Divalent metal ion complexes of S100B in the absence and presence of pentamidine

As part of an effort to inhibit S100B, structures of pentamidine (Pnt) bound to Ca²⁺-loaded and Zn²⁺,Ca²⁺-loaded S100B were determined by X-ray crystallography at 2.15 Å (R_free = 0.266) and 1.85 Å (R_free = 0.243) resolution, respectively. These data were compared to X-ray structures solved in the absence of Pnt, including Ca²⁺-loaded S100B and Zn²⁺,Ca²⁺-loaded S100B determined here (1.88 Å; R_free = 0.267). In the presence and absence of Zn²⁺, electron density corresponding to two Pnt molecules per S100B subunit was mapped for both drug-bound structures. One Pnt binding site (site 1) was adjacent to a p53 peptide binding site on S100B (± Zn²⁺), and the second Pnt molecule was mapped to the dimer interface (site 2; ± Zn²⁺) and in a pocket near residues that define the Zn²⁺ binding site on S100B. In addition, a conformational change in S100B was observed upon the addition of Zn²⁺ to Ca²⁺-S100B, which changed the conformation and orientation of Pnt bound to sites 1 and 2 of Pnt-Zn²⁺,Ca²⁺-S100B when compared to Pnt-Ca²⁺-S100B. That Pnt can adapt to this Zn²⁺-dependent conformational change was unexpected and provides a new mode for S100B inhibition by this drug. These data will be useful for developing novel inhibitors of both Ca²⁺- and Ca²⁺,Zn²⁺-bound S100B.     

A Combination of Proteomic Approaches Identifies A Panel of Circulating Extracellular Vesicle Proteins Related to the Risk of Suffering Cardiovascular Disease in Obese Patients

Plasma‐derived extracellular vesicles (EVs) have been extensively described as putative biomarkers in different diseases. Interestingly, increased levels of EVs subpopulations are well known to associate with obesity. The goal of this study is to identify EVs‐derived biomarkers in plasma from obese patients in order to predict the development of pathological events associated with obesity. Samples are obtained from 22 obese patients and their lean‐matched controls are divided into two cohorts: one for a 2D fluorescence difference gel electrophoresis (2D‐DIGE)‐based study, and the other one for a label free LC–MS/MS‐based approach. EVs are isolated following a serial ultracentrifugation protocol. Twenty‐two and 23 differentially regulated features are detected from 2D‐DIGE and label free LC–MS/MS, respectively; most of them involve in the coagulation and complement cascades. Remarkably, there is an upregulation of complement C4, complement C3, and fibrinogen in obese patients following both approaches, the latter two also validated by 2D‐western‐blotting in an independent cohort. These results correlate with a proinflammatory and prothrombotic state of those individuals. On the other hand, a downregulation of adiponectin leading to an increased risk of suffering cardiovascular diseases has been shown. The results suggest the relevance of plasma‐derived‐EVs proteins as a source of potential biomarkers for the development of atherothrombotic events in obesity.     

Genetic selection for a highly functional cysteine-less membrane protein using site saturation mutagenesis

We describe an efficient method for generating highly functional membrane proteins with variant amino acids at defined positions that couples a modified site saturation strategy with functional genetic selection. We applied this method to the production of a cysteine-less variant of the Crithidia fasciculata inosine-guanosine permease CfNT2 to facilitate biochemical studies using thiol-specific modifying reagents. Of 10 endogenous cysteine residues in CfNT2, two cannot be replaced with serine or alanine without loss of function. High-quality single- and double-mutant libraries were produced by combining a previously reported site saturation mutagenesis scheme based on the Stratagene Quikchange method with a novel gel purification step that effectively eliminated template DNA from the products. Following selection for functional complementation in Saccharomyces cerevisiae cells auxotrophic for purines, several highly functional noncysteine substitutions were efficiently identified at each desired position, allowing the construction of cysteine-less variants of CfNT2 that retained wild-type affinity for inosine. This combination of an improved site saturation mutagenesis technique and positive genetic selection provides a simple and efficient means to identify functional and perhaps unexpected amino acid variants at a desired position.     

Altitudinal distribution limits of aquatic macroinvertebrates: an experimental test in a tropical alpine stream

1. Temperature and oxygen are recognised as the main drivers of altitudinal limits of species distributions. However, the two factors are linked, and both decrease with altitude, why their effects are difficult to disentangle. 2. This was experimentally addressed using aquatic macroinvertebrates; larvae of Andesiops (Ephemeroptera), Claudioperla, (Plecoptera), Scirtes (Coleoptera) and Anomalocosmoecus (Trichoptera), and the amphipod Hyalella in an Ecuadorian glacier‐fed stream (4100–4500 m a.s.l.). The following were performed: (i) quantitative benthic sampling at three sites to determine altitudinal patterns in population densities, (ii) transplants of the five taxa upstream of their natural altitudinal limit to test the short‐term (14 days) effect on survival, and (iii) in situ experiments of locomotory activity as a proxy for animal response to relatively small differences in temperature (5 °C vs. 10 °C) and oxygen saturation (55% vs. 62%). 3. The transplant experiment reduced survival to a varying degree among taxa, but Claudioperla survived well at a site where it did not naturally occur. In the in situ experiment, Scirtes and Hyalella decreased their activity at lower oxygen saturation, whereas Andesiops and Anomalocosmoecus did so at a low temperature. The decrease in activity from a high to a low temperature and oxygen for the five taxa was significantly correlated with their mortality in the transplant experiment. 4. Together the present experiments indicate that even relatively small differences in temperature and oxygen may produce effects explaining ecological patterns, and depending on the taxon, either water temperature or oxygen saturation, without clear interacting effects, are important drivers of altitudinal limits.     

GVC-Net: Global Vascular Context Network for Cerebrovascular Segmentation Using Sparse Labels

ObjectivesCerebrovascular disease is a serious threat to human health. Because of its high mortality and disability rate, early diagnosis and prevention are very important. The performance of existing cerebrovascular segmentation methods based on deep learning depends on the integrity of labels. However, manual labels are usually of low quality and poor connectivity at small blood vessels, which directly affects the cerebrovascular segmentation results.Material and methodIn this paper, we propose a new segmentation network to segment cerebral vessels from MRA images by using sparse labels. The long-distance dependence between vascular structures is captured by the global vascular context module, and the topology is constrained by the hybrid loss function to segment the cerebral vessels with good connectivity.ResultExperiments show that our method performed with a sensitivity, precision, dice similarity coefficient, intersection over union and centerline dice similarity coefficient of 61.24%, 75.58%, 67.66%, 51.13% and 83.79% respectively.ConclusionThe obtained results reveal that the proposed cerebrovascular segmentation network has better segmentation performance for cerebrovascular segmentation under sparse labels, and can suppress the noise of background to a certain extent.     

离子导入法和渗透促进剂并用对裸鼠皮肤角质层影响的ESR研究

通过测定５－唑烷氮氧自由基硬脂酸（５－ＤＳＡ）标记裸鼠皮肤角质层的ＥＳＲ谱,研究离子导入法与渗透促进剂１００％月桂氮酮（１００％ＡＺ）、５％月桂氨　酮／丙二醇溶液（５％　ＡＺ／ＰＧ）和１０％油酸／丙二醇溶液（１０％ＯＡ／ＰＧ）并用对裸鼠皮肤角质层的影响。离子导入法处理皮肤后,标记物序参数降低,各向同性超精细分裂偶合常数增大,表明低密度电流能够引起皮肤角质层细胞间脂质排列有序性降低,流动性增大,极性增大;离子导入法与渗透促进剂并用处理皮肤后,序参数进一步降低,各向同性超精细分裂偶合常数进一步增大,表明二者对皮肤角质层的影响具有协同作用。     

Electron paramagnetic resonance study on calmodulin: conformational change and interaction with divalent cations

Biologically active spin labelled derivatives of calmodulin were prepared and used to study CA²⁺- and Mg²⁺-induced conformational changes of the protein. The rotational correlation time of the spin labelled residues increased upon addition of divalent cations. Two calcium ions per spin labelled calmodulin were found to induce a 75% conformational change, whereas four calcium ions were necessary for a maximum conformational change. The increase in rotational correlation time induced by Mg²⁺ is less pronounced. Two different covalently attached spin labels (iodoacetamide and maleimide) were compared and marked differences were found in their chemical stability. The binding of manganese ions to calmodulin could be observed directly from the electron paramagnetic resonance spectra of these paramagnetic ions. Two specific classes of binding sites, each binding two manganese ions with $\text{k}{}_{\mathrm{<mtext>D</mtext>}}\text{= 0.6 × 10}{}^{\mathrm{?6}}\text{m}\text{and}\text{k}{}_{\mathrm{D}}\text{= 3 × 10}{}^{\mathrm{?5}}\text{m}$, respectively, were determined. Further ion binding occurs at non-specific sites.     

Soil temperature affects carbon allocation within arbuscular mycorrhizal networks and carbon transport from plant to fungus

How soil carbon balance will be affected by plant–mycorrhizal interactions under future climate scenarios remains a significant unknown in our ability to forecast ecosystem carbon storage and fluxes. We examined the effects of soil temperature (14, 20, 26 °C) on the structure and extent of a multispecies community of arbuscular mycorrhizal (AM) fungi associated with Plantago lanceolata. To isolate fungi from roots, we used a mesh‐divided pot system with separate hyphal compartments near and away from the plant. A ¹³C pulse label was then used to trace the flow of recently fixed photosynthate from plants into belowground pools and respiration. Temperature significantly altered the structure and allocation of the AM hyphal network, with a switch from more vesicles (storage) in cooled soils to more extensive extraradical hyphal networks (growth) in warmed soils. As soil temperature increased, we also observed an increase in the speed at which plant photosynthate was transferred to and respired by roots and AM fungi coupled with an increase in the amount of carbon respired per unit hyphal length. These differences were largely independent of plant size and rates of photosynthesis. In a warmer world, we would therefore expect more carbon losses to the atmosphere from AM fungal respiration, which are unlikely to be balanced by increased growth of AM fungal hyphae.     

Maximal ecological diversity exceeds evolutionary diversity in model ecosystems

Understanding community saturation is fundamental to ecological theory. While investigations of the diversity of evolutionary stable states (ESSs) are widespread, the diversity of communities that have yet to reach an evolutionary endpoint is poorly understood. We use Lotka–Volterra dynamics and trait-based competition to compare the diversity of randomly assembled communities to the diversity of the ESS. We show that, with a large enough founding diversity (whether assembled at once or through sequential invasions), the number of long-time surviving species exceeds that of the ESS. However, the excessive founding diversity required to assemble a saturated community increases rapidly with the dimension of phenotype space. Additionally, traits present in communities resulting from random assembly are more clustered in phenotype space compared to random, although still markedly less ordered than the ESS. By combining theories of random assembly and ESSs we bring a new viewpoint to both the saturation and random assembly literature.