Host–microbe interactions as a driver of acclimation to salinity gradients in brown algal cultures

Like most eukaryotes, brown algae live in association with bacterial communities that frequently have beneficial effects on their development. Ectocarpus is a genus of small filamentous brown algae, which comprises a strain that has recently colonized freshwater, a rare transition in this lineage. We generated an inventory of bacteria in Ectocarpus cultures and examined the effect they have on acclimation to an environmental change, that is, the transition from seawater to freshwater medium. Our results demonstrate that Ectocarpus depends on bacteria for this transition: cultures that have been deprived of their associated microbiome do not survive a transfer to freshwater, but restoring their microflora also restores the capacity to acclimate to this change. Furthermore, the transition between the two culture media strongly affects the bacterial community composition. Examining a range of other closely related algal strains, we observed that the presence of two bacterial operational taxonomic units correlated significantly with an increase in low salinity tolerance of the algal culture. Despite differences in the community composition, no indications were found for functional differences in the bacterial metagenomes predicted to be associated with algae in the salinities tested, suggesting functional redundancy in the associated bacterial community. Our study provides an example of how microbial communities may impact the acclimation and physiological response of algae to different environments, and thus possibly act as facilitators of speciation. It paves the way for functional examinations of the underlying host–microbe interactions, both in controlled laboratory and natural conditions.     

Detection of Anti-Pentraxin-3 Autoantibodies in ANCA-Associated Vasculitis

Objectives

Pentraxin 3 (PTX3), in common with myeloperoxidase and proteinase 3, is stored in human neutrophil granules and is expressed on apoptotic neutrophil surface. We therefore investigated the presence of anti-PTX3 autoantibodies (aAbs) in the sera of antineutrophil cytoplasmic antibodies (ANCA)-associated vasculitis (AAV) patients.

Methods

Presence of anti-PTX3 autoantibodies was analysed by a specific enzyme-linked immunosorbent assay in sera from 150 patients with microscopic polyangiitis (MPA), granulomatosis with polyangiitis (GPA), and eosinophilic granulomatosis with polyangiitis (EGPA), and in sera of 227 healthy subjects (HS), 40 systemic sclerosis (SSc) patients, and 25 giant cell arteritis patients (GCA). Using indirect immunofluorescence on fixed human neutrophils, we also analyzed the staining pattern associated with the presence of anti-PTX3 aAbs.

Results

Anti-PTX3 aAbs were detected in 56 of 150 (37.3%) of the AAV patients (versus 12 of 227 (5.3%) of HS, p<0.001) and, interestingly, in 7 of 14 MPO and PR3 ANCA negative AAV patients. Moreover, by indirect immunofluorescence on fixed neutrophils, anti-PTX3 aAbs gave rise to a specific cytoplasmic fluorescence pattern distinct from the classical cytoplasmic (c-ANCA), perinuclear (p-ANCA), and atypical (a-ANCA) pattern. Anti-PTX3 aAbs levels were higher in patients with active AAV as compared to patients with inactive disease.

Conclusion

Our work suggests that PTX3 is as a novel ANCA antigen. Anti-PTX3 aAbs appear thus as a promising novel biomarker in the diagnosis of AAV, including in patients without detectable MPO and PR3 ANCA.     

External K+ modulates the activity of the Arabidopsis potassium channel SKOR via an unusual mechanism

Plant outward-rectifying K+ channels mediate K+ efflux from guard cells during stomatal closure and from root cells into the xylem for root-shoot allocation of potassium (K). Intriguingly, the gating of these channels depends on the extracellular K+ concentration, although the ions carrying the current are derived from inside the cell. This K+ dependence confers a sensitivity to the extracellular K+ concentration ([K+]) that ensures that the channels mediate K+ efflux only, regardless of the [K+] prevailing outside. We investigated the mechanism of K+-dependent gating of the K+ channel SKOR of Arabidopsis by site-directed mutagenesis. Mutations affecting the intrinsic K+ dependence of gating were found to cluster in the pore and within the sixth transmembrane helix (S6), identifying an 'S6 gating domain' deep within the membrane. Mapping the SKOR sequence to the crystal structure of the voltage-dependent K+ channel KvAP from Aeropyrum pernix suggested interaction between the S6 gating domain and the base of the pore helix, a prediction supported by mutations at this site. These results offer a unique insight into the molecular basis for a physiologically important K+-sensory process in plants.     

Hierarchy of factors affecting behavioural signs used for oestrus detection of Holstein and Normande dairy cows in a seasonal calving system

As oestrous expression of dairy cows has decreased over the last decades oestrus detection has become more difficult. The objective of this study is to identify the main factors that affect oestrus detection in seasonal calving dairy cows, and to establish their relative importance. In each of 5 years 36 Normande and 36 Holstein cows were assigned to a Low or High winter-feeding level group. Half of each group was then assigned to a Low or High pasture-feeding group. The Low-Low strategy resulted in the lowest milk yield and the greatest body condition (BC) loss from calving to nadir BC score (6302 kg; -0.98 unit). The High-High strategy had the converse effect (7549 kg; -0.75 units). Low-High and High-Low strategies had intermediate values. The Normande cows had lower milk yield and BC loss than Holstein cows (6153 kg versus 7620 kg; -0.82 unit versus -1.20 unit). A database of 415 observed spontaneous oestruses was created. Oestruses were classified according to detection signs: (1) standing to be mounted, (2) mounting without standing, (3) other signs without standing or mounting (slight signs). Presence of another cow in oestrus, access to pasture, Normande breed and Low-Low strategy increased standing detection. In the Normande breed, 97% of oestruses were detected by standing while combining the presence of a herdmate in oestrus and access to pasture with a milk production of less than 6550 kg. Holstein cows had a higher frequency of slight signs oestruses than Normande ones, which was associated with a decreased subsequent calving rate (P<0.05). In multiparous Holstein cows, the odds of slight signs detection was multiplied by 7.8 for the High-High group in comparison with the Low-Low group (P<0.05). In our study milk yield had an effect on oestrus detection which was not explained by BC loss. As High-High cows produced more milk than others, we logically found that an increase in milk yield increased slight signs detection. Conversely, as they lost less BC than others, BC loss improved the chance of standing or mounting detection. These two results show that an increase in milk yield may reduce oestrous behaviour even if BC loss is moderate. Oestrus detection is crucial in seasonal compact calving systems. High phenotypic milk yields appear unsuitable with such systems in regard to depressed oestrous behaviour.     

Neuroanatomical study of the A11 diencephalospinal pathway in the non-human primate

Background

The A11 diencephalospinal pathway is crucial for sensorimotor integration and pain control at the spinal cord level. When disrupted, it is thought to be involved in numerous painful conditions such as restless legs syndrome and migraine. Its anatomical organization, however, remains largely unknown in the non-human primate (NHP). We therefore characterized the anatomy of this pathway in the NHP.

Methods and Findings

In situ hybridization of spinal dopamine receptors showed that D1 receptor mRNA is absent while D2 and D5 receptor mRNAs are mainly expressed in the dorsal horn and D3 receptor mRNA in both the dorsal and ventral horns. Unilateral injections of the retrograde tracer Fluoro-Gold (FG) into the cervical spinal enlargement labeled A11 hypothalamic neurons quasi-exclusively among dopamine areas. Detailed immunohistochemical analysis suggested that these FG-labeled A11 neurons are tyrosine hydroxylase-positive but dopa-decarboxylase and dopamine transporter-negative, suggestive of a L-DOPAergic nucleus. Stereological cell count of A11 neurons revealed that this group is composed by 4002±501 neurons per side. A 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) intoxication with subsequent development of a parkinsonian syndrome produced a 50% neuronal cell loss in the A11 group.

Conclusion

The diencephalic A11 area could be the major source of L-DOPA in the NHP spinal cord, where it may play a role in the modulation of sensorimotor integration through D2 and D3 receptors either directly or indirectly via dopamine formation in spinal dopa-decarboxylase-positives cells.     

Short pyrimidine stretches containing mixed base PNAs are versatile tools to induce translation elongation arrest and truncated protein synthesis

Recently, we showed that antisense peptide nucleic acids (PNA) containing a short pyrimidine stretch (C(4)TC(3)) invade Ha-ras mRNA hairpin structures to form highly stable duplex and triplex complexes that contribute to the arrest of translation elongation. The antisense PNA targeted to codon 74 of Ha-ras was designed to bind in antiparallel configuration (the N-terminal of the PNA faces the 3'-end of target mRNA), as PNA/RNA duplexes are most stable in this configuration. In order to show that different sequences in the coding region could be targeted successfully with antisense PNAs, we extended our study to three other purine-rich targets. We show that the tridecamer PNA (targeted to codon 149) containing a CTC(3)T pyrimidine stretch forms with the complementary oligoribonucleotide (ORN) a stable (PNA)(2)/ORN triplex at neutral pH (T(m) = 50 degrees C) and arrests Ha-ras mRNA translation elongation. Interestingly, the thermal stability of triplexes formed with PNAs designed to bind to the complementary ORN in a parallel orientation (the N-terminal of the PNA faces the 5'-end of target) was higher than that formed with antiparallel oriented PNAs (T(m) = 58 degrees C). Because parallel and antiparallel PNAs form stable triplexes with target sequence, they act as translation elongation blockers. These duplex-forming and partly triplex-forming PNAs targeted to Ha-ras mRNA also arrested translation elongation at specific polypurine sites contained in the mRNA coding for HIV-integrase protein. Furthermore, the tridecamer PNA containing the C(3)TC(4) motif was more active than a bis-PNA in which the Hoogsteen recognizing strand was linked to the Watson-Crick recognizing strand by a flexible linker. Pyrimidine-rich, short PNAs that form very stable duplexes with target Ha-ras mRNA inhibit translation by a mechanism that does not involve ribosome elongation arrest, whereas PNAs forming duplex and triplex structures arrest ribosome elongation. The remarkable efficacy of the tridecamer PNAs in arresting translation elongation of HIV-1 integrase mRNA is explained by their ability to form stable triplexes at neutral pH with short purine sequences.     

Breaking conceptual locks in modelling root absorption of nutrients: reopening the thermodynamic viewpoint of ion transport across the root

BackgroundThe top-down analysis of nitrate influx isotherms through the Enzyme-Substrate interpretation has not withstood recent molecular and histochemical analyses of nitrate transporters. Indeed, at least four families of nitrate transporters operating at both high and/or low external nitrate concentrations, and which are located in series and/or parallel in the different cellular layers of the mature root, are involved in nitrate uptake. Accordingly, the top-down analysis of the root catalytic structure for ion transport from the Enzyme-Substrate interpretation of nitrate influx isotherms is inadequate. Moreover, the use of the Enzyme-Substrate velocity equation as a single reference in agronomic models is not suitable in its formalism to account for variations in N uptake under fluctuating environmental conditions. Therefore, a conceptual paradigm shift is required to improve the mechanistic modelling of N uptake in agronomic models.ScopeAn alternative formalism, the Flow-Force theory, was proposed in the 1970s to describe ion isotherms based upon biophysical ‘flows and forces’ relationships of non-equilibrium thermodynamics. This interpretation describes, with macroscopic parameters, the patterns of N uptake provided by a biological system such as roots. In contrast to the Enzyme-Substrate interpretation, this approach does not claim to represent molecular characteristics. Here it is shown that it is possible to combine the Flow-Force formalism with polynomial responses of nitrate influx rate induced by climatic and in planta factors in relation to nitrate availability.ConclusionsApplication of the Flow-Force formalism allows nitrate uptake to be modelled in a more realistic manner, and allows scaling-up in time and space of the regulation of nitrate uptake across the plant growth cycle.     

High frequency stimulation of the entopeduncular nucleus has no effect on striatal dopaminergic transmission

High frequency stimulation (HFS) of the subthalamic nucleus (STN) is thought to be superior to stimulation of the internal pallidum (GPi) in alleviating symptoms of Parkinson's disease (PD). However, preliminary controlled studies comparing the effectiveness of both targets have not found significant differences in the improvement of parkinsonian symptoms, but have shown that STN stimulation allows a dramatic decrease in dopaminergic medication. We have previously shown that STN-HFS increases striatal extracellular dopamine (DA) metabolites, but not DA, in both naive and 6-hydroxydopamine (6-OHDA)-lesioned rats, whereas stimulation of the entopeduncular nucleus (EP), the rodent equivalent of the internal pallidum, does not affect DA or metabolite levels. Intriguingly, STN-HFS increases striatal DA release after inhibition of DA reuptake or metabolism, suggesting that this observation may have been obscured in non-drug treated animals by rapid and effective DA reuptake. Since STN-HFS further enhances DA metabolism after DA reuptake inhibition or depletion it has been proposed that STN-HFS increases both, striatal DA release and metabolism, independently. Therefore, the present study assesses the impact of EP-HFS on striatal DA release and metabolism in normal rats after inhibition of DA reuptake or metabolism, using microdialysis. In summary, our data demonstrate that, contrary to STN stimulation, EP-HFS has no effect on striatal DA release and metabolism. Thus, the present study provides a partial explanation for the reported clinical differences, and experimental evidence for differential mechanisms of action between HFS of the internal pallidum and the STN, that are most likely related to differences in functional anatomy.