Top-down impact of bacterivorous nematodes on the bacterial community structure: a microcosm study

The influence of bacterivorous nematodes (Diplolaimelloides meyli, Diplolaimelloides oschei, Diplolaimella dievengatensis, Panagrolaimus paetzoldi) on the development of a bacterial community growing on decaying cordgrass detritus was studied in laboratory microcosm experiments. Cordgrass leaves were incubated on a sediment surface with a natural bacterial mixture containing bacteria from sediment, cordgrass detritus and habitat water. The four nematode species were applied separately to the microcosms; controls remained without nematodes. Samples were taken seven times over a 65-day period. The bacterial community structure was analysed by means of DGGE of the 16S rRNA genes. Multi Dimensional Scaling showed grouping of the samples per treatment. Analysis of Similarities indicated that the differences between treatments were significantly larger than differences within treatments. Our results suggest that nematodes can have a significant structuring top-down influence on the 'pool' of bacteria growing on the detritus, even at low densities. Dissimilarities were similar between all treatments. Differences in bacterial community composition within the treatments with monhysterids (D. meyli, D. oschei and D. dievengatensis) can be explained by species-specific food preferences. Panagrolaimus paetzoldi on the other hand feeds unselectively, and thus affects the bacterial community differently. A top-down effect of the nematodes on the diversity of the bacterial community was only evident under high grazing pressure, i.e. in the presence of P. paetzoldi.     

A Novel TRAF6 binding site in MALT1 defines distinct mechanisms of NF-kappaB activation by API2middle dotMALT1 fusions

The recurrent translocation t(11;18)(q21;q21) associated with mucosa-associated lymphoid tissue (MALT) lymphoma results in the expression of an API2.MALT1 fusion protein that constitutively activates NF-kappaB. The first baculovirus IAP repeat (BIR) domain of API2 and the C terminus of MALT1, which contains its caspase-like domain, are present in all reported fusion variants and interact with TRAF2 and TRAF6, respectively, suggesting their contribution to NF-kappaB signaling by API2.MALT1. Also, the involvement of BCL10 has been suggested via binding to BIR1 of API2 and via its interaction with the immunoglobulin domains of MALT1, present in half of the fusion variants. However, conflicting reports exist concerning their roles in API2.MALT1-induced NF-kappaB signaling. In this report, streptavidin pulldowns of biotinylated API2.MALT1 fusion variants showed that none of the fusion variants interacted with endogenous BCL10; its role in NF-kappaB signaling by API2.MALT1 was further questioned by RNA interference experiments. In contrast, TRAF6 was essential for NF-kappaB activation by all fusion variants, and we identified a novel TRAF6 binding site in the second immunoglobulin domain of MALT1, which enhanced NF-kappaB activation when present in the fusion protein. Furthermore, inclusion of both immunoglobulin domains in API2.MALT1 further enhanced NF-kappaB signaling via intramolecular TRAF6 activation. Finally, binding of TRAF2 to BIR1 contributed to NF-kappaB activation by API2.MALT1, although additional mechanisms involving BIR1-mediated raft association are also important. Taken together, these data reveal distinct mechanisms of NF-kappaB activation by the different API2.MALT1 fusion variants with an essential role for TRAF6.     

Nod factor perception during infection thread growth fine-tunes nodulation

Bacterial nodulation factors (NFs) are essential signaling molecules for the initiation of a nitrogen-fixing symbiosis in legumes. NFs are perceived by the plant and trigger both local and distant responses, such as curling of root hairs and cortical cell divisions. In addition to their requirement at the start, NFs are produced by bacteria that reside within infection threads. To analyze the role of NFs at later infection stages, several phases of nodulation were studied by detailed light and electron microscopy after coinoculation of adventitious root primordia of Sesbania rostrata with a mixture of Azorhizobium caulinodans mutants ORS571-V44 and ORS571-X15. These mutants are deficient in NF production or surface polysaccharide synthesis, respectively, but they can complement each other, resulting in functional nodules occupied by ORS571-V44. The lack of NFs within the infection threads was confirmed by the absence of expression of an early NF-induced marker, leghemoglobin 6 of S. rostrata. NF production within the infection threads is shown to be necessary for proper infection thread growth and for synchronization of nodule formation with bacterial invasion. However, local production of NFs by bacteria that are taken up by the plant cells at the stage of bacteroid formation is not required for correct symbiosome development.     

The preclinical pharmacology of the high affinity anti-IL-6R Nanobody? ALX-0061 supports its clinical development in rheumatoid arthritis

IntroductionThe pleiotropic cytokine interleukin-6 (IL-6) plays an important role in the pathogenesis of different diseases, including rheumatoid arthritis (RA). ALX-0061 is a bispecific Nanobody® with a high affinity and potency for IL-6 receptor (IL-6R), combined with an extended half-life by targeting human serum albumin. We describe here the relevant aspects of its in vitro and in vivo pharmacology.MethodsALX-0061 is composed of an affinity-matured IL-6R-targeting domain fused to an albumin-binding domain representing a minimized two-domain structure. A panel of different in vitro assays was used to characterize the biological activities of ALX-0061. The pharmacological properties of ALX-0061 were examined in cynomolgus monkeys, using plasma levels of total soluble (s)IL-6R as pharmacodynamic marker. Therapeutic effect was evaluated in a human IL-6-induced acute phase response model in the same species, and in a collagen-induced arthritis (CIA) model in rhesus monkeys, using tocilizumab as positive control.ResultsALX-0061 was designed to confer the desired pharmacological properties. A 200-fold increase of target affinity was obtained through affinity maturation of the parental domain. The high affinity for sIL-6R (0.19 pM) translated to a concentration-dependent and complete neutralization of sIL-6R in vitro. In cynomolgus monkeys, ALX-0061 showed a dose-dependent and complete inhibition of hIL-6-induced inflammatory parameters, including plasma levels of C-reactive protein (CRP), fibrinogen and platelets. An apparent plasma half-life of 6.6 days was observed after a single intravenous administration of 10 mg/kg ALX-0061 in cynomolgus monkeys, similar to the estimated expected half-life of serum albumin. ALX-0061 and tocilizumab demonstrated a marked decrease in serum CRP levels in a non-human primate CIA model. Clinical effect was confirmed in animals with active drug exposure throughout the study duration.ConclusionsALX-0061 represents a minimized bispecific biotherapeutic of 26 kDa, nearly six times smaller than monoclonal antibodies. High in vitro affinity and potency was demonstrated. Albumin binding as a half-life extension technology resulted in describable and expected pharmacokinetics. Strong IL-6R engagement was shown to translate to in vivo effect in non-human primates, demonstrated via biomarker deregulation as well as clinical effect. Presented results on preclinical pharmacological properties of ALX-0061 are supportive of clinical development in RA.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-015-0651-0) contains supplementary material, which is available to authorized users.     

Efficient secretory expression of functional barley limit dextrinase inhibitor by high cell-density fermentation of Pichia pastoris

The limit dextrinase inhibitor (LDI) from barley seeds acts specifically on limit dextrinase (LD), an endogenous starch debranching enzyme. LDI is a 14 kDa hydrophobic protein containing four disulfide bonds and one unpaired thiol group previously found to be either glutathionylated or cysteinylated. It is a member of the so-called CM-protein family that includes α-amylase and serine protease inhibitors, which have been extremely challenging to produce recombinantly in functional form and in good yields. Here, LDI is produced in very high yields by secretory expression by Pichia pastoris applying high cell-density fermentation in a 5L fed-batch bioreactor. Thus about 200mg of LDI, which showed twofold higher inhibitory activity towards LD than LDI from barley seeds, was purified from 1L of culture supernatant by His-tag affinity chromatography and gel filtration. Electrospray ionization mass spectrometry verified the identity of the produced glutathionylated LDI-His(6). At a 1:1M ratio the recombinant LDI completely inhibited hydrolysis of pullulan catalyzed by 5-10 nM LD. LDI retained stability in the pH 2-12 range and at pH 6.5 displayed a half-life of 53 and 33 min at 90 and 93°C, respectively. The efficient heterologous production of LDI suggests secretory expression by P. pastoris to be a promising strategy to obtain other recombinant CM-proteins.     

Dynamic Changes in ANGUSTIFOLIA3 Complex Composition Reveal a Growth Regulatory Mechanism in the Maize Leaf

Most molecular processes during plant development occur with a particular spatio-temporal specificity. Thus far, it has remained technically challenging to capture dynamic protein-protein interactions within a growing organ, where the interplay between cell division and cell expansion is instrumental. Here, we combined high-resolution sampling of the growing maize (Zea mays) leaf with tandem affinity purification followed by mass spectrometry. Our results indicate that the growth-regulating SWI/SNF chromatin remodeling complex associated with ANGUSTIFOLIA3 (AN3) was conserved within growing organs and between dicots and monocots. Moreover, we were able to demonstrate the dynamics of the AN3-interacting proteins within the growing leaf, since copurified GROWTH-REGULATING FACTORs (GRFs) varied throughout the growing leaf. Indeed, GRF1, GRF6, GRF7, GRF12, GRF15, and GRF17 were significantly enriched in the division zone of the growing leaf, while GRF4 and GRF10 levels were comparable between division zone and expansion zone in the growing leaf. These dynamics were also reflected at the mRNA and protein levels, indicating tight developmental regulation of the AN3-associated chromatin remodeling complex. In addition, the phenotypes of maize plants overexpressing miRNA396a-resistant GRF1 support a model proposing that distinct associations of the chromatin remodeling complex with specific GRFs tightly regulate the transition between cell division and cell expansion. Together, our data demonstrate that advancing from static to dynamic protein-protein interaction analysis in a growing organ adds insights in how developmental switches are regulated.     

Strigolactones fine-tune the root system

Strigolactones were originally discovered to be involved in parasitic weed germination, in mycorrhizal association and in the control of shoot architecture. Despite their clear role in rhizosphere signaling, comparatively less attention has been given to the belowground function of strigolactones on plant development. However, research has revealed that strigolactones play a key role in the regulation of the root system including adventitious roots, primary root length, lateral roots, root hairs and nodulation. Here, we review the recent progress regarding strigolactone regulation of the root system and the antagonism and interplay with other hormones.     

Projected climate and land use change alter western blacklegged tick phenology,seasonal host‐seeking suitability and human encounter risk in California

Global environmental change is having profound effects on the ecology of infectious disease systems, which are widely anticipated to become more pronounced under future climate and land use change. Arthropod vectors of disease are particularly sensitive to changes in abiotic conditions such as temperature and moisture availability. Recent research has focused on shifting environmental suitability for, and geographic distribution of, vector species under projected climate change scenarios. However, shifts in seasonal activity patterns, or phenology, may also have dramatic consequences for human exposure risk, local vector abundance and pathogen transmission dynamics. Moreover, changes in land use are likely to alter human–vector contact rates in ways that models of changing climate suitability are unlikely to capture. Here we used climate and land use projections for California coupled with seasonal species distribution models to explore the response of the western blacklegged tick (Ixodes pacificus), the primary Lyme disease vector in western North America, to projected climate and land use change. Specifically, we investigated how environmental suitability for tick host‐seeking changes seasonally, how the magnitude and direction of changing seasonal suitability differs regionally across California, and how land use change shifts human tick‐encounter risk across the state. We found vector responses to changing climate and land use vary regionally within California under different future scenarios. Under a hotter, drier scenario and more extreme land use change, the duration and extent of seasonal host‐seeking activity increases in northern California, but declines in the south. In contrast, under a hotter, wetter scenario seasonal host‐seeking declines in northern California, but increases in the south. Notably, regardless of future scenario, projected increases in developed land adjacent to current human population centers substantially increase potential human–vector encounter risk across the state. These results highlight regional variability and potential nonlinearity in the response of disease vectors to environmental change.     

Influence of consumption of a high-protein vs. high-carbohydrate meal on the physiological cortisol and psychological mood response in men and women

Consumption of meals with different macronutrient contents, especially high in carbohydrates, may influence the stress-induced physiological and psychological response. The objective of this study was to investigate effects of consumption of a high-protein vs. high-carbohydrate meal on the physiological cortisol response and psychological mood response. Subjects (n = 38, 19 m/19f, age =25 ± 9 yrs, BMI = 25.0 ± 3.3 kg/m2) came to the university four times, fasted, for either condition: rest-protein, stress-protein, rest-carbohydrate, stress-carbohydrate (randomized cross-over design). Stress was induced by means of a psychological computer-test. The test-meal was either a high-protein meal (En% P/C/F 65/5/30) or a high-carbohydrate meal (En% P/C/F 6/64/30), both meals were matched for energy density (4 kJ/g) and daily energy requirements (30%). Per test-session salivary cortisol levels, appetite profile, mood state and level of anxiety were measured. High hunger, low satiety (81 ± 16, 12 ± 15 mm VAS) confirmed the fasted state. The stress condition was confirmed by increased feelings of depression, tension, anger, anxiety (AUC stress vs. rest p < 0.02). Consumption of the high-protein vs. high-carbohydrate meal did not affect feelings of depression, tension, anger, anxiety. Cortisol levels did not differ between the four test-sessions in men and women (AUC nmol·min/L p > 0.1). Consumption of the test-meals increased cortisol levels in men in all conditions (p < 0.01), and in women in the rest-protein and stress-protein condition (p < 0.03). Men showed higher cortisol levels than women (AUC nmol·min/L p < 0.0001). Consumption of meals with different macronutrient contents, i.e. high-protein vs. high-carbohydrate, does not influence the physiological and psychological response differentially. Men show a higher meal-induced salivary cortisol response compared with women.