External push and internal pull forces recruit curvature-sensing N-BAR domain proteins to the plasma membrane

Many of the more than 20 mammalian proteins with N-BAR domains control cell architecture and endocytosis by associating with curved sections of the plasma membrane. It is not well understood whether N-BAR proteins are recruited directly by processes that mechanically curve the plasma membrane or indirectly by plasma-membrane-associated adaptor proteins that recruit proteins with N-BAR domains that then induce membrane curvature. Here, we show that externally induced inward deformation of the plasma membrane by cone-shaped nanostructures (nanocones) and internally induced inward deformation by contracting actin cables both trigger recruitment of isolated N-BAR domains to the curved plasma membrane. Markedly, live-cell imaging in adherent cells showed selective recruitment of full-length N-BAR proteins and isolated N-BAR domains to plasma membrane sub-regions above nanocone stripes. Electron microscopy confirmed that N-BAR domains are recruited to local membrane sites curved by nanocones. We further showed that N-BAR domains are periodically recruited to curved plasma membrane sites during local lamellipodia retraction in the front of migrating cells. Recruitment required myosin-II-generated force applied to plasma-membrane-connected actin cables. Together, our results show that N-BAR domains can be directly recruited to the plasma membrane by external push or internal pull forces that locally curve the plasma membrane.     

Genome-wide identification and characterization of replication origins by deep sequencing

Background

DNA replication initiates at distinct origins in eukaryotic genomes, but the genomic features that define these sites are not well understood.

Results

We have taken a combined experimental and bioinformatic approach to identify and characterize origins of replication in three distantly related fission yeasts: Schizosaccharomyces pombe, Schizosaccharomyces octosporus and Schizosaccharomyces japonicus. Using single-molecule deep sequencing to construct amplification-free high-resolution replication profiles, we located origins and identified sequence motifs that predict origin function. We then mapped nucleosome occupancy by deep sequencing of mononucleosomal DNA from the corresponding species, finding that origins tend to occupy nucleosome-depleted regions.

Conclusions

The sequences that specify origins are evolutionarily plastic, with low complexity nucleosome-excluding sequences functioning in S. pombe and S. octosporus, and binding sites for trans-acting nucleosome-excluding proteins functioning in S. japonicus. Furthermore, chromosome-scale variation in replication timing is conserved independently of origin location and via a mechanism distinct from known heterochromatic effects on origin function. These results are consistent with a model in which origins are simply the nucleosome-depleted regions of the genome with the highest affinity for the origin recognition complex. This approach provides a general strategy for understanding the mechanisms that define DNA replication origins in eukaryotes.     

Evaluation of a composite test of kicking performance

The aim of this study was to evaluate the reliability and sensitivity of variables for the direct assessment of kicking performance in young soccer players. One hundred and six elite young soccer players were divided into 4 age groups (12-15 years). Absolute error (AE), variable error (VE), and constant error (CE) were evaluated as the variables of kicking accuracy, whereas the kicking velocity variables involved the maximum ball velocity (BVmax) and the ball velocity during accurate kicks (BVacc). Results suggested low-to-moderate reliability of the kicking accuracy (intraclass correlation coefficient [ICC] = 0.00-0.67) and high reliability of the kicking velocity variables (ICC = 0.87-0.94). Regarding the sensitivity, most of the variables detected the differences both between the dominant and nondominant legs and among the age groups. Because the evaluated variables should have a property of face validity, the findings obtained generally suggest that AE (and perhaps VE, as the measures of kicking accuracy) and both BVmax and BVacc (as the measures of kicking velocity) could be used within a routine composite test of kicking performance in young elite soccer players. Further development of the evaluated composite test of kicking performance could be based on the involvement of other kicking techniques and on testing the athletes of different ages, levels of skill, or sport specialization.     

Effects of loading on maximum vertical jumps: Selective effects of weight and inertia

A novel loading method was applied to explore selective effects of externally added weight (W), weight and inertia (W+I), and inertia (I) on maximum counter-movement jumps (CMJ) performed with arm swing. Externally applied extended rubber bands and/or loaded vest added W, W+I, and I corresponding to 10-40% of subjects' body mass. As expected, an increase in magnitude of all types of load was associated with an increase in ground reaction forces (GRF), as well as with a decrease in both the jumping performance and power output. However, of more importance could be that discernible differences among the effects of W, W+I, and I were recorded despite a relatively narrow loading range. In particular, an increase in W was associated with the minimal changes in movement kinematic pattern and smallest reduction of jumping performance, while also allowing for the highest power output. Conversely, W+I was associated with the highest ground reaction forces. Finally, the lowest maxima of GRF and power were associated with I. Although further research is apparently needed, the obtained finding could be of potential importance not only for understanding fundamental properties of the neuromuscular system, but also for optimization of loading in standard athletic training and rehabilitation procedures.     

Studies of hyperlipidemia in drug-induced diabetic rats by high-performance liquid chromatography

A hyperlipidemic condition is often associated with diabetes. The possibility that specific serum lipids (i.e., individual triglycerides or cholesterol esters) may be altered in the diabetic state was investigated. Serum lipids from both controls and streptozotocin- and alloxantreated rats were separated into approximately twenty chromatographic fractions by reversed-phase high-performance liquid chromatography; a number of individual triglycerides and cholesterol esters were identified. The methodology described allowed subtle changes in individual lipid components to be detected. Only minor variations in the cholesterol and cholesterol ester fractions were observed between the control and diabetic samples. While not uniform throughout, elevations in the triglyceride fractions occurred in the diabetics. Also, differences in triglyceride content were found to exist between the groups of streptozotocin- and alloxan-treated animals.     

Molecular and chemical characterization of the most widespread Ocimum species

DNA fingerprinting (AFLP) and chemical analyses of essential oils were utilized to define the extent of variation existing in the genus Ocimum. Research was carried out on 22 Ocimum accessions representing seven species. Concerning the essential oil composition of all investigated accessions, 115 compounds were identified. UPGMA cluster analysis, based on Euclidian distances of essential oil constituents between all pairs of accessions, showed four well-supported clusters (O. tenuiflorum, O. basilicum/O. africanum, O. basilicum, and O. americanum/O. africanum). Relating to the essential oil composition of all of the investigated accessions, 17 compounds were identified as the main ones, and according to them 13 chemotypes were determined. AFLP relationships were determined by neighbor-joining (NJ) cluster analysis based on Dice??s distance matrix and by maximum parsimony (MP) analysis. O. basilicum, O. americanum/O. africanum, O. tenuiflorum, and O. gratissimum represented four clusters supported with high bootstrap values. A neighbor-net diagram allowed the visualization of apparently conflicting data by revealing relationships between genotypes and chemotypes. Concerning the O. africanum species, two distinct chemotypes, geranial/neral (accession 11) and estragol (accession 10), have been established, while all the studied O. americanum accessions belong to the geranial/neral chemotype. This could be additional evidence that O. americanum is one of the parents of O. africanum. Furthermore, the fact that the O. africanum accession (10) as well as O. basilicum ??Purpurascens?? and O. basilicum ??Erevanskii?? accessions belong to the estragol chemotype supports the theory that O. africanum is one of the parents of these two O. basilicum accessions.     

Chemical characterization and genetic relationships among Ocimum basilicum L. cultivars

Twenty-seven Ocimum basilicum cultivars were subjected to a chemical characterization of essential oil components by gas chromatography/mass spectrometry (GC/MS) and a genetic characterization using the amplified fragment-length polymorphism (AFLP) technique. Since the same 27 accessions had previously been classified into six morphotypes, these analyses allowed us to make detailed comparisons of chemistry, genetics, and morphology. The chemical composition and morphology of the studied cultivars appeared to have a strong genetic component. The AFLP analysis revealed a distinction between the green and purple morphotypes. The green morphotypes predominantly utilized the terpene biosynthetic pathway, while most purple morphotypes primarily utilized the phenylpropene biosynthetic pathway. The GC/MS analysis led to identification of 87 volatiles. Among the 27 cultivars, five chemotypes were identified. A detailed characterization of the essential oil constituents indicated the existence of both specific combinations of compounds and 'private' compounds with the potential to be used in many aspects of human life. The established relationship between a genetic profile, chemical composition, and morphology represents an important step in future breeding programs and in the cultivation of this species.