Spinitectus osorioi n. sp. (Nematoda: Cystidicolidae) from Chirostoma spp. (Osteichthyes: Atherinidae) in Lake Pátzcuaro, Michoacán, México.

Spinitectus osorioi (Nematoda: Cystidicolidae) is described from the freshwater atherinids Chirostoma estor and Chirostoma attenuatum from Lake Pátzcuaro in the Mesa Central, Michoacán State, México. This nematode is characterized by a conspicuous protuberance on the ventral surface of the distal end of the long spicule that distinguishes it from its congeners in North America and in the neotropics. In addition, the species can be readily distinguished from 4 of the 5 nominal species of North American freshwater Spinitectus by the absence of either a terminal barb or heel on the short spicule and from Spinitectus mexicanus by the spination. Previous records of Spinitectus carolini from Chirostoma spp. in México (Lakes Pátzcuaro and Zirahuén) refer to S. osorioi, and the species appears to be specific to Chirostoma spp. The geological history of the Mesa Central drainages and the historical biogeography of freshwater atherinids in this region suggest that the origin of S. osorioi may be associated with either the marine history of their hosts or with host-switching from more distantly related freshwater hosts after colonization of freshwater environments by atherinids.     

Dose-finding based on feasibility and toxicity in T-cell infusion trials

A new modality for treatment of cancer involves the ex vivo growth of cancer-specific T-cells for subsequent infusion into the patient. The therapeutic aim is selective destruction of cancer cells by the activated infused cells. An important problem in the early phase of developing such a treatment is to determine a maximal tolerated dose (MTD) for use in a subsequent phase II clinical trial. Dose may be quantified by the number of cells infused per unit body weight, and determination of an MTD may be based on the probability of infusional toxicity as a function of dose. As in a phase I trial of a new chemotherapeutic agent, this may be done by treating successive cohorts of patients at different dose levels, with each new level chosen adaptively based on the toxicity data of the patients previously treated. Such a dose-finding strategy is inadequate in T-cell infusion trials because the number of cells grown ex vivo for a given patient may be insufficient for infusing the patient at the current targeted dose. To address this problem, we propose an algorithm for trial conduct that determines a feasible MTD based on the probabilities of both infusibility and toxicity as functions of dose. The method is illustrated by application to a dendritic cell activated lymphocyte infusion trial in the treatment of acute leukemia. A simulation study indicates that the proposed methodology is both safe and reliable.     

Recanalization of chronic total coronary occlusions: the impact of a new specific guidewire on primary success rate

BACKGROUND: Although chronic total occlusions are encountered frequently in patients with coronary artery disease, an effective strategy to deal with them has yet to be devised. Various new guidewires have been designed in an attempt to negotiate chronic occlusions successfully. The authors have analysed the impact of the Athlete guidewire on procedural success in this lesion subset. METHODS: Sixty-two consecutive patients undergoing percutaneous intervention for chronic total occlusions over a two-year period were retrospectively studied. For the initial attempt, conventional guidewires were used. In case of failure, further attempts were made using the Athlete guidewire. Procedural success rates with the use of conventional and Athlete guidewires were assessed. RESULTS: Failure of the first attempt with the conventional guidewire occurred in 32 (51.6%) patients and success was achieved in 30 (48.4%) patients. In the former patients, a second attempt was made using the Athlete guidewire to cross the occlusion. The second attempt was successful in 20 patients (60%) in whom the first attempt was unsuccessful, while in the remaining 12 (40%) patients the occlusion could not be crossed even during the second attempt and the procedure was then terminated. Following the use of the Athlete guidewire, the success rate increased to 62% (p < 0.001). No complication occurred during the first attempt, while one patient had a coronary perforation using the Athlete guidewire, which was managed successfully without the need for bypass surgery. CONCLUSION: The use of the Athlete guidewire is feasible and safe, and enhances the chances of successfully treating chronic total occlusions during percutaneous coronary revascularization procedures.     

The chloride channel ClC-4 co-localizes with cystic fibrosis transmembrane conductance regulator and may mediate chloride flux across the apical membrane of intestinal epithelia.

Cystic fibrosis (CF) causing mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) lead to mislocalization of CFTR protein from the brush border membrane of epithelial tissues and/or its dysfunction as a chloride channel. In initial reports, it was proposed that certain channels from the ClC family of chloride channels may provide compensatory or alternative pathways for epithelial chloride secretion in tissues from cystic fibrosis patients. In the present work, we provide the first evidence that ClC-4 protein is functionally expressed on the surface of the intestinal epithelium and hence, is appropriately localized to act as a therapeutic target in this CF-affected tissue. We show using confocal and electron microscopy that ClC-4 co-localizes with CFTR in the brush border membrane of the epithelium lining intestinal crypts in mouse and human tissues. In Caco-2 cells, a cell line thought to model human enterocytes, ClC-4 protein is expressed on the cell surface and also partially co-localizes with EEA1 and transferrin, marker molecules of early and recycling endosomes, respectively. Hence, like CFTR, ClC-4 may cycle between the plasma membrane and endosomal compartment. Furthermore, we show that ClC-4 functions as a chloride channel on the surface of these epithelial cells as antisense ClC-4 cDNA expression reduced the amplitude of endogenous chloride currents by 50%. These studies provide the first evidence that ClC-4 is endogenously expressed and may be functional in the brush border membrane of enterocytes and hence should be considered as a candidate channel to provide an alternative pathway for chloride secretion in the gastrointestinal tract of CF patients.     

Synergistic effect of TRM2/RNC1 and EXO1 in DNA double-strand break repair in Saccharomyces cerevisiae

In our recently published study, we provided in vitro as well as in vivo data demonstrating the involvement of TRM2/RNC1 in homologous recombination based repair (HRR) of DNA double strand breaks (DSBs), in support of such claims reported earlier. To further validate its role in DNA DSB processing, our present study revealed that the trm2 single mutant displays higher sensitivity to persistent induction of specific DSBs at the MAT locus by HO-endonuclease with higher sterility rate among the survivors compared to wild type (wt) or exo1 single mutants. Intriguingly, both sensitivity and sterility rate increased dramatically in trm2exo1 double mutants lacking both endo-exonucleases with a progressively increased sterility rate in trm2exo1 double mutants with short-induction periods, reaching a very high level of sterility with persistent DSB inductions. Mutation analysis of the mating type (MAT) locus among the sterile survivors with persistent HO-induction in trm2 and exo1 single mutants as well as in trm2exo1 double mutants revealed a similar small insertions and deletions events, characteristic of non-homologous end joining (NHEJ) that might have occurred due to the lack of proper processing function in these mutants. In addition, trm2ku80 and trm2rad52 double mutants also displayed significantly higher sterility with persistent DSB induction compared to ku80 and rad52 single mutants, respectively, exhibiting a mutation spectra that shifted from base substitution (in ku80 and rad52 single mutants) to small insertions and deletions in the double mutants (in trm2ku80 and trm2rad52 mutants). These data indicate a defective processing in absence of TRM2, with a synergistic effect of TRM2, and EXO1 in such processing.     

Differential distribution of pigment-protein complexes in the Thylakoid membranes of Synechocystis 6803

Thylakoids in Synechocystis 6803, though apparently uniform in appearance in ultrastructure, were found to consist of segments which were functionally dissimilar and had distinct proteomes. These thylakoid segments can be isolated from Synechocystis 6803 by successive ultracentrifugation of cell free extracts at 40,000×g (40?k segments), 90,000×g (90?k segments) and 150,000×g (150?k segments). Electron microscopy showed differences in their appearance. 40?k segments looked feathery and fluffy, whereas the 90?k and 150?k thylakoid membrane segments appeared tiny and less fluffy. The absorption spectra showed heterogeneous distribution of pigment-protein complexes in the three types of segments. The photochemical activities of Photosystem I (PSI) and Photosystem II (PSII) showed unequal distributions in 40?k, 90?k and 150?k segments which were substantiated with low temperature fluorescence measurements. The ratio of PSII/PSI fluorescence emission at 77?K (λ(ex)?=?435?nm) was highest in 150?k segments indicating higher PSII per unit PSI in these segments. The chlorophyll fluorescence lifetimes in the membranes, determined with a time-correlated single-photon counting technique, could be resolved in three components: τ(1) (=)?<40?ps, τ(2) (=)?425-900?ps and τ(3) (=)?2.4-3.2?ns. The percentage contribution of the fastest component (τ(1)) decreased in the order 40?k?>?90?k?>?150?k segments whereas that of the other two components showed a reversed trend. These studies indicated differential distribution of pigment-protein complexes in the three membrane segments suggesting heterogeneity in the thylakoids of Synechocystis 6803.     

Structural analysis of a peptide fragment of transmembrane transporter protein bilitranslocase

Using a combination of genomic and post-genomic approaches is rapidly altering the number of identified human influx carriers. A transmembrane protein bilitranslocase (TCDB 2.A.65) has long attracted attention because of its function as an organic anion carrier. It has also been identified as a potential membrane transporter for cellular uptake of several drugs and due to its implication in drug uptake, it is extremely important to advance the knowledge about its structure. However, at present, only the primary structure of bilitranslocase is known. In our work, transmembrane subunits of bilitranslocase were predicted by a previously developed chemometrics model and the stability of these polypeptide chains were studied by molecular dynamics (MD) simulation. Furthermore, sodium dodecyl sulfate (SDS) micelles were used as a model of cell membrane and herein we present a high-resolution 3D structure of an 18 amino acid residues long peptide corresponding to the third transmembrane part of bilitranslocase obtained by use of multidimensional NMR spectroscopy. It has been experimentally confirmed that one of the transmembrane segments of bilitranslocase has alpha helical structure with hydrophilic amino acid residues oriented towards one side, thus capable of forming a channel in the membrane.     

Secretion of Soluble Vascular Endothelial Growth Factor Receptor 1 (sVEGFR1/sFlt1) Requires Arf1, Arf6, and Rab11 GTPases

The soluble form of vascular endothelial growth factor receptor 1 (sVEGFR-1/sFlt1) is generated by alternative splicing of the FLT1 gene. Secretion of sFlt1 from endothelial cells plays an important role in blood vessel sprouting and morphogenesis. However, excess sFlt1 secretion is associated with diseases such as preeclampsia and chronic kidney disease. To date, the secretory transport process involved in the secretion of sFlt1 is poorly understood. In the present study, we investigated the itinerary of sFlt1 trafficking along the secretory pathway. To understand the timecourse of sFlt1 secretion, endothelial cells stably expressing sFlt1 were metabolically radiolabeled with [(35)S]-methionine and cysteine. Our results indicate that after initial synthesis the levels of secreted [(35)S]-sFlt1 in the extracellular medium peaks at 8 hours. Treatment with brefeldin A (BFA), a drug which blocks trafficking between the endoplasmic reticulum (ER) and the Golgi complex, inhibited extracellular release of sFlt1 suggesting that ER to Golgi and intra-Golgi trafficking of sFlt1 are essential for its secretion. Furthermore, we show that ectopic expression of dominant-negative mutant forms of Arf1, Arf6, and Rab11 as well as siRNA-mediated knockdown of these GTPases block secretion of sFlt1 during normoxic and hypoxic conditions suggesting role for these small GTPases. This work is the first to report role of regulatory proteins involved in sFlt1 trafficking along the secretory pathway and may provide insights and new molecular targets for the modulation of sFlt-1 release during physiological and pathological conditions.     

Hydrogen sulfide inhibits high glucose-induced matrix protein synthesis by activating AMP-activated protein kinase in renal epithelial cells

Hydrogen sulfide, a signaling gas, affects several cell functions. We hypothesized that hydrogen sulfide modulates high glucose (30 mm) stimulation of matrix protein synthesis in glomerular epithelial cells. High glucose stimulation of global protein synthesis, cellular hypertrophy, and matrix laminin and type IV collagen content was inhibited by sodium hydrosulfide (NaHS), an H(2)S donor. High glucose activation of mammalian target of rapamycin (mTOR) complex 1 (mTORC1), shown by phosphorylation of p70S6 kinase and 4E-BP1, was inhibited by NaHS. High glucose stimulated mTORC1 to promote key events in the initiation and elongation phases of mRNA translation: binding of eIF4A to eIF4G, reduction in PDCD4 expression and inhibition of its binding to eIF4A, eEF2 kinase phosphorylation, and dephosphorylation of eEF2; these events were inhibited by NaHS. The role of AMP-activated protein kinase (AMPK), an inhibitor of protein synthesis, was examined. NaHS dose-dependently stimulated AMPK phosphorylation and restored AMPK phosphorylation reduced by high glucose. Compound C, an AMPK inhibitor, abolished NaHS modulation of high glucose effect on events in mRNA translation as well as global and matrix protein synthesis. NaHS induction of AMPK phosphorylation was inhibited by siRNA for calmodulin kinase kinase β, but not LKB1, upstream kinases for AMPK; STO-609, a calmodulin kinase kinase β inhibitor, had the same effect. Renal cortical content of cystathionine β-synthase and cystathionine γ-lyase, hydrogen sulfide-generating enzymes, was significantly reduced in mice with type 1 diabetes or type 2 diabetes, coinciding with renal hypertrophy and matrix accumulation. Hydrogen sulfide is a newly identified modulator of protein synthesis in the kidney, and reduction in its generation may contribute to kidney injury in diabetes.     

Estimating the global distribution of field size using crowdsourcing

There is an increasing evidence that smallholder farms contribute substantially to food production globally, yet spatially explicit data on agricultural field sizes are currently lacking. Automated field size delineation using remote sensing or the estimation of average farm size at subnational level using census data are two approaches that have been used. However, both have limitations, for example, automatic field size delineation using remote sensing has not yet been implemented at a global scale while the spatial resolution is very coarse when using census data. This paper demonstrates a unique approach to quantifying and mapping agricultural field size globally using crowdsourcing. A campaign was run in June 2017, where participants were asked to visually interpret very high resolution satellite imagery from Google Maps and Bing using the Geo‐Wiki application. During the campaign, participants collected field size data for 130 K unique locations around the globe. Using this sample, we have produced the most accurate global field size map to date and estimated the percentage of different field sizes, ranging from very small to very large, in agricultural areas at global, continental, and national levels. The results show that smallholder farms occupy up to 40% of agricultural areas globally, which means that, potentially, there are many more smallholder farms in comparison with the two different current global estimates of 12% and 24%. The global field size map and the crowdsourced data set are openly available and can be used for integrated assessment modeling, comparative studies of agricultural dynamics across different contexts, for training and validation of remote sensing field size delineation, and potential contributions to the Sustainable Development Goal of Ending hunger, achieve food security and improved nutrition and promote sustainable agriculture.