An Extracellular Ion Pathway Plays a Central Role in the Cooperative Gating of a K2P K+ Channel by Extracellular pH

Proton-gated TASK-3 K⁺ channel belongs to the K_2P family of proteins that underlie the K⁺ leak setting the membrane potential in all cells. TASK-3 is under cooperative gating control by extracellular [H⁺]. Use of recently solved K_2P structures allows us to explore the molecular mechanism of TASK-3 cooperative pH gating. Tunnel-like side portals define an extracellular ion pathway to the selectivity filter. We use a combination of molecular modeling and functional assays to show that pH-sensing histidine residues and K⁺ ions mutually interact electrostatically in the confines of the extracellular ion pathway. K⁺ ions modulate the pK_a of sensing histidine side chains whose charge states in turn determine the open/closed transition of the channel pore. Cooperativity, and therefore steep dependence of TASK-3 K⁺ channel activity on extracellular pH, is dependent on an effect of the permeant ion on the channel pH_o sensors.     

S100A6 Amyloid Fibril Formation Is Calcium-modulated and Enhances Superoxide Dismutase-1 (SOD1) Aggregation

S100A6 is a small EF-hand calcium- and zinc-binding protein involved in the regulation of cell proliferation and cytoskeletal dynamics. It is overexpressed in neurodegenerative disorders and a proposed marker for Amyotrophic Lateral Sclerosis (ALS). Following recent reports of amyloid formation by S100 proteins, we investigated the aggregation properties of S100A6. Computational analysis using aggregation predictors Waltz and Zyggregator revealed increased propensity within S100A6 helices H_I and H_IV. Subsequent analysis of Thioflavin-T binding kinetics under acidic conditions elicited a very fast process with no lag phase and extensive formation of aggregates and stacked fibrils as observed by electron microscopy. Ca²⁺ exerted an inhibitory effect on the aggregation kinetics, which could be reverted upon chelation. An FT-IR investigation of the early conformational changes occurring under these conditions showed that Ca²⁺ promotes anti-parallel β-sheet conformations that repress fibrillation. At pH 7, Ca²⁺ rendered the fibril formation kinetics slower: time-resolved imaging showed that fibril formation is highly suppressed, with aggregates forming instead. In the absence of metals an extensive network of fibrils is formed. S100A6 oligomers, but not fibrils, were found to be cytotoxic, decreasing cell viability by up to 40%. This effect was not observed when the aggregates were formed in the presence of Ca²⁺. Interestingly, native S1006 seeds SOD1 aggregation, shortening its nucleation process. This suggests a cross-talk between these two proteins involved in ALS. Overall, these results put forward novel roles for S100 proteins, whose metal-modulated aggregation propensity may be a key aspect in their physiology and function.     

Distribution of fitness effects of cross-species transformation reveals potential for fast adaptive evolution

Bacterial transformation, a common mechanism of horizontal gene transfer, can speed up adaptive evolution. How its costs and benefits depend on the growth environment is poorly understood. Here, we characterize the distributions of fitness effects (DFE) of transformation in different conditions and test whether they predict in which condition transformation is beneficial. To determine the DFEs, we generate hybrid libraries between the recipient Bacillus subtilis and different donor species and measure the selection coefficient of each hybrid strain. In complex medium, the donor Bacillus vallismortis confers larger fitness effects than the more closely related donor Bacillus spizizenii. For both donors, the DFEs show strong effect beneficial transfers, indicating potential for fast adaptive evolution. While some transfers of B. vallismortis DNA show pleiotropic effects, various transfers are beneficial only under a single growth condition, indicating that the recipient can benefit from a variety of donor genes to adapt to varying growth conditions. We scrutinize the predictive value of the DFEs by laboratory evolution under different growth conditions and show that the DFEs correctly predict the condition at which transformation confers a benefit. We conclude that transformation has a strong potential for speeding up adaptation to varying environments by profiting from a gene pool shared between closely related species.Subject terms: Molecular evolution, Bacterial genetics     

In vitro selection of high affinity HspR-binding sites within the genome of Helicobacter pylori

The major chaperone genes of Helicobacter pylori are negatively regulated by HspR, a homologue of the repressor of the dnaK operon of Streptomyces coelicolor. Using an in vitro selection and amplification approach we identified two new chromosomal binding sites of the HspR protein. Both binding sites were characterized by footprinting analysis with purified HspR protein. Intriguingly, these HspR binding sites are located at the 3prime prime or minute ends of two genes coding for predicted proteins with functions unrelated to those of chaperones. This suggests that H. pylori HspR may regulate the expression of genes encoding proteins with diverse functions. Nucleotide sequence alignment of HspR-binding sites highlights conserved nucleotides extending outside the previously proposed consensus binding sequence with structural features predicting geometry of HspR binding as an oligomer.     

A second-generation diagnostic single nucleotide polymorphism (SNP)-based assay, optimized to distinguish among eight poplar (Populus L.) species and their early hybrids

Rapid identification of Populus L. species and hybrids can be achieved with relatively little effort through the use of primer extension-based single nucleotide polymorphism (SNP) genotyping assays. We present an optimized set of 36 SNP markers from 28 gene regions that diagnose eight poplar species (Populus angustifolia James, Populus balsamifera L., Populus deltoides Bartram, Populus fremontii Watson, Populus laurifolia Ledeb., Populus maximowiczii Henry, Populus nigra L., and Populus trichocarpa Torr. & Gray). A total of 700 DNA sequences from six Populus species (1–15 individuals per species) were used to construct the array. A set of flanking and probe oligonucleotides was developed and tested. The accuracy of the SNP assay was validated by genotyping 448 putatively “pure” individuals from 14 species of Populus. Overall, the SNP assay had a high success rate (97.6 %) and will prove useful for the identification of all Aigeiros Duby and Tacamahaca Spach. species and their early-generation hybrids within natural populations and breeding programs. Null alleles and intraspecific polymorphisms were detected for a few locus/species combinations in the Aigeiros and Tacamahaca sections. When we attempted to genotype aspens of the section Populus (Populus alba L., Populus grandidentata Michx., Populus tremula L., and Populus tremuloides Michx.), the success rate of the SNP array decreased by 13 %, demonstrating moderate cross-sectional transferability.     

Social Motility of African Trypanosomes Is a Property of a Distinct Life-Cycle Stage That Occurs Early in Tsetse Fly Transmission

The protozoan pathogen Trypanosoma brucei is transmitted between mammals by tsetse flies. The first compartment colonised by trypanosomes after a blood meal is the fly midgut lumen. Trypanosomes present in the lumen—designated as early procyclic forms—express the stage-specific surface glycoproteins EP and GPEET procyclin. When the trypanosomes establish a mature infection and colonise the ectoperitrophic space, GPEET is down-regulated, and EP becomes the major surface protein of late procyclic forms. A few years ago, it was discovered that procyclic form trypanosomes exhibit social motility (SoMo) when inoculated on a semi-solid surface. We demonstrate that SoMo is a feature of early procyclic forms, and that late procyclic forms are invariably SoMo-negative. In addition, we show that, apart from GPEET, other markers are differentially expressed in these two life-cycle stages, both in culture and in tsetse flies, indicating that they have different biological properties and should be considered distinct stages of the life cycle. Differentially expressed genes include two closely related adenylate cyclases, both hexokinases and calflagins. These findings link the phenomenon of SoMo in vitro to the parasite forms found during the first 4–7 days of a midgut infection. We postulate that ordered group movement on plates reflects the migration of parasites from the midgut lumen into the ectoperitrophic space within the tsetse fly. Moreover, the process can be uncoupled from colonisation of the salivary glands. Although they are the major surface proteins of procyclic forms, EP and GPEET are not essential for SoMo, nor, as shown previously, are they required for near normal colonisation of the fly midgut.     

Identification of the pgmG Gene, Encoding a Bifunctional Protein with Phosphoglucomutase and Phosphomannomutase Activities, in the Gellan Gum-Producing Strain Sphingomonas paucimobilis ATCC 31461

The pgmG gene of Sphingomonas paucimobilis ATCC 31461, the industrial gellan gum-producing strain, was cloned and sequenced. It encodes a 50,059-Da polypeptide that has phosphoglucomutase (PGM) and phosphomannomutase (PMM) activities and is 37 to 59% identical to other bifunctional proteins with PGM and PMM activities from gram-negative species, including Pseudomonas aeruginosa AlgC. Purified PgmG protein showed a marked preference for glucose-1-phosphate (G1P); the catalytic efficiency was about 50-fold higher for G1P than it was for mannose-1-phosphate (M1P). The estimated apparent K_m values for G1P and M1P were high, 0.33 and 1.27 mM, respectively. The pgmG gene allowed the recovery of alginate biosynthetic ability in a P. aeruginosa mutant with a defective algC gene. This result indicates that PgmG protein can convert mannose-6-phosphate into M1P in the initial steps of alginate biosynthesis and, together with other results, suggests that PgmG may convert glucose-6-phosphate into G1P in the gellan pathway.     

Actualización en el manejo de la degeneración macular asociada a la edad

Age-related macular degeneration is the leading cause of legal blindness in people over 50 in developed countries. It is a multifactorial disease resulting from the interaction of genetic and environmental factors, and the age is the only worldwide admitted risk factor. The socioeconomic impact of the disease reaches enormous proportions, if we take into account the high cost of the available antiangiogenic therapy, the strict schedule of medical visits that it requires, and the impairment that it gives rise to. The response to treatment and the visual outcomes improve with early management of the retinal lesions, thus the early diagnosis of the disease in its initial phases, based on self-control with an Amsler grid and with regular ophthalmologic assessments, is essential.