Characterization of T cell responses to co-administered hookworm vaccine candidates Na-GST-1 and Na-APR-1 in healthy adults in Gabon

Two hookworm vaccine candidates, Na-GST-1 and Na-APR-1, formulated with Glucopyranosyl Lipid A (GLA-AF) adjuvant, have been shown to be safe, well tolerated, and to induce antibody responses in a Phase 1 clinical trial (Clinicaltrials.gov {"type":"clinical-trial","attrs":{"text":"NCT02126462","term_id":"NCT02126462"}}NCT02126462) conducted in Gabon. Here, we characterized T cell responses in 24 Gabonese volunteers randomized to get vaccinated three times with Na-GST-1 and Na-APR-1 at doses of 30μg (n = 8) or 100μg (n = 10) and as control Hepatitis B (n = 6). Blood was collected pre- and post-vaccination on days 0, 28, and 180 as well as 2-weeks after each vaccine dose on days 14, 42, and 194 for PBMCs isolation. PBMCs were stimulated with recombinant Na-GST-1 or Na-APR-1, before (days 0, 28 and 180) and two weeks after (days 14, 42 and 194) each vaccination and used to characterize T cell responses by flow and mass cytometry. A significant increase in Na-GST-1 -specific CD4⁺ T cells producing IL-2 and TNF, correlated with specific IgG antibody levels, after the third vaccination (day 194) was observed. In contrast, no increase in Na-APR-1 specific T cell responses were induced by the vaccine. Mass cytometry revealed that, Na-GST-1 cytokine producing CD4⁺ T cells were CD161⁺ memory cells expressing CTLA-4 and CD40-L. Blocking CTLA-4 enhanced the cytokine response to Na-GST-1.In Gabonese volunteers, hookworm vaccine candidate, Na-GST-1, induces detectable CD4⁺ T cell responses that correlate with specific antibody levels. As these CD4⁺ T cells express CTLA-4, and blocking this inhibitory molecules resulted in enhanced cytokine production, the question arises whether this pathway can be targeted to enhance vaccine immunogenicity.     

Gene targeting in plants using theAgrobacterium vector system

In the past decade several methods have been developed for the introduction of foreign DNA into plant cells to obtain transgenic plants. In some of these methods, purified DNA is directly introduced into protoplasts that for some species can be regenerated into mature plants. The more commonly used protocols, however, employ the natural capacity ofAgrobacterium tumefaciens to transfer a defined peice of DNa, called T-DNA, to the nucleus of plant cells that are more easy to regenerate than protoplasts. In plant cells, like in animal cells, foreign DNA (including T-DNA) is readily inserted into the genome via illegitimates recombination. In contrast, targeted integration via homologous recombination, referred to as ‘gene targeting’, can only be obtained at relatively low frequencies. Nevertheless, gene targeting has become a standard strategy for reverse genetics studies in animals. In plants, the occurrence of gene targeting was only reported recently. This review focuses on the use of theAgrobacterium vector system to achieve gene targeting in plants. Recent experimental data concerning gene targeting in plants are presented and the overall suitability ofAgrobacterium T-DNA transfer for this purpose is assessed in light of contemporary views on the mechanism of T-DNA transfer.     

Unique gene organization of thioredoxin and thioredoxin reductase in Mycobacterium leprae

The thioredoxin system comprising thioredoxin (Trx), thioredoxin reductase (TR) and NADPH operates via redox-active disulphides and provides electrons for a wide variety of different metabolic processes in prokaryotic and eukaryotic cells. Thioredoxin is also a general protein disulphide reductase involved in redox regulation. In bacteria, the Trx and TR proteins previously identified were encoded by separate genes (trxA and trxB). In this study, we report a novel genomic organization of TR and Trx in mycobacteria and show that at least three modes of organization of TR and Trx genes can exist within a single bacterial genus: (i) in the majority of mycobacterial strains the genes coding for TR and Trx are located on separate sites of the genome; (ii) interestingly, in all pathogenic Mycobacterium tuberculosis complex mycobacteria both genes are found on the same locus, overlapping in one nucleotide; (iii) in the pathogen Mycobacterium leprae, TR and Trx are encoded by a single gene. Sequence analysis of the M. leprae gene demonstrated that the N-terminal part of the protein corresponds to TR and the C-terminal part to Trx. A corresponding single protein product of approximately 49 kDa was detected in cell extracts of M. leprae. These findings demonstrate the very unusual phenomenon of a single gene coding for both the substrate (thioredoxin) and the enzyme (thioredoxin reductase), which seems to be unique to M. leprae.     

Canopy leaf area of a mature evergreen Eucalyptus woodland does not respond to elevated atmospheric [CO2] but tracks water availability

Canopy leaf area, quantified by the leaf area index (L), is a crucial driver of forest productivity, water use and energy balance. Because L responds to environmental drivers, it can represent an important feedback to climate change, but its responses to rising atmospheric [CO₂] and water availability of forests have been poorly quantified. We studied canopy leaf area dynamics for 28 months in a native evergreen Eucalyptus woodland exposed to free‐air CO₂ enrichment (the EucFACE experiment), in a subtropical climate where water limitation is common. We hypothesized that, because of expected stimulation of productivity and water‐use efficiency, L should increase with elevated [CO₂]. We estimated L from diffuse canopy transmittance, and measured monthly leaf litter production. Contrary to expectation, L did not respond to elevated [CO₂]. We found that L varied between 1.10 and 2.20 across the study period. The dynamics of L showed a quick increase after heavy rainfall and a steady decrease during periods of low rainfall. Leaf litter production was correlated to changes in L, both during periods of decreasing L (when no leaf growth occurred) and during periods of increasing L (active shedding of old foliage when new leaf growth occurred). Leaf lifespan, estimated from mean L and total annual litter production, was up to 2 months longer under elevated [CO₂] (1.18 vs. 1.01 years; P = 0.05). Our main finding that L was not responsive to elevated CO₂ is consistent with other forest FACE studies, but contrasts with the positive response of L commonly predicted by many ecosystem models.     

Modeling of product removal during enzymatic conversions by using affinity molecules

The feasibility of using magnetic particles for in-line product isolation during enzymatic conversion was studied. A comparison was made between a process based on magnetic particles and a conventional adsorption column. The enzymatic reaction was described by two consecutive first-order reactions (synthesis and subsequent hydrolysis), while the adsorption of substrate and product was described by multicomponent Langmuir isotherms. The yield as well as synthesis/hydrolysis ratio were calculated for various system characteristics. The results show that magnetic particles are very effective when the affinity with the particles is specific and for enzymatic conversions involving low ratios of the rate of synthesis versus the rate of hydrolysis. For slow conversions and for low specific affinity molecules column separations are more appropriate.     

Auxin-induced Fruit Set in Capsicum annuum L. Requires Downstream Gibberellin Biosynthesis

A hierarchical scheme for the central role of the plant hormones auxin and gibberellins in fruit set and development has been established for the model plants Arabidopsis and tomato. In the fruit crop Capsicum annuum, the importance of auxin as an early signal in fruit set has also been recognized; however, the effect of gibberellins and their interaction with auxin has not yet been studied. The aim of this study was to determine the role of gibberellin and the hierarchy between auxin and gibberellin. We applied gibberellin alone or in combination with auxin or with the gibberellin biosynthesis inhibitor paclobutrazol on stigmas of emasculated flowers. Gibberellin application enhanced fruit set, whereas application of paclobutrazol reduced fruit set. The effect of paclobutrazol treatment could be counteracted by coapplication of gibberellin but not by auxin_. These results indicate that in C. annuum, like in Arabidopsis and tomato, auxin is the major inducer of fruit set that acts in part by inducing gibberellin biosynthesis. Interestingly, gibberellin does not significantly contribute to the final fruit size but seems to play an important role in preventing flower and fruit abscission, a major determinant of production loss in C. annuum. At the same time, gibberellin together with auxin seems to balance cell division and cell expansion during fruit growth.     

A kinetic model to explain the maximum in alpha-amylase activity measurements in the presence of small carbohydrates

The effect of the presence of several small carbohydrates on the measurement of the alpha-amylase activity was determined over a broad concentration range. At low carbohydrate concentrations, a distinct maximum in the alpha-amylase activity versus concentration curves was observed in several cases. At higher concentrations, all carbohydrates show a decreasing alpha-amylase activity at increasing carbohydrate concentrations. A general kinetic model has been developed that can be used to describe and explain these phenomena. This model is based on the formation of a carbohydrate-enzyme complex that remains active. It is assumed that this complex is formed when a carbohydrate binds to alpha-amylase without blocking the catalytic site and its surrounding subsites. Furthermore, the kinetic model incorporates substrate inhibition and substrate competition. Depending on the carbohydrate type and concentration, the measured alpha-amylase activity can be 75% lower than the actual alpha-amylase activity. The model that has been developed can be used to correct for these effects in order to obtain the actual amount of active enzyme.     

Analysing Population Numbers of the House Sparrow in the Netherlands With a Matrix Model and Suggestions for Conservation Measures

The House Sparrow (Passer domesticus), formerly a common bird species, has shown a rapid decline in Western Europe over recent decades. In The Netherlands, its decline is apparent from 1990 onwards. Many causes for this decline have been suggested that all decrease the vital rates, i.e. survival and reproduction, but their actual impact remains unknown. Although the House Sparrow has been dominant in The Netherlands, data on life history characteristics for this bird species are scarce: data on reproduction are non-existent, and here we first present survival estimates based on live encounters and dead recoveries of marked individuals over the period 1976–2003, 14 years before and 14 years during the decline, reported to the Dutch Ringing Centre. We show that there is an indication that both juvenile and adult survival are lower during the period of decline. Secondly, to be able to analyse the relative impact of changes in the vital rates, we formulated a general matrix model based on a range of survival values between zero and one with a step size of 0.01 (both juvenile and adult yearly survival) and a range of realistic reproduction values (one, three or five fledglings per pair per year). With the matrix model, we calculated the finite rate of population change (λ) and applied elasticity analysis. To diagnose the cause of the decline in the Dutch House Sparrow, we parameterised the model with estimates of survival values before and during the decline and present the resulting λ. With the survival estimates from the declining period, λ < 1 only if reproduction is relatively low. We discuss this result within the light of available literature data on survival in the House Sparrow. Finally, we evaluate which of the suggested causes of population decline should be reversed to mitigate the decline and how this can be achieved.