Chemically-induced affinity star restriction specificity: a novel TspGWI/sinefungin endonuclease with theoretical 3-bp cleavage frequency

The type IIS/IIC restriction endonuclease TspGWI recognizes the sequence 5'-ACGGA-3', cleaving DNA 11/9 nucleotides downstream. Here we show that sinefungin, a cofactor analog of S-adenosyl methionine, induces a unique type of relaxation in DNA recognition specificity. In the presence of sinefungin, TspGWI recognizes and cleaves at least 12 degenerate variants of the original recognition sequence that vary by single base pair changes from the original 5-bp restriction site with only a single degeneracy per variant appearing to be allowed. In addition, sinefungin was found to have a stimulatory effect on cleavage at these nondegenerate TspGWI recognition sites, irrespective of their number or the DNA topology. Interestingly, no fixed "core" could be identified among the new recognition sequences. Theoretically, TspGWI cleaves DNA every 1024 bp, while sinefungin-induced activity cleaves every 78.8 bp, corresponding to a putative 3-bp long recognition site. Thus, the combination of sinefungin and TspGWI represents a novel frequent cutter, next only to CviJI/CviJI*, that should prove useful in DNA cloning methodologies.     

African Y chromosome and mtDNA divergence provides insight into the history of click languages

BACKGROUND: About 30 languages of southern Africa, spoken by Khwe and San, are characterized by a repertoire of click consonants and phonetic accompaniments. The Jumid R:'hoansi (!Kung) San carry multiple deeply coalescing gene lineages. The deep genetic diversity of the San parallels the diversity among the languages they speak. Intriguingly, the language of the Hadzabe of eastern Africa, although not closely related to any other language, shares click consonants and accompaniments with languages of Khwe and San. RESULTS: We present original Y chromosome and mtDNA variation of Hadzabe and other ethnic groups of Tanzania and Y chromosome variation of San and peoples of the central African forests: Biaka, Mbuti, and Lisongo. In the context of comparable published data for other African populations, analyses of each of these independently inherited DNA segments indicate that click-speaking Hadzabe and Jumid R:'hoansi are separated by genetic distance as great or greater than that between any other pair of African populations. Phylogenetic tree topology indicates a basal separation of the ancient ancestors of these click-speaking peoples. That genetic divergence does not appear to be the result of recent gene flow from neighboring groups. CONCLUSIONS: The deep genetic divergence among click-speaking peoples of Africa and mounting linguistic evidence suggest that click consonants date to early in the history of modern humans. At least two explanations remain viable. Clicks may have persisted for tens of thousands of years, independently in multiple populations, as a neutral trait. Alternatively, clicks may have been retained, because they confer an advantage during hunting in certain environments.     

The effects of 2-substituted oestrogen sulphamates on the growth of prostate and ovarian cancer cells

The human endogenous metabolite 2-methoxyoestradiol (2-MeOE2) has been shown to inhibit the proliferation of breast cancer cells. We have previously shown that sulphamoylation of a series of 2-substituted oestrogens greatly enhances their ability to inhibit breast cancer cell proliferation and induce apoptosis. In this study, we have investigated the ability of a number of 2-substituted oestrogens and their sulphamoylated derivatives to inhibit the proliferation of two prostate cancer cell lines, an ovarian cancer cell line and its drug-resistant derivatives. 2-Methoxyoestrone, 2-ethyloestrone and 2-ethyloestradiol had little effect on the growth of the cell lines tested (IC(50)>10 microM). 2-MeOE2 did inhibit the growth of the cells (IC(50)<10 microM), but to a lesser extent than any of the sulphamoylated derivatives tested (IC(50)<1.0 microM). Cells treated with the sulphamoylated derivatives became detached and rounded, displaying a characteristic apoptotic appearance. FACS analysis revealed induced G(2)/M cell cycle arrest. Treatment of cells and subsequent drug removal indicated that the effects of the drugs on the cells were irreversible. Immunoblot analysis indicated that apoptosis may be induced by phosphorylation of BCL-2. From these studies, 2-substituted oestrogen sulphamates are emerging as a potent new class of drug that may be effective against AR+/AR- prostate and ovarian tumours, and against tumours that are resistant to conventional chemotherapeutic regimens.     

The Effects of l-Arginine, Alone and Combined with Vitamin C, on Mineral Status in Relation to its Antidiabetic, Anti-Inflammatory, and Antioxidant Properties in Male Rats on a High-Fat Diet

The aim of this study was to evaluate the influence of the intake of l-arginine alone and of l-arginine with vitamin C on mineral concentration in rats fed with a high-fat diet, and to assess the lipid glucose, insulin, and total antioxidant status (TAS) and tumor necrosis factor (TNF) alpha serum levels that result. Wistar rats were assigned to groups fed with either a standard control diet (C), a diet high in fat (FD), a diet high in fat with l-arginine, or a diet high in fat with l-arginine and vitamin C. After 6 weeks, the length and weight of the rats were measured, and the animals were euthanized. The liver, spleen, kidneys, pancreas, heart, and gonads were collected, as were blood samples. The total serum cholesterol, triglyceride, fasting glucose, insulin, TAS, and TNF alpha levels were measured. The tissue calcium, magnesium, iron, zinc, and copper concentrations were determined. It was found that l-arginine supplementation diminished the effect of the modified diet on the concentration of iron in the liver and spleen and of copper in heart. At the same time, it was observed that l-arginine supplementation reduced the effect of the high-fat diet on insulin, TNF alpha, and TAS. The combination of l-arginine and vitamin C produced a similar effect on the mineral levels in the tissues as did l-arginine used alone. Moreover, positive correlations between serum insulin and iron in the liver, between TNF alpha and iron in the liver, and between TNF alpha and copper in the heart were observed. The level of TAS in serum was inversely correlated with the copper level in the heart and the iron level in the liver. We concluded that the beneficial influence of l-arginine on insulin, TAS, and TNF alpha serum level is associated with changes in the iron and copper status in rats fed with a high-fat diet. No synergistic effect of l-arginine and vitamin C in the biochemical parameters or in the mineral status in rats fed with the modified diet was observed.     

Acceptability of Animal-Assisted Therapy: Attitudes toward AAT,Psychotherapy, and Medication for the Treatment of Child Disruptive Behavioral Problems

Animal-assisted therapies (AATs) are not widely promoted in routine mental healthcare but represent a viable treatment option given positive perceptions of pets and growing evidence that animals provide meaningful contribution to psychological wellbeing. Relatively little is known about the general public's attitude toward AATs, especially in relation to more commonly used alternatives. This study compared the acceptability of four different treatment options (AAT, medication, psychotherapy, and no active treatment) for common externalizing behaviors in children. Parents from a community sample (N = 189) were presented with vignettes describing a child with symptoms of an externalizing behavior disorder and were asked to rate the acceptability of the four different treatment options. Participants rated AAT as a highly acceptable form of treatment and more acceptable than no active treatment and medication. However, AAT was rated as less acceptable than psychotherapy. Experiences with animals (both positive and negative) were unrelated to the acceptability of AAT. These findings suggest that AAT is viewed as a highly acceptable form of treatment for common externalizing behaviors and should be more systematically investigated as a treatment for children's mental health problems.     

Quantitative analysis of extracted phycobilin pigments in cyanobacteria—an assessment of spectrophotometric and spectrofluorometric methods

Phycobilins are an important group of pigments that through complementary chromatic adaptation optimize the light-harvesting process in phytoplankton cells, exhibiting great potential as cyanobacteria species biomarkers. In their extracted form, concentrations of these water-soluble molecules are not easily determined using the chromatographic methods well suited to solvent-soluble pigments. Insights regarding the quantitative spectroscopic analysis of extracted phycobilins also remain limited. Here, we present an in-depth study of two methods that utilize the spectral properties of phycobilins in aqueous extracts. The technical work was carried out using high-purity standards of phycocyanin, phycoerythrin, and allophycocyanin. Calibration parameters for the spectrofluorometer and spectrophotometer were established. This analysis indicated the possibility of detecting pigments in concentrations ranging from 0.001 to 10 μg cm^?3. Fluorescence data revealed a reproducibility of 95 %. The differences in detection limits between the two methods enable the presence of phycobilins to be investigated and their amounts to be monitored from oligotrophic to eutrophic aquatic environments.     

Life cycle thinking in small and medium enterprises: the results of research on the implementation of life cycle tools in Polish SMEs—Part 3: LCC-related aspects

Purpose

This article is the third of a series of articles presenting the results of research on the implementation of life cycle management tools in small- and medium-sized companies in Poland. The purpose of the three-part series of articles is to present the results of research on the implementation of life cycle tools in Polish small and medium enterprises (SMEs). This work is part of a project financed by the Polish Agency for Enterprise Development (PAED) which began in February 2011. It was carried out by the Wielkopolska Quality Institute—a business environment institution associated with the Polish Centre for LCA (PCLCA). The main practical objective of the project was to support SMEs in their business development, e.g. by expanding their horizons beyond the sphere of their operation and identifying new areas for the improvement and promotion of the products and services on offer. The specific objective of the analysis involving the assessment of life-cycle costs of products and services was an attempt to answer the question to determine whether the assessment carried out in accordance with the life-cycle cost (LCC) methodology is a good tool for cost management in this type of business. Part 3 describes the results of studies on the assessment of the implementation of LCC in SMEs conducted in 50 companies involved in the project.

Methods

In order to assess the effectiveness of the project and the effectiveness of the implementation of LCA and LCC, a survey was conducted of small- and medium-sized businesses where the implementation works had been fully completed. In total, 50 organisations agreed to participate in the LCC survey (while 46 in the LCA—part 2 paper), which was 71 % of all the companies where the LCA and LCC studies had been carried out within the project. The survey was conducted using individual in-depth interviews. Questions to the representatives of the companies referred both to aspects of their operating in the market (characteristics of a company, its market share, management systems, environmental policy, suppliers, clients) and the implementation of their environmental service (assessment of its effectiveness, motivation, difficulties in its implementation), as well as opinions on the potential applications of LCA in their current operations.

Results and discussion

The experience and observations of LCC experts resulting from their cooperation with the analysed organisations are largely supported by the results of the survey. The overall impression gained from the project is that the small- and medium-sized enterprises considered have a problem with accepting and understanding the life-cycle perspective, and show limited interest in taking liability for environmental and cost aspects beyond the mandatory legal standards and boundaries of their business operations. Nevertheless, the LCC analyses aroused much bigger interest among the companies than the environmental due to the fact that the cost aspects in companies undergoing normal development are seen as an important source of information about the structure of the costs generated with respect to the products or services provided. It is important to note that a very important factor encouraging businesses to join the studies was the fact that they were cost-free. Moreover, the planned introduction of a new product onto the market was the argument that often influenced the decision to implement the LCC. The survey has shown that companies rarely perform cost analyses including all stages of the life cycle of a product or service. Although the awareness of the importance of conducting economic researches for the entire life cycle of a product or service is great, it turned out to be problematic to unambiguously define the practical use of such an analysis, at least at the present stage of development of the companies surveyed.

Conclusions

The results obtained in the survey indicate that in the case of simple products, with a short life cycle, complex cost analyses may seem less useful. For more complex products or services, with long periods of use, high reliability required, and high operating costs, the analyses presented are useful tools that increase the economic efficiency of the projects implemented. It appears that from the point of view of polish SMEs, the usefulness of an LCA is seen mainly from the angle of opportunities for cost reduction (preferably in business) and increased sales (marketing). A good solution would be to conduct relatively simple, but integrated LCA/LCC analyses in SMEs so that the companies would clearly see the economic effects of the proposed environmental improvements.     

Genomic inference accurately predicts the timing and severity of a recent bottleneck in a nonmodel insect population

The analysis of molecular data from natural populations has allowed researchers to answer diverse ecological questions that were previously intractable. In particular, ecologists are often interested in the demographic history of populations, information that is rarely available from historical records. Methods have been developed to infer demographic parameters from genomic data, but it is not well understood how inferred parameters compare to true population history or depend on aspects of experimental design. Here, we present and evaluate a method of SNP discovery using RNA sequencing and demographic inference using the program δaδi, which uses a diffusion approximation to the allele frequency spectrum to fit demographic models. We test these methods in a population of the checkerspot butterfly Euphydryas gillettii. This population was intentionally introduced to Gothic, Colorado in 1977 and has as experienced extreme fluctuations including bottlenecks of fewer than 25 adults, as documented by nearly annual field surveys. Using RNA sequencing of eight individuals from Colorado and eight individuals from a native population in Wyoming, we generate the first genomic resources for this system. While demographic inference is commonly used to examine ancient demography, our study demonstrates that our inexpensive, all‐in‐one approach to marker discovery and genotyping provides sufficient data to accurately infer the timing of a recent bottleneck. This demographic scenario is relevant for many species of conservation concern, few of which have sequenced genomes. Our results are remarkably insensitive to sample size or number of genomic markers, which has important implications for applying this method to other nonmodel systems.     

COOH-terminal collagen Q (COLQ) mutants causing human deficiency of endplate acetylcholinesterase impair the interaction of ColQ with proteins of the basal lamina

Collagen Q (ColQ) is a key multidomain functional protein of the neuromuscular junction (NMJ), crucial for anchoring acetylcholinesterase (AChE) to the basal lamina (BL) and accumulating AChE at the NMJ. The attachment of AChE to the BL is primarily accomplished by the binding of the ColQ collagen domain to the heparan sulfate proteoglycan perlecan and the COOH-terminus to the muscle-specific receptor tyrosine kinase (MuSK), which in turn plays a fundamental role in the development and maintenance of the NMJ. Yet, the precise mechanism by which ColQ anchors AChE at the NMJ remains unknown. We identified five novel mutations at the COOH-terminus of ColQ in seven patients from five families affected with endplate (EP) AChE deficiency. We found that the mutations do not affect the assembly of ColQ with AChE to form asymmetric forms of AChE or impair the interaction of ColQ with perlecan. By contrast, all mutations impair in varied degree the interaction of ColQ with MuSK as well as basement membrane extract (BME) that have no detectable MuSK. Our data confirm that the interaction of ColQ to perlecan and MuSK is crucial for anchoring AChE to the NMJ. In addition, the identified COOH-terminal mutants not only reduce the interaction of ColQ with MuSK, but also diminish the interaction of ColQ with BME. These findings suggest that the impaired attachment of COOH-terminal mutants causing EP AChE deficiency is in part independent of MuSK, and that the COOH-terminus of ColQ may interact with other proteins at the BL.