Observing the primary steps of ion solvation in helium droplets.

Solvation is a ubiquitous phenomenon in the natural sciences. At the macroscopic level, it is well understood through thermodynamics and chemical reaction kinetics1,2. At the atomic level, the primary steps of solvation are the attraction and binding of individual molecules or atoms of a solvent to molecules or ions of a solute1. These steps have, however, never been observed in real time. Here we instantly create a single sodium ion at the surface of a liquid helium nanodroplet3,4, and measure the number of solvent atoms that successively attach to the ion as a function of time. We found that the binding dynamics of the first five helium atoms is well described by a Poissonian process with a binding rate of 2.0 atoms per picosecond. This rate is consistent with time-dependent density-functional-theory simulations of the solvation process. Furthermore, our measurements enable an estimate of the energy removed from the region around the sodium ion as a function of time, revealing that half of the total solvation energy is dissipated after four picoseconds. Our experimental method opens possibilities for benchmarking theoretical models of ion solvation and for time-resolved measurements of cation-molecule complex formation.

Cell surface nucleocapsid protein expression: A betacoronavirus immunomodulatory strategy.

We recently reported that SARS-CoV-2 nucleocapsid (N) protein is abundantly expressed on the surface of both infected and neighboring uninfected cells, where it enables activation of Fc receptor-bearing immune cells with anti-N antibodies (Abs) and inhibits leukocyte chemotaxis by binding chemokines (CHKs). Here, we extend these findings to N from the common cold human coronavirus (HCoV)-OC43, which is also robustly expressed on the surface of infected and noninfected cells by binding heparan sulfate/heparin (HS/H). HCoV-OC43 N binds with high affinity to the same set of 11 human CHKs as SARS-CoV-2 N, but also to a nonoverlapping set of six cytokines. As with SARS-CoV-2 N, HCoV-OC43 N inhibits CXCL12ß-mediated leukocyte migration in chemotaxis assays, as do all highly pathogenic and common cold HCoV N proteins. Together, our findings indicate that cell surface HCoV N plays important evolutionarily conserved roles in manipulating host innate immunity and as a target for adaptive immunity.

Quantitative analysis of the GABAergic innervation of the soma and axon initial segment of pyramidal cells in the human and mouse neocortex.

Perisomatic GABAergic innervation in the cerebral cortex is carried out mostly by basket and chandelier cells, which differentially participate in the control of pyramidal cell action potential output and synchronization. These cells establish multiple synapses with the cell body (and proximal dendrites) and the axon initial segment (AIS) of pyramidal neurons, respectively. Using multiple immunofluorescence, confocal microscopy and 3D quantification techniques, we have estimated the number and density of GABAergic boutons on the cell body and AIS of pyramidal neurons located through cortical layers of the human and mouse neocortex. The results revealed, in both species, that there is clear variability across layers regarding the density and number of perisomatic GABAergic boutons. We found a positive linear correlation between the surface area of the soma, or the AIS, and the number of GABAergic terminals in apposition to these 2 neuronal domains. Furthermore, the density of perisomatic GABAergic boutons was higher in the human cortex than in the mouse. These results suggest a selectivity for the GABAergic innervation of the cell body and AIS that might be related to the different functional attributes of the microcircuits in which neurons from different layers are involved in both human and mouse.

Microanatomical study of pyramidal neurons in the contralesional somatosensory cortex after experimental ischemic stroke.

At present, many studies support the notion that after stroke, remote regions connected to the infarcted area are also affected and may contribute to functional outcome. In the present study, we have analyzed possible microanatomical alterations in pyramidal neurons from the contralesional hemisphere after induced stroke. We performed intracellular injections of Lucifer yellow in pyramidal neurons from layer III in the somatosensory cortex of the contralesional hemisphere in an ischemic stroke mouse model. A detailed 3-dimensional analysis of the neuronal complexity and morphological alterations of dendritic spines was then performed. Our results demonstrate that pyramidal neurons from layer III in the somatosensory cortex of the contralesional hemisphere show selective changes in their dendritic arbors, namely, less dendritic complexity of the apical dendritic arbor-but no changes in the basal dendritic arbor. In addition, we found differences in spine morphology in both apical and basal dendrites comparing the contralesional hemisphere with the lesional hemisphere. Our results show that pyramidal neurons of remote areas connected to the infarct zone exhibit a series of selective changes in neuronal complexity and morphological distribution of dendritic spines, supporting the hypothesis that remote regions connected to the peri-infarcted area are also affected after stroke.

Relative Match Load in Young Professional Soccer Players during Soccer-7 and Soccer-11.

The aim of this study was to analyze the differences in internal and external load during Soccer-7 and Soccer-11, comparing positional requirements and neuromuscular fatigue in both modalities. Twenty-four young soccer players were monitored in Soccer-7 and Soccer-11 matches using global positioning systems. Total distance covered (TD), distance covered at high speed (HSR), distance covered at very high speed (VHSR), peak speed, accelerations (Acc) and decelerations (Dec) and rate of perceived exertion (RPE) were recorded differentiating between central backs (CB), midfielders (MF), external players (EX) and forwards (FW). Neuromuscular fatigue were assessed using a jump test. During Soccer-11, players showed significantly higher TD, HSR and VHSR, with low Acc and greater RPE compared with Soccer-7. During Soccer-11, all positions recorded significantly greater TD, distance at HSR and at VHSR than Soccer-7. In terms of playing position, CB, MF and FW achieved significantly higher Peak Speed during Soccer-1, but there was no difference for EX. During Soccer-7 all positions performed significantly higher numbers of Acc. Although the Soccer-7 modality is considered an optimal format for the development of young soccer players, there is a significant difference in match running activity for all playing positions with respect to the Soccer-11 format.

Influence of the Number of Players on the Load of Soccer Players During Transition Games.

The aims of this study were to determine the effect of different compositions in transition games (TGs) on the load of soccer players and to evaluate their performance in physical tests. Using a GPS system, 18 players were monitored during: 3vs2, 2vs1 and 1vs1. Distance covered (DC), DC 18-20.9 km·h-1, 21-23.9 km·h-1,>24 km·h-1, peak speed, accelerations (Acc) and decelerations (Dec)>1.0 m·s-2 and>2.5 m·s-2 and rate of perceived exertion (RPE) were recorded. Before and after each TG, countermovement-jump (CMJ), 15- (S15) and 30 m (S30) speed tests were assessed. TG3vs2 showed greater DC and Dec>1.0 m·s-2 than TG2vs1, and DC, DC 18.0-23.9 km·h-1, Acc>1.0 m·s-2 and Dec>2.5 m·s-2 than TG1vs1 (p<0.01). TG2vs1 achieved higher DC, DC 18.0-23.9 km·h-1, and Acc>2.5 m·s-2 (p<0.01) but lower peak speed (p=0.02) and RPE (p=0.02) than TG1vs1. Post-intervention, TG1vs1 showed lower CMJ and higher S15 (p=0.02), while TG3vs2, showed improvements in CMJ (p<0.01). The three tasks showed large variations for DC>24 km·h-1, Acc>1.0 m·s-2, Dec>1.0 m·s-2 and Dec>2.5 m·s-2. The load of TGs is sensitive to their player composition.

An update on vitamin D signaling and cancer.

A low vitamin D status is associated with an increased risk of various cancers, such as of colon, breast, prostate and hematological cells. The biologically most active vitamin D metabolite 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) is a high affinity ligand of the transcription factor vitamin D receptor (VDR). 1,25(OH)2D3 induces via VDR changes to the epigenome of healthy and neoplastic cells and in this way influences their transcriptome. Ligand-activated VDR binds to more than 10,000 loci within the human genome and affects the transcription of some 1000 target genes in a large proportion of human tissues and cell types. From the evolutionary perspective, the prime role of vitamin D was probably the control of energy metabolism later shifting to modulate innate and adaptive immunity as well as to regulate calcium and bone homeostasis. Since rapidly growing immune and cancer cells both use the same pathways and genes for controlling their proliferation, differentiation and apoptosis, not surprisingly, vitamin D signaling changes these processes also in neoplastic cells. Thus, anti-cancer effects of vitamin D may derive from managing growth and differentiation in immunity. This review provides an update on the molecular basis of vitamin D signaling, i.e., the effects of 1,25(OH)2D3 on the epigenome and transcriptome, and its relationship to cancer prevention and therapy.

Variations in Cell Surface ACE2 Levels Alter Direct Binding of SARS-CoV-2 Spike Protein and Viral Infectivity: Implications for Measuring Spike Protein Interactions with Animal ACE2 Orthologs.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19), the most severe pandemic in a century. The virus gains access to host cells when the viral spike protein (S-protein) binds to the host cell surface receptor angiotensin-converting enzyme 2 (ACE2). Studies have attempted to understand SARS-CoV-2 S-protein interactions with vertebrate orthologs of ACE2 by expressing ACE2 orthologs in mammalian cells and measuring viral infection or S-protein binding. Often, these cells only transiently express ACE2 proteins, and the levels of ACE2 at the cell surface are not quantified. Here, we describe a cell-based assay that uses stably transfected cells expressing ACE2 proteins in a bicistronic vector with an easy-to-quantify reporter protein, Thy1.1. We found that both the binding of the S-protein receptor-binding domain (RBD) and infection with a SARS-CoV-2 pseudovirus are proportional to the amount of human ACE2 expressed at the cell surface, which can be inferred by quantifying the level of Thy1.1. We also compared different ACE2 orthologs, which were expressed in stably transfected cells expressing equivalent levels of Thy1.1. When ranked for either viral infectivity or RBD binding, mouse ACE2 had a weak to undetectable affinity for S-protein, while human ACE2 had the highest level detected, and feline ACE2 had an intermediate phenotype. The generation of stably transfected cells whose ACE2 level can be normalized for cross-ortholog comparisons allows us to create a reusable cellular library useful for measuring emerging SARS-CoV-2 variants' abilities to potentially infect different animals. IMPORTANCE SARS-CoV-2 is a zoonotic virus responsible for the worst global pandemic in a century. An understanding of how the virus can infect other vertebrate species is important for controlling viral spread and understanding the natural history of the virus. Here, we describe a method to generate cells stably expressing different orthologs of ACE2, the receptor for SARS-CoV-2, on the surface of a human cell line. We find that both the binding of the viral spike protein receptor-binding domain (RBD) and infection of cells with a SARS-CoV-2 pseudovirus are proportional to the ACE2 levels at the cell surface. This method will allow the creation of a library of stably transfected cells expressing similar levels of different vertebrate ACE2 orthologs, which can be used repeatedly for identifying vertebrate species that may be susceptible to infection with SARS-CoV-2 and its many variants.

Herpes simplex virus 2 (HSV-2) evolves faster in cell culture than HSV-1 by generating greater genetic diversity.

Herpes simplex virus type 1 and 2 (HSV-1 and HSV-2, respectively) are prevalent human pathogens of clinical relevance that establish long-life latency in the nervous system. They have been considered, along with the Herpesviridae family, to exhibit a low level of genetic diversity during viral replication. However, the high ability shown by these viruses to rapidly evolve under different selective pressures does not correlates with that presumed genetic stability. High-throughput sequencing has revealed that heterogeneous or plaque-purified populations of both serotypes contain a broad range of genetic diversity, in terms of number and frequency of minor genetic variants, both in vivo and in vitro. This is reminiscent of the quasispecies phenomenon traditionally associated with RNA viruses. Here, by plaque-purification of two selected viral clones of each viral subtype, we reduced the high level of genetic variability found in the original viral stocks, to more genetically homogeneous populations. After having deeply characterized the genetic diversity present in the purified viral clones as a high confidence baseline, we examined the generation of de novo genetic diversity under culture conditions. We found that both serotypes gradually increased the number of de novo minor variants, as well as their frequency, in two different cell types after just five and ten passages. Remarkably, HSV-2 populations displayed a much higher raise of nonconservative de novo minor variants than the HSV-1 counterparts. Most of these minor variants exhibited a very low frequency in the population, increasing their frequency over sequential passages. These new appeared minor variants largely impacted the coding diversity of HSV-2, and we found some genes more prone to harbor higher variability. These data show that herpesviruses generate de novo genetic diversity differentially under equal in vitro culture conditions. This might have contributed to the evolutionary divergence of HSV-1 and HSV-2 adapting to different anatomical niche, boosted by selective pressures found at each epithelial and neuronal tissue.

Demographic and spatially explicit landscape genomic analyses in a tropical oak reveal the impacts of late Quaternary climate change on Andean montane forests.

The tropical Andes are one of the most important biodiversity hotspots on Earth, yet our understanding of how their biotas have responded to Quaternary climatic oscillations is extraordinarily limited and the alternative models proposed to explain their demographic dynamics have been seldom formally evaluated. Here, we test the hypothesis that the interplay between the spatial configuration of geographical barriers to dispersal and elevational displacements driven by Quaternary cooling-warming cycles has shaped the demographic trajectories of montane oak forests (Quercus humboldtii) from the Colombian Andes. Specifically, we integrate genomic data and environmental niche modelling at fine temporal resolution to test competing spatially explicit demographic and coalescent models, including scenarios considering (i) isotropic gene flow through the landscape, (ii) the hypothetical impact of contemporary barriers to dispersal (i.e., inter-Andean valleys), and (iii) distributional shifts of montane oak forests from the Last Glacial Maximum to the present. Although our data revealed a marked genetic fragmentation of montane oak forests, statistical support for isolation-with-migration models indicates that geographically separated populations from the different Andean Cordilleras regularly exchange gene flow. Accordingly, spatiotemporally explicit demographic analyses supported a model of flickering connectivity, with scenarios considering isotropic gene flow or currently unsuitable habitats as persistent barriers to dispersal providing a comparatively worse fit to empirical genomic data. Overall, these results emphasize the role of landscape heterogeneity on shaping spatial patterns of genomic variation in montane oak forests, rejecting the hypothesis of genetic continuity and supporting a significant impact of Quaternary climatic oscillations on their demographic trajectories.

Baseline Severity and Inflammation Would Influence the Effect of Simvastatin on Clinical Outcomes in Cirrhosis Patients.

BACKGROUND: Simvastatin administration to decompensated cirrhosis patients improved Child-Pugh (CP) at the end of a safety trial (EST). AIM: To evaluate whether simvastatin reduces cirrhosis severity through a secondary analysis of the safety trial. METHODS: Thirty patients CP class (CPc) CPc A (n = 6), CPc B (n = 22), and CPc C (n = 2) received simvastatin for one year. PRIMARY ENDPOINT: cirrhosis severity. Secondary endpoints: health-related quality of life (HRQoL) and hospitalizations for cirrhosis complications. RESULTS: Cirrhosis severity decreased baseline versus EST only across CP score (7.3 ± 1.3 versus 6.7 ± 1.7, P = 0.041), and CPc: 12 patients lessened from CPc B to CPc A, and three patients increased from CPc A to CPc B (P = 0.029). Due to cirrhosis severity changes and differences in clinical outcomes, 15 patients completed the trial as CPc AEST and another 15 as CPc B/C. At baseline, CPc AEST showed greater albumin and high-density lipoprotein cholesterol concentrations than CPc B/C (P = 0.036 and P = 0.028, respectively). Comparing EST versus baseline, only in CPc AEST, there was a reduction in white-cell blood (P = 0.012), neutrophils (P = 0.029), monocytes (P = 0.035), and C-reactive protein (P = 0.046); an increase in albumin (P = 0.011); and a recovery in HRQoL (P < 0.030). Finally, admissions for cirrhosis complications decreased in CPc AEST versus CPc B/C (P = 0.017). CONCLUSIONS: Simvastatin would reduce cirrhosis severity only in CPc B at baseline in a suitable protein and lipid milieu, possibly due to its anti-inflammatory effects. Furthermore, only in CPc AEST would improve HRQoL and reduce admissions by cirrhosis complications. However, as these outcomes were not primary endpoints, they require validation.

Germline heterozygous exons 8-11 pathogenic BARD1 gene deletion reported for the first time in a family with suspicion of a hereditary colorectal cancer syndrome: more than an incidental finding?

BACKGROUND: Colorectal cancer (CRC) is a highly prevalent disease in developed countries. Inherited Mendelian causes account for approximately 5% of CRC cases, with Lynch syndrome and familial adenomatous polyposis being the most prevalent forms. Scientific efforts are focused on the discovery of new candidate genes associated with CRC and new associations of phenotypes with well-established cancer-related genes. BRCA1-associated ring domain (BARD1) gene deleterious germline variants are associated with a moderate increase in the relative risk of breast cancer, but their association with other neoplasms, such as CRC, remains unclear. CASE PRESENTATION: We present the case of a 49-year-old male diagnosed with rectal adenocarcinoma whose maternal family fulfilled Amsterdam clinical criteria for Lynch syndrome. Genetic test confirmed the presence in heterozygosis of a germline pathogenic deletion of exons 8-11 in BARD1 gene. The predictive genetic study of the family revealed the presence of this pathogenic variant in his deceased cancer affected relatives, confirming co-segregation of the deletion with the disease. CONCLUSIONS: To the best of our knowledge, this is the first published work in which this BARD1 deletion is detected in a family with familial colorectal cancer type X (FCCTX) syndrome, in which the clinical criteria for Lynch syndrome without alteration of the DNA mismatch repair (MMR) system are fulfilled. Whether this incidental germline finding is the cause of familial colorectal aggregation remains to be elucidated in scientific forums. Patients should be carefully assessed in specific cancer genetic counseling units to account for hypothetical casual findings in other genes, in principle unrelated to the initial clinical suspicion, but with potential impact on their health.

Effects of Bout Duration on Load, Sprint, and Jump Ability During a One-on-one Transition Task.

This study examined the training load on professional soccer players during transition games performed with different bout durations and their effects on speed and jump tests. Fourteen young soccer players played a transition game of different bout durations: 15 (TG15), 30 (TG30), and 60 (TG60) seconds. Total distance covered (DC), accelerations and decelerations above 1.0 and 2.5 m·s-2, rate of perceived exertion (RPE), maximal heart rate (HRmax) and above 90% (HR>90), distance covered between 18.0-20.9 km·h-1 (DC 18.0-20.9 km·h-1), 21.0-23.9 km·h-1 (DC 21.0-23.9 km·h-1), above 24.0 km·h-1 (DC>24.0 km·h-1), peak speed, sprint profile, sprint, and countermovement jump tests were recorded. TG15 obtained greater DC, DC>21.0 km·h-1, Player load, Acc>2.5 m·s-2 than TG30 and TG60 (p<0.01) and Acc<2.5 m·s-2, Dec>2.5 m·s-2 than TG60 (p<0.01). TG30 showed more HR>90 and RPE (p<0.01) than TG15, and DC, DC>18.0 km·h-1, Player load, Acc>2.5 m·s-2, Dec>2.5 m·s-2, HR>90 and RPE than TG60 (p<0.01 and<0.05). Transition games showed lower sprint and jump results after the intervention (p<0.01). Bout duration has been configured as an important constraint that influences the transition games and the performance of soccer players.

Autonomous Underwater Vehicles: Identifying Critical Issues and Future Perspectives in Image Acquisition.

Underwater imaging has been present for many decades due to its relevance in vision and navigation systems. In recent years, advances in robotics have led to the availability of autonomous or unmanned underwater vehicles (AUVs, UUVs). Despite the rapid development of new studies and promising algorithms in this field, there is currently a lack of research toward standardized, general-approach proposals. This issue has been stated in the literature as a limiting factor to be addressed in the future. The key starting point of this work is to identify a synergistic effect between professional photography and scientific fields by analyzing image acquisition issues. Subsequently, we discuss underwater image enhancement and quality assessment, image mosaicking and algorithmic concerns as the last processing step. In this line, statistics about 120 AUV articles fro recent decades have been analyzed, with a special focus on state-of-the-art papers from recent years. Therefore, the aim of this paper is to identify critical issues in autonomous underwater vehicles encompassing the entire process, starting from optical issues in image sensing and ending with some issues related to algorithmic processing. In addition, a global underwater workflow is proposed, extracting future requirements, outcome effects and new perspectives in this context.

Analysis of Connectome Graphs Based on Boundary Scale.

The purpose of this work is to advance in the computational study of connectome graphs from a topological point of view. Specifically, starting from a sequence of hypergraphs associated to a brain graph (obtained using the Boundary Scale model, BS2), we analyze the resulting scale-space representation using classical topological features, such as Betti numbers and average node and edge degrees. In this way, the topological information that can be extracted from the original graph is substantially enriched, thus providing an insightful description of the graph from a clinical perspective. To assess the qualitative and quantitative topological information gain of the BS2 model, we carried out an empirical analysis of neuroimaging data using a dataset that contains the connectomes of 96 healthy subjects, 52 women and 44 men, generated from MRI scans in the Human Connectome Project. The results obtained shed light on the differences between these two classes of subjects in terms of neural connectivity.

Novel therapeutic avenues for the study of chronic liver disease and regeneration: The foundation of the Iberoamerican Consortium for the study of liver Cirrhosis.

Unfortunately, there is a gap of understanding in the pathophysiology of chronic liver disease due to the lack of experimental models that exactly mimic the human disease. Additionally, the diagnosis of patients is very poor due to the lack of biomarkers than can detect the disease in early stages. Thus, it is of utmost interest the generation of a multidisciplinary consortium from different countries with a direct translation. The present reports the meeting of the 2021 Iberoamerican Consortium for the study of liver Cirrhosis, held online, in October 2021. The meeting, was focused on the recent advancements in the field of chronic liver disease and cirrhosis with a specific focus on cell pathobiology and liver regeneration, molecular and cellular targets involved in non-alcoholic hepatic steatohepatitis, alcoholic liver disease (ALD), both ALD and western diet, and end-stage liver cirrhosis and hepatocellular carcinoma. In addition, the meeting highlighted recent advances in targeted novel technology (-omics) and opening therapeutic avenues in this field of research.

Different pitch configurations constrain the external and internal loads of young professional soccer players during transition games.

The aims of this study were to compare the influence of transition game (TG) size on the external and internal loads of young professional soccer players and to describe the high-speed profile of these drills in response to pitch dimensions. Eighteen young professional soccer players (age: 16.1 ± 0.3 years; height: 178.3 ± 5.4 cm; weight: 70.1 ± 6.2 kg) performed a 3vs2 TG on pitches measuring 40 × 30 m (TG30), 40 × 50 m (TG50) and 40 × 70 m (TG70) m. Distance covered (DC); accelerations-decelerations above 1.0 m · s-2 and 2.5 m · s-2; rate of perceived exertion (RPE); maximal heart rate and time above 90%; DC at 18.0 to 21.0 km · h-1 (DC 18-20.9 km · h-1); DC at 21.0 to 23.9 km · h-1 (DC 21-23.9 km · h-1); DC above 24.0 km · h-1 (DC > 24 km · h-1); and peak speed and sprint profile (duration, distance and maximal speed) were measured. TG30 achieved lower DC, DC above 18 km · h-1, peak speed, sprint distance and RPE than TG50 and TG70 (p < 0.01 and p < 0.05) and lower sprint duration and maximal speed sprint than TG70 (p < 0.01). TG30 and TG50 achieved higher Acc > 1.0 and > 2.5 m · s-2 respectively than TG70 (p < 0.05). TG70 showed greater DC above 21 km · h-1, peak speed, sprint distance and maximal speed sprint than TG50 (p < 0.01). Soccer coaches should use larger TGs to overload variables related to high speed and sprint demands during training and smaller TG formats to stimulate the accelerations of the soccer players.

High-throughput engineering of cytoplasmic- and nuclear-replicating large dsDNA viruses by CRISPR/Cas9.

The application of CRISPR/Cas9 to improve genome engineering efficiency for large dsDNA viruses has been extensively described, but a robust and versatile method for high-throughput generation of marker-free recombinants for a desired locus has not yet been reported. Cytoplasmic-replicating viruses use their own repair enzymes for homologous recombination, while nuclear-replicating viruses use the host repair machinery. This is translated into a wide range of Cas9-induced homologous recombination efficiencies, depending on the virus replication compartment and viral/host repair machinery characteristics and accessibility. However, the use of Cas9 as a selection agent to target parental virus genomes robustly improves the selection of desired recombinants across large dsDNA viruses. We used ectromelia virus (ECTV) and herpes simplex virus (HSV) type 1 and 2 to optimize a CRISPR/Cas9 method that can be used versatilely for efficient genome editing and selection of both cytoplasmic- and nuclear-replicating viruses. We performed a genome-wide genetic variant analysis of mutations located at predicted off-target sequences for 20 different recombinants, showing off-target-free accuracy by deep sequencing. Our results support this optimized method as an efficient, accurate and versatile approach to enhance the two critical factors of high-throughput viral genome engineering: generation and colour-based selection of recombinants. This application of CRISPR/Cas9 reduces the time and labour for screening of desired recombinants, allowing for high-throughput generation of large collections of mutant dsDNA viruses for a desired locus, optimally in less than 2 weeks.

High-speed Training in a Specific Context in Soccer: Transition Games.

The aims of this study were to compare the load of three tasks designed to train features of soccer: a transition game, a small-sided game with a change of playing area, and a large-sided game. Twenty young elite players performed these tasks. Variables measured were total distance covered (DC), distance covered at 14.0-17.9 km·h-1 (DC 14.0-17.9 km·h-1), distance covered at 18.0-21 km·h-1 (DC 18-21 km·h-1), distance covered > 21 km·h-1 (DC>21 km·h-1), peak speed, accelerations and decelerations > 1.0 and > 2.5 m·s-2, player load, and rate of perceived exertion. Transition games produced greater DC 18-21 km·h-1, DC>21 km·h-1, peak speed and Acc>2.5 m·s-2 than the other drills (p<0.01) and higher DC 14-17.9 km·h-1 (p<0.01), Dec>1 m·s-2 (p<0.05) and Dec>2.5 m·s-2 (p<0.01) than the large-sided game. Both sided games produced more DC (p<0.01), Acc>1 m·s-2 (p<0.01) and player load (p<0.01 and p<0.05, respectively) than the transition game. During the small-sided game, significantly higher DC 14-17.9 km·h-1, DC 18-21 km·h-1 and DC>21 km·h-1 were recorded in comparison with the large-sided game (p<0.01). The studied parameters showed lower variation in the transition game. Coaches could use transition games to train high speed running in counter-attack contexts.

Calcium homeostasis plays important roles in the internalization and activities of the small synthetic antifungal peptide PAF26.

Fungal diseases are responsible for the deaths of over 1.5 million people worldwide annually. Antifungal peptides represent a useful source of antifungals with novel mechanisms-of-action, and potentially provide new methods of overcoming resistance. Here we investigate the mode-of-action of the small, rationally designed synthetic antifungal peptide PAF26 using the model fungus Neurospora crassa. Here we show that the cell killing activity of PAF26 is dependent on extracellular Ca2+ and the presence of fully functioning fungal Ca2+ homeostatic/signaling machinery. In a screen of mutants with deletions in Ca2+ -signaling machinery, we identified three mutants more tolerant to PAF26. The Ca2+ ATPase NCA-2 was found to be involved in the initial interaction of PAF26 with the cell envelope. The vacuolar Ca2+ channel YVC-1 was shown to be essential for its accumulation and concentration within the vacuolar system. The Ca2+ channel CCH-1 was found to be required to prevent the translocation of PAF26 across the plasma membrane. In the wild type, Ca2+ removal from the medium resulted in the peptide remaining trapped in small vesicles as in the Δyvc-1 mutant. It is, therefore, apparent that cell killing by PAF26 is complex and unusually dependent on extracellular Ca2+ and components of the Ca2+ -regulatory machinery.