Genetics of phenological development and implications for seed yield in lentil.

Understanding phenology, its genetics and agronomic consequences, is critical for crop adaptation. Here we aim at (1) characterising lentil response to photoperiod with a focus on five loci: the lentil ELF3 ortholog Sn, two loci linked to clusters of lentil FT orthologs and two loci without candidates in chromosomes 2 and 5 (exp. 1: 36 lines, short and long day in phytotron); (2) establishing phenology-yield relationship (exp. 2: 25 lines, 11 field environments). A vintage perspective, where we quantify time trends in phenotype over three decades of breeding, links both experiments. Yield increased linearly from older to newer varieties at 29 kg ha-1 yr-1 or 1.5% yr-1, correlated negatively with flowering time in both winter- and summer-rainfall regimes, and decoupled from biomass in favourable environments. Time to flowering shortened from older to newer varieties at -0.56 % yr-1 in the field, and -0.42 % yr-1 (short day) and -0.99 % yr-1 (long day) in the phytotron. Early-flowering lines of diverse origin carried multiple early alleles for the five loci, indicating that at least some of these loci affect phenology additively. Current germplasm primarily features the early flowering haplotype for an FTb cluster region, hence the potential to increase phenological diversity with yield implications.

Child health, infant formula funding and South African health professionals: Eliminating conflict of interest.

Letters by Van Niekerk and Khan on article by Lake et al. (Lake L, Kroon M, Sanders D, et al. Child health, infant formula funding and South African health professionals: Eliminating conflict of interest. S Afr Med J 2019;109(12):902-906. https://doi.org/10.7196/SAMJ.2019.v109i12.14336); and response by Lake et al.

Child health, infant formula funding and South African health professionals: Eliminating conflict of interest.

Despite clear evidence of the benefits of exclusive and continued breastfeeding for children, women and society, far too few children in South Africa (SA) are breastfed. One of the major impediments to improving this situation is the continued and aggressive marketing of breastmilk substitutes (BMSs) and infiltration of the BMS industry into contexts with exposure to health professionals. In this article we, as academics, practitioners and child health advocates, describe contraventions of the regulations that protect breastfeeding in SA and argue that bold, proactive leadership to eliminate conflict of interest in respect of the BMS industry is urgently required, together with far greater investments in proven interventions to promote and support breastfeeding.

Characterization of multiple life stages of two Australian Fasciola hepatica isolates in sheep.

Information on the susceptibility status of Fasciola hepatica isolates is lacking in the literature, even for those isolates considered to be laboratory reference strains. Four controlled efficacy studies were conducted on two Fasciola hepatica isolates from Australia, viz. 'Oberon' and 'Sunny Corner' with treatment at either 2, 6 or 10 weeks post-infection (wpi) as defined in each study. Fluke burdens and examination of livers occurred at necropsy in weeks 12 (Study 1) or 13 (Studies 2, 3 and 4) post-infection. The triclabendazole (TCBZ) resistance status of the Oberon isolate was confirmed in 6 and 10-week old F. hepatica, utilizing the drug alone (Fasinex; 71.5% and 31.1%, respectively) and in combination with oxfendazole (Flukazole C; 79.9% and 0%, respectively). The susceptibility of this isolate to albendazole, as well as salicylanilide and sulphonamide drugs was confirmed. The Sunny Corner isolate was confirmed as susceptible to TCBZ (>99% all stages) and closantel (>90% at ≥6 wpi).

Application of a coproantigen ELISA as an indicator of efficacy against multiple life stages of Fasciola hepatica infections in sheep.

At present diagnosis of true resistance and determination of drug efficacy in Fasciola hepatica infection rely solely on terminal experiments. The coproantigen ELISA (cELISA) has been reported previously as a sensitive and specific tool appropriate to detect treatment failure, and potentially drug resistance. Two studies were conducted to determine whether the cELISA was appropriate for on-farm efficacy and resistance testing in Australian Merino sheep. In Study 1 sheep were infected orally with 50 F. hepatica metacercariae on three occasions, twelve, six and two weeks prior to a single flukicide treatment with triclabendazole, closantel or albendazole. Sheep were sampled weekly for a further seven weeks prior to necropsy. Following effective treatment, no faecal antigen was detected from 1 week. When immature stages (≤6 weeks) survived treatment, coproantigen reappeared from 6 weeks post-treatment. Therefore, cELISA conducted 1-4 weeks after treatment will demonstrate obvious treatment failure against adult F. hepatica, but is not sufficiently sensitive to detect survival of immature fluke until these reach maturity. In study 2, fluke burdens of sheep necropsied 13 weeks post single infection were compared to fecal worm egg counts (FWEC) and cELISA at necropsy. Regression analysis demonstrated that cELISA correlated strongly with fluke burden, whilst FWEC correlated weakly with cELISA. The correlation between FWEC and fluke burden was also weak, although stronger than that of FWEC with cELISA. The cELISA is an appropriate tool for monitoring effectiveness of treatments against Fasciola hepatica if an adult infection is present, however when immature stages of the parasite are present it is not as reliable. Where immature parasites are present it is recommended that initial cELISA be followed with a secondary cELISA at least 6 weeks after treatment to ensure resistance to immature stages is detected. Further testing is justified for monitoring the effectiveness of control programs by detecting adult populations that have survived a treatment regime.

Automated processing integrated with a microflow cytometer for pathogen detection in clinical matrices.

A spinning magnetic trap (MagTrap) for automated sample processing was integrated with a microflow cytometer capable of simultaneously detecting multiple targets to provide an automated sample-to-answer diagnosis in 40 min. After target capture on fluorescently coded magnetic microspheres, the magnetic trap automatically concentrated the fluorescently coded microspheres, separated the captured target from the sample matrix, and exposed the bound target sequentially to biotinylated tracer molecules and streptavidin-labeled phycoerythrin. The concentrated microspheres were then hydrodynamically focused in a microflow cytometer capable of 4-color analysis (two wavelengths for microsphere identification, one for light scatter to discriminate single microspheres and one for phycoerythrin bound to the target). A three-fold decrease in sample preparation time and an improved detection limit, independent of target preconcentration, was demonstrated for detection of Escherichia coli 0157:H7 using the MagTrap as compared to manual processing. Simultaneous analysis of positive and negative controls, along with the assay reagents specific for the target, was used to obtain dose-response curves, demonstrating the potential for quantification of pathogen load in buffer and serum.

Outbreak of tuberculosis in a homeless population involving multiple sites of transmission.

SETTING: During 2002-2003, a large outbreak of tuberculosis (TB) occurred among persons using multiple homeless facilities in King County, Washington. OBJECTIVE: To control the transmission of TB in multiple settings. DESIGN: In 2002, contacts exposed to patients in homeless facilities were screened using tuberculin skin tests (TSTs) and symptom review. Based on these screening results, sites of transmission were identified and prioritised, and exposed cohorts at these sites were offered intensive screening tests in 2003 (e.g., symptom review, TST, chest radiograph [CXR], sputum examination and culture). Mycobacterium tuberculosis isolates from patients were genotyped using PCR-based methods to identify outbreak-associated patients quickly. RESULTS: During 2002-2003, 48 (15%) of 313 patients diagnosed in King County were outbreak-associated; 47 culture-positive patients had isolates that matched the outbreak strain by genotyping. Three facilities visited by >12 patients in 2002 had a higher prevalence of TST positive results (approximately 30%) among clients compared with the background rate (7%) in the homeless community. Screening contacts with one sputum culture was as sensitive as CXR in detecting TB disease (77% vs. 62%, respectively). CONCLUSIONS: A comprehensive, resource-intensive approach likely helped to control transmission. This outbreak highlights the vulnerability of homeless populations and the need to maintain robust TB programs in urban settings.

A portable array biosensor for detecting multiple analytes in complex samples.

The Multi-Analyte Array Biosensor (MAAB) has been developed at the Naval Research Laboratory (NRL) with the goal of simultaneously detecting and identifying multiple target agents in complex samples with minimal user manipulation. This paper will focus on recent improvements in the biochemical and engineering aspects of this instrument. These improvements have enabled the expansion of the repertoire of analytes detected to include Salmonella typhimurium and Listeria monocytogenes, and also expanded the different sample matrices tested. Furthermore, all components of the biochemical assays could be prepared well in advance of sample testing, resulting in a "plug-and-play" methodology. Simultaneous detection of three toxins (ricin, staphylococcal enterotoxin B, and cholera toxin) was demonstrated using a novel fluidics cube module that limits the number of manipulations to only the initial sample loading. This work demonstrates the utility of the MAAB for rapid analysis of complex samples with multianalyte capability, with a minimum of operator manipulations required for either sample preparation or final analysis.

Explosives detection in soil using a field-portable continuous flow immunosensor.

A field method for quantitative analysis of explosives in contaminated soil samples is described. The method is based on a displacement immunoassay performed in a commercial instrument, the FAST 2000, engineered by Research International Inc. The method can be used on-site to measure 2,4,6-trinitrotoluene (TNT) and hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) within 5min. For this study, replicate analyses were performed on soil extracts prepared from each field sample as well as appropriate controls, blanks, and laboratory standards. Statistical analyses were done to assess accuracy, bias, and predictability of the method. The results demonstrated that the immunosensor could be used effectively to screen environmental samples for the presence or absence of explosives. In most samples, the method also provided quantitative values that were in good agreement with standard laboratory analyses using HPLC. A limited number of sample matrices interfered with the immunoassay and produced results that varied significantly from the laboratory data. In each case, the compounds causing the problem have been identified and efforts are being made to minimize these matrix interferences in future field evaluations.

Antibody immobilization using heterobifunctional crosslinkers.

Covalent attachment of functional proteins to a solid support is important for biosensors. One method employs thiol-terminal silanes and heterobifunctional crosslinkers such as N-succinimidyl 4-maleimidobutyrate (GMBS) to immobilize proteins through amino groups onto glass, silica, silicon or platinum surfaces. In this report, several heterobifunctional crosslinkers are compared to GMBS for their ability to immobilize active antibodies onto glass cover slips at a high density. Antibodies were immobilized at densities of 74-220 ng/cm2 with high levels of specific antigen binding. Carbohydrate-reactive crosslinkers were also compared to GMBS using a fiber optic biosensor to detect fluorescently-labeled antigen. At the concentrations tested, the antibodies immobilized with carbohydrate-reactive crosslinkers bound more antigen than GMBS immobilized antibodies as indicated by the fluorescence signal.

Regeneration of immobilized antibodies on fiber optic probes.

The regeneration of antibodies covalently immobilized to an optical fibre surface was investigated by dissociation of the antibody-antigen complex with three different solvents: (a) an acidic solution (0.1 M glycine hydrochloride in 50% (v/v) ethylene glycol, pH 1.75), (b) a basic solution (0.05 M tetraethylamine in 50% (v/v) ethylene glycol, pH 11.0) and (c) 50% (v/v) ethanol in PBS. The fibres coated with polyclonal rabbit anti-goat antibody against a large protein retained 70% and 65% of the original signal after five consecutive regenerations with acidic and basic solvent systems, respectively. The fibres coated with monoclonal mouse anti-trinitrobenzene antibody specific for a small organic molecule, retained over 90% of the original signal when regenerated with basic and ethanol solutions. This study evaluated regeneration and reuse of antibody-coated fibre optic biosensors as a means of reducing routine laboratory analysis costs and time.

Fabrication of surfaces resistant to protein adsorption and application to two-dimensional protein patterning.

Proteins were attached in defined geometric patterns on a surface. A prerequisite to making a pattern of proteins is generation of surfaces resistant to nonspecific protein adsorption. This was accomplished via oxidation of the thiol terminus of an organosilane self-assembled monolayer film by deep ultraviolet (DUV) irradiation. The resultant surface exhibited marked resistance to protein adsorption. Using a mask to protect regions of the silanized surface from irradiation, proteins were selectively adsorbed or attached via covalent linkage at locations protected from the DUV light. Antibodies immobilized in patterns using this procedure retained their antigen-binding capability. Thus chemistry and DUV lithography were combined to create patterns of active biomolecules which could be used in the microfabrication of electronic devices and biosensors.

Detection of Clostridium botulinum toxin A using a fiber optic-based biosensor.

A rapid, sensitive, analytical method for the detection of Clostridium botulinum toxin has been developed. The fiber optic-based biosensor utilizes the evanescent wave of a tapered optical fiber for signal discrimination. A 50 mW argon-ion laser, which generates laser light at 514 nm, is used in conjunction with an optical fiber probe that is tapered at the distal end. Antibodies specific for C. botulinum are covalently attached to the surface of the tapered fiber. The principle of the system is a sandwich immunoassay using rhodamine-labeled polyclonal anti-toxin A immunoglobin G (IgG) antibodies for generation of the specific fluorescent signal. Various anti-toxin antibodies were immobilized to the fibers. Affinity-purified polyclonal horse anti-toxin A antibodies performed better than the IgG fraction from the same horse serum or than the monoclonal anti-toxin A antibody BA11-3. Botulinum toxin could be detected within a minute, at concentrations as low as 5 ng/ml. The reaction was highly specific and no response was observed against tetanus toxin.

Use of thiol-terminal silanes and heterobifunctional crosslinkers for immobilization of antibodies on silica surfaces.

A procedure for covalent immobilization of functional proteins on silica substrates was developed using thiol-terminal silanes and heterobifunctional cross-linkers. Using this procedure, a high density of functional antibodies was bound to glass cover slips and silica fibers. The amount of anti-IgG antibody immobilized was determined to be in the range of 0.66 to 0.96 ng/mm2 using radiolabeled antibody. The relative amount of IgG antigen bound by the immobilized antibody (0.37 to 0.55 mol antigen/mol antibody) was three to five times greater than other investigators have reported. In addition, the amount of protein nonspecifically adsorbed to the antibody-coated surface was further reduced by the addition of blocking agents so that nonspecific adsorption of protein antigens represented only 2-6% of the total antigen binding. With this low background, IgG antigen binding could be measured at levels as low as 150 fmol when an antigen concentration of 3 pmol/ml was applied. The process for antibody immobilization is straightforward, easy to perform, and adaptable for modifying mass quantities of biosensor components.

Effects of 17 beta-estradiol and progesterone on the metabolism of benz[a]pyrene by rabbit endometrial cells in primary culture.

The direct effects of estrogen (17 beta-estradiol) and progesterone on the metabolism of [3H]benzo[a]pyrene (BP) by primary cultures of rabbit endometrium epithelial cells were studied. 17 beta-Estradiol (10(-7) M) did not affect the metabolism of [3H]BP, expressed as the formation of water soluble metabolites of [3H]BP, during a 24-h incubation period. However, progesterone (10(-7) M) inhibited the formation of water-soluble metabolites of [3H]BP and the inhibitory effect was not prevented by 17 beta-estradiol. Progesterone did not appear to affect the relative distribution of the major conjugate types (sulfate esters and glutathiones) that comprised the water-soluble metabolites of [3H]BP. The results, which showed that progesterone acted to inhibit the metabolism of BP by endometrial cells, suggest additional evidence of a role for the ovarian hormone in the modulation of the carcinogenic efficacy of chemical carcinogens in a hormone-responsive tissue.

Restrained eating: measuring an elusive construct.

As a measure of restrained eating, Herman's Restraint Scale (1978) reliably predicts laboratory food consumption in college students regardless of their weight. However, the generality and psychometric properties of the scale have not been established. In the present study, 136 male and female adults were cross-classified as obese and normal and as dieting or non-dieting. The subjects were administered a single questionnaire containing items of the Lie, Social Desirability, and Restraint scales presented in randomized order. Unlike previous reports by Herman, the three adult groups differed significantly on the Restraint Scale in the following order: Obese dieters greater than Obese non-dieters greater than normals. Also, alpha reliability coefficients varied across groups and corrected item-total correlations also displayed considerable variability with no uniformity apparent for individual item correlations. The factor analysis identified three factors within the ten item scale, and for the obese dieters, the scale was not independent of social desirability. These results indicate that the Restraint Scale has limited usefulness beyond laboratory settings with college students.

Inhibition by 12-O-tetradecanoylphorbol-13-acetate of 17 beta-estradiol-induced initiation of DNA synthesis in rabbit endometrial cells in culture.

The initial effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) and 17 beta-estradiol on initiation of DNA synthesis by primary cultures of estrogen-responsive rabbit uterine epithelial cells were determined by autoradiography after a [3H]thymidine pulse. In contrast to the stimulatory response produced by estradiol, TPA (1.6 x 10(-7) M) reduced the fraction of cells synthesizing DNA for up to 48 h. Cells synthesizing DNA at the time TPA was added were found to be less sensitive to its inhibitory effect, suggesting that TPA blocked the cells from entering the S phase of the cell cycle. The cells were unresponsive to estradiol during the TPA-induced inhibitory period, although TPA did not interfere with the binding of [3H]estradiol to the hormonal whole-cell receptors. The results indicted that TPA acted to reduce the fraction of cells initiating DNa synthesis by a mechanism that appears to be independent of estrogen involvement.