Multiplex site-directed mutagenesis strategy including high-efficiency selection of the mutant PCR products

Site-directed mutagenesis is of great importance for probing the structure/function relationship of proteins. Developing our previous method (Nagy et al. Anal Biochem 324:301–303, 2004), here we report a multiplex strategy for site-directed mutagenesis using PCR in one tube to introduce a single mutation into three or more genes at the same time. DNA fragments carrying the desired mutation can be distinguished from each other in a standard antibiotic selection step of the transformed bacteria. Due to this strategy the mutagenesis procedure for several genes can be accelerated.     

Efficacy and safety of long pulse 1064 and 2940 nm lasers in noninvasive lipolysis and skin tightening

Noninvasive body shaping is becoming a growing demand. The aim of this study was to investigate the efficacy and safety of the combined treatments of 1064 nm Nd:YAG and 2940 nm Er:YAG in noninvasive lipolysis and skin tightening. Ten females were enrolled, and all women's side of the waist or the lower part of the abdomen were treated. In the first step, the 1064 nm Nd:YAG was used. As a second step, the 2940 nm Er:YAG laser was applied. Each woman was treated four times, once every 2 weeks. The effects were determined by comparative photo documentation, waist circumference measurement, two‐dimensional B‐mode ultrasonography and low‐dose native computer tomography (CT), whereas body fat was monitored with bioelectric impedance. The tissue firmness was measured by ultrasound shear wave elastography. Combined laser treatment significantly reduced waist circumference and total body fat. Ultrasonography has revealed that the treatment considerably decreased fat thickness and improved skin stiffness in the treated region. Subcutaneous fat volume, measured by low‐dose CT, displayed a moderate decrease in the waist region. The combined 1064 nm Nd:YAG and 2940 nm Er:YAG laser treatment results in the reduction of fat tissue and tightens the skin as confirmed by objective measurements.      

Kinematic Responses to Changes in Walking Orientation and Gravitational Load in Drosophila melanogaster

Walking behavior is context-dependent, resulting from the integration of internal and external influences by specialized motor and pre-motor centers. Neuronal programs must be sufficiently flexible to the locomotive challenges inherent in different environments. Although insect studies have contributed substantially to the identification of the components and rules that determine locomotion, we still lack an understanding of how multi-jointed walking insects respond to changes in walking orientation and direction and strength of the gravitational force. In order to answer these questions we measured with high temporal and spatial resolution the kinematic properties of untethered Drosophila during inverted and vertical walking. In addition, we also examined the kinematic responses to increases in gravitational load. We find that animals are capable of shifting their step, spatial and inter-leg parameters in order to cope with more challenging walking conditions. For example, flies walking in an inverted orientation decreased the duration of their swing phase leading to increased contact with the substrate and, as a result, greater stability. We also find that when flies carry additional weight, thereby increasing their gravitational load, some changes in step parameters vary over time, providing evidence for adaptation. However, above a threshold that is between 1 and 2 times their body weight flies display locomotion parameters that suggest they are no longer capable of walking in a coordinated manner. Finally, we find that functional chordotonal organs are required for flies to cope with additional weight, as animals deficient in these proprioceptors display increased sensitivity to load bearing as well as other locomotive defects.     

Complex formation of EphB1/Nck/Caskin1 leads to tyrosine phosphorylation and structural changes of the Caskin1 SH3 domain

Background

Scaffold proteins have an important role in the regulation of signal propagation. These proteins do not possess any enzymatic activity but can contribute to the formation of multiprotein complexes. Although scaffold proteins are present in all cell types, the nervous system contains them in the largest amount. Caskin proteins are typically present in neuronal cells, particularly, in the synapses. However, the signaling mechanisms by which Caskin proteins are regulated are largely unknown.

Results

Here we demonstrate that EphB1 receptor tyrosine kinase can recruit Caskin1 through the adaptor protein Nck. Upon activation of the receptor kinase, the SH2 domain of Nck binds to one of its tyrosine residues, while Nck SH3 domains interact with the proline-rich domain of Caskin1. Complex formation of the receptor, adaptor and scaffold proteins results in the tyrosine phosphorylation of Caskin1 on its SH3 domain. The phosphorylation sites were identified by mass-spectrometry as tyrosines 296 and 336. To reveal the structural consequence of this phosphorylation, CD spectroscopy was performed. This measurement suggests that upon tyrosine phosphorylation the structure of the Caskin1 SH3 domain changes significantly.

Conclusion

Taken together, we propose that the scaffold protein Caskin1 can form a complex with the EphB1 tyrosine kinase via the Nck protein as a linker. Complex formation results in tyrosine phosphorylation of the Caskin1 SH3 domain. Although we were not able to identify any physiological partner of the SH3 domain so far, we could demonstrate that phosphorylation on conserved tyrosine residues results in marked changes in the structure of the SH3 domain.

     

Effects of Growth and Mutation on Pattern Formation in Tissues

In many developing tissues, neighboring cells enter different developmental pathways, resulting in a fine-grained pattern of different cell states. The most common mechanism that generates such patterns is lateral inhibition, for example through Delta-Notch coupling. In this work, we simulate growth of tissues consisting of a hexagonal arrangement of cells laterally inhibiting their neighbors. We find that tissue growth by cell division and cell migration tends to produce ordered patterns, whereas lateral growth leads to disordered, patchy patterns. Ordered patterns are very robust to mutations (gene silencing or activation) in single cells. In contrast, mutation in a cell of a disordered tissue can produce a larger and more widespread perturbation of the pattern. In tissues where ordered and disordered patches coexist, the perturbations spread mostly at boundaries between patches. If cell division occurs on time scales faster than the degradation time, disordered patches will appear. Our work suggests that careful experimental characterization of the disorder in tissues could pinpoint where and how the tissue is susceptible to large-scale damage even from single cell mutations.     

Species history masks the effects of human-induced range loss--unexpected genetic diversity in the endangered giant mayfly Palingenia longicauda

Freshwater biodiversity has declined dramatically in Europe in recent decades. Because of massive habitat pollution and morphological degradation of water bodies, many once widespread species persist in small fractions of their original range. These range contractions are generally believed to be accompanied by loss of intraspecific genetic diversity, due to the reduction of effective population sizes and the extinction of regional genetic lineages. We aimed to assess the loss of genetic diversity and its significance for future potential reintroduction of the long-tailed mayfly Palingenia longicauda (Olivier), which experienced approximately 98% range loss during the past century. Analysis of 936 bp of mitochondrial DNA of 245 extant specimens across the current range revealed a surprisingly large number of haplotypes (87), and a high level of haplotype diversity (Hd = 0.875). In contrast, historic specimens (6) from the lost range (Rhine catchment) were not differentiated from the extant Rába population (F(ST) = 0.02, p = 0.61), despite considerable geographic distance separating the two rivers. These observations can be explained by an overlap of the current with the historic (Pleistocene) refugia of the species. Most likely, the massive recent range loss mainly affected the range which was occupied by rapid post-glacial dispersal. We conclude that massive range losses do not necessarily coincide with genetic impoverishment and that a species' history must be considered when estimating loss of genetic diversity. The assessment of spatial genetic structures and prior phylogeographic information seems essential to conserve once widespread species.     

Recovery of native grass biodiversity by sowing on former croplands: Is weed suppression a feasible goal for grassland restoration?

Grassland restoration on former croplands offers good opportunity to mitigate the loss of grassland biodiversity. Weed suppression can be another benefit, which becomes increasingly important because of the high recent rate of abandonment of arable lands in Central and Eastern Europe. Our aim was to evaluate the usefulness of sowing two low-diversity seed mixtures followed by annual mowing, a frequently used restoration technique, in weed suppression. We found that rapidly forming cover of sown grasses effectively suppressed short-lived weeds and their germination except in the first year. The detected dense seed bank of short-lived weeds points out the possibility and threat of later weed infestation. In the short run perennial weeds cannot be suppressed easily by sowing and annual mowing. We found that the effectiveness of seed sowing followed by mowing in weed suppression can be different on sites with different history or seed mixture. Rapidly establishing perennial weeds, such as Agropyron species were only detected in former alfalfa fields; Cirsium arvense was found in former cereal and sunflower fields but not in former alfalfa fields. We found that the rate of weed suppression and success was influenced by the seed mixtures used. In several alkali restorations the high proportion of perennial weeds was detected in year 3. In loess restorations, much lower scores were typical. This was likely caused by the different seed mixture used. The loess seed mixture contained seeds of a clonally spreading tall-grass, Bromus inermis, which could compete more effectively with clonally spreading weeds, than could short grass species with or without tussock forming. Our findings indicate that post-restoration management require carefully designed actions that are fine-tuned addressing specific threats at the site level.     

Protein kinase C protects from DNA damage-induced necrotic cell death by inhibiting poly(ADP-ribose) polymerase-1

The goal of the current study, conducted in freshly isolated thymocytes was (1) to investigate the possibility that the activation of poly(ADP-ribose) polymerase-1 (PARP-1) in an intact cell can be regulated by protein kinase C (PKC) mediated phosphorylation and (2) to examine the consequence of this regulatory mechanism in the context of cell death induced by the genotoxic agent. In cells stimulated by the PKC activating phorbol esters, DNA breakage was unaffected, PARP-1 was phosphorylated, 1-methyl-3-nitro-1-nitrosoguanidine-induced PARP activation and cell necrosis were suppressed, with all these effects attenuated by the PKC inhibitors GF109203X or G?6976. Inhibition of cellular PARP activity by PKC-mediated phosphorylation may provide a plausible mechanism for the previously observed cytoprotective effects of PKC activators.