit a wider range of mineralogies would be the next step in carefully evaluating the new filter sets

Biosignature detection by Mars rover equivalent instruments in samples from the CanMars Mars Sample Return Analogue Deployment

The University of Winnipeg's HOSERLab was established with funding from the Canada Foundation for Innovation, the Manitoba Research Innovations Fund and the Canadian Space Agency, whose support is gratefully acknowledged. This study was supported with grants from the Canadian Space Agency through their FAST program, NSERC, and UWinnipeg.This work details the laboratory analysis of a suite of 10 samples collected from an inverted fluvial channel near Hanksville, Utah, USA as a part of the CanMars Mars Sample Return Analogue Deployment (MSRAD). The samples were acquired along the rover traverse for detailed off-site analysis to evaluate the TOC and astrobiological significance of the samples selected based on site observations, and to address one of the science goals of the CanMars mission: to evaluate the ability of different analytical techniques being employed by the Mars2020 mission to detect and characterize any present biosignatures. Analytical techniques analogous to those on the ExoMars, MSL and the MER rovers were also applied to the samples. The total organic carbon content of the samples was <0.02% for all but 4 samples, and organic biosignatures were detected in multiple samples by UV–Vis–NIR reflectance spectroscopy and Raman spectroscopy (532 nm, time-resolved, and UV), which was the most effective of the techniques. The total carbon content of the samples is < 0.3 wt% for all but one calcite rich sample, and organic C was not detectable by FTIR. Carotene and chlorophyll were detected in two samples which also contained gypsum and mineral phases of astrobiological importance for paleoenvironment/habitability and biomarker preservation (clays, gypsum, calcite) were detected and characterized by multiple techniques, of which passive reflectance was most effective. The sample selected in the field (S2) as having the highest potential for TOC did not have the highest TOC values, however, when considering the sample mineralogy in conjunction with the detection of organic carbon, it is the most astrobiologically relevant. These results highlight importance of applying multiple techniques for sample characterization and provide insights into their strengths and limitations.PostprintPeer reviewe

Use of a High Resolution Melting (HRM) Assay to Compare Gag, Pol, and Env Diversity in Adults with Different Stages of HIV Infection

Cross-sectional assessment of HIV incidence relies on laboratory methods to discriminate between recent and non-recent HIV infection. Because HIV diversifies over time in infected individuals, HIV diversity may serve as a biomarker for assessing HIV incidence. We used a high resolution melting (HRM) diversity assay to compare HIV diversity in adults with different stages of HIV infection. This assay provides a single numeric HRM score that reflects the level of genetic diversity of HIV in a sample from an infected individual.HIV diversity was measured in 203 adults: 20 with acute HIV infection (RNA positive, antibody negative), 116 with recent HIV infection (tested a median of 189 days after a previous negative HIV test, range 14-540 days), and 67 with non-recent HIV infection (HIV infected >2 years). HRM scores were generated for two regions in gag, one region in pol, and three regions in env.Median HRM scores were higher in non-recent infection than in recent infection for all six regions tested. In multivariate models, higher HRM scores in three of the six regions were independently associated with non-recent HIV infection.The HRM diversity assay provides a simple, scalable method for measuring HIV diversity. HRM scores, which reflect the genetic diversity in a viral population, may be useful biomarkers for evaluation of HIV incidence, particularly if multiple regions of the HIV genome are examined

Performance of a Limiting-Antigen Avidity Enzyme Immunoassay for Cross-Sectional Estimation of HIV Incidence in the United States

Background: A limiting antigen avidity enzyme immunoassay (HIV-1 LAg-Avidity assay) was recently developed for cross-sectional HIV incidence estimation. We evaluated the performance of the LAg-Avidity assay alone and in multi-assay algorithms (MAAs) that included other biomarkers. Methods and Findings: Performance of testing algorithms was evaluated using 2,282 samples from individuals in the United States collected 1 month to >8 years after HIV seroconversion. The capacity of selected testing algorithms to accurately estimate incidence was evaluated in three longitudinal cohorts. When used in a single-assay format, the LAg-Avidity assay classified some individuals infected >5 years as assay positive and failed to provide reliable incidence estimates in cohorts that included individuals with long-term infections. We evaluated >500,000 testing algorithms, that included the LAg-Avidity assay alone and MAAs with other biomarkers (BED capture immunoassay [BED-CEIA], BioRad-Avidity assay, HIV viral load, CD4 cell count), varying the assays and assay cutoffs. We identified an optimized 2-assay MAA that included the LAg-Avidity and BioRad-Avidity assays, and an optimized 4-assay MAA that included those assays, as well as HIV viral load and CD4 cell count. The two optimized MAAs classified all 845 samples from individuals infected >5 years as MAA negative and estimated incidence within a year of sample collection. These two MAAs produced incidence estimates that were consistent with those from longitudinal follow-up of cohorts. A comparison of the laboratory assay costs of the MAAs was also performed, and we found that the costs associated with the optimal two assay MAA were substantially less than with the four assay MAA. Conclusions: The LAg-Avidity assay did not perform well in a single-assay format, regardless of the assay cutoff. MAAs that include the LAg-Avidity and BioRad-Avidity assays, with or without viral load and CD4 cell count, provide accurate incidence estimates

A Comparison of Two Measures of HIV Diversity in Multi-Assay Algorithms for HIV Incidence Estimation

Background: Multi-assay algorithms (MAAs) can be used to estimate HIV incidence in cross-sectional surveys. We compared the performance of two MAAs that use HIV diversity as one of four biomarkers for analysis of HIV incidence. Methods: Both MAAs included two serologic assays (LAg-Avidity assay and BioRad-Avidity assay), HIV viral load, and an HIV diversity assay. HIV diversity was quantified using either a high resolution melting (HRM) diversity assay that does not require HIV sequencing (HRM score for a 239 base pair env region) or sequence ambiguity (the percentage of ambiguous bases in a 1,302 base pair pol region). Samples were classified as MAA positive (likely from individuals with recent HIV infection) if they met the criteria for all of the assays in the MAA. The following performance characteristics were assessed: (1) the proportion of samples classified as MAA positive as a function of duration of infection, (2) the mean window period, (3) the shadow (the time period before sample collection that is being assessed by the MAA), and (4) the accuracy of cross-sectional incidence estimates for three cohort studies. Results: The proportion of samples classified as MAA positive as a function of duration of infection was nearly identical for the two MAAs. The mean window period was 141 days for the HRM-based MAA and 131 days for the sequence ambiguity-based MAA. The shadows for both MAAs were <1 year. Both MAAs provided cross-sectional HIV incidence estimates that were very similar to longitudinal incidence estimates based on HIV seroconversion. Conclusions: MAAs that include the LAg-Avidity assay, the BioRad-Avidity assay, HIV viral load, and HIV diversity can provide accurate HIV incidence estimates. Sequence ambiguity measures obtained using a commercially-available HIV genotyping system can be used as an alternative to HRM scores in MAAs for cross-sectional HIV incidence estimation

Planetary science and exploration in the deep subsurface: results from the MINAR Program, Boulby Mine, UK

The subsurface exploration of other planetary bodies can be used to unravel their geological history and assess their habitability. On Mars in particular, present-day habitable conditions may be restricted to the subsurface. Using a deep subsurface mine, we carried out a program of extraterrestrial analog research – MINe Analog Research (MINAR). MINAR aims to carry out the scientific study of the deep subsurface and test instrumentation designed for planetary surface exploration by investigating deep subsurface geology, whilst establishing the potential this technology has to be transferred into the mining industry. An integrated multi-instrument suite was used to investigate samples of representative evaporite minerals from a subsurface Permian evaporite sequence, in particular to assess mineral and elemental variations which provide small-scale regions of enhanced habitability. The instruments used were the Panoramic Camera emulator, Close-Up Imager, Raman spectrometer, Small Planetary Linear Impulse Tool, Ultrasonic drill and handheld X-ray diffraction (XRD). We present science results from the analog research and show that these instruments can be used to investigate in situ the geological context and mineralogical variations of a deep subsurface environment, and thus habitability, from millimetre to metre scales. We also show that these instruments are complementary. For example, the identification of primary evaporite minerals such as NaCl and KCl, which are difficult to detect by portable Raman spectrometers, can be accomplished with XRD. By contrast, Raman is highly effective at locating and detecting mineral inclusions in primary evaporite minerals. MINAR demonstrates the effective use of a deep subsurface environment for planetary instrument development, understanding the habitability of extreme deep subsurface environments on Earth and other planetary bodies, and advancing the use of space technology in economic mining