PocketOptimizer 2.0: A modular framework for computer-aided ligand-binding design

The ability to design customized proteins to perform specific tasks is of great interest. We are particularly interested in the design of sensitive and specific small molecule ligand-binding proteins for biotechnological or biomedical applications. Computational methods can narrow down the immense combinatorial space to find the best solution and thus provide starting points for experimental procedures. However, success rates strongly depend on accurate modeling and energetic evaluation. Not only intra- but also intermolecular interactions have to be considered. To address this problem, we developed PocketOptimizer, a modular computational protein design pipeline, that predicts mutations in the binding pockets of proteins to increase affinity for a specific ligand. Its modularity enables users to compare different combinations of force fields, rotamer libraries, and scoring functions. Here, we present a much-improved version––PocketOptimizer 2.0. We implemented a cleaner user interface, an extended architecture with more supported tools, such as force fields and scoring functions, a backbone-dependent rotamer library, as well as different improvements in the underlying algorithms. Version 2.0 was tested against a benchmark of design cases and assessed in comparison to the first version. Our results show how newly implemented features such as the new rotamer library can lead to improved prediction accuracy. Therefore, we believe that PocketOptimizer 2.0, with its many new and improved functionalities, provides a robust and versatile environment for the design of small molecule-binding pockets in proteins. It is widely applicable and extendible due to its modular framework. PocketOptimizer 2.0 can be downloaded at https://github.com/Hoecker-Lab/pocketoptimizer .     

Characterization of 24 porcine (dA-dC)n-(dT-dG)n microsatellites: genotyping of unrelated animals from four breeds and linkage studies

Twenty-four PCR primer pairs were designed for the detection of porcine microsatellites. Polymorphism was investigated in 76 unrelated animals from four different breeds: Duroc, Landrace, Hampshire, and Yorkshire. Compared with human microsatellites, a general lower heterozygosity was detected; however, for each microsatellite a significant variation between breeds in number of alleles and heterozygosity was seen. Mean heterozygosity was found to be significantly higher (P<0.01%) in the Yorkshire breed than in the other three breeds. Linkage analyses with the CEPH linkage packet were performed in a backcross family comprising 45 animals, of which 43 had informative meioses. Ten of the microsatellites could be assigned to six different linkage groups, demonstrating that linkage mapping with microsatellites can be carried out with great efficiency in a relatively small number of animals. Four of the linkage groups represent Chromosomes (Chrs) 4, 6, 7, and 8 respectively, while two linkage groups are unassigned.The nucleotide sequence data reported in this paper have been submitted to GenBank and have been assigned the accession numbers listed in Table 1.     

Anti-predator defences of a web-building spider, Holocnemus pluchei (Araneae, Pholcidae)

Anti-predator defence behaviour of Holocnemus pluchei (Araneae, Pholcidae) was studied in the laboratory and the field. Bouncing, in which this web-building spider moves its body rapidly up and down while keeping its legs on the silk, was the most frequent response to disturbance. Less often, H. pluchei left the web when disturbed. Experiments were carried out to determine the types of stimuli that elicited bouncing and leaving the web: tactile stimulation (striking the spider with a brush) and air movement (blowing on the spider) elicited bouncing and leaving the web, but there was no evidence that chemical stimuli from potential predators were important. Tactile stimulation elicited stronger responses than air movement. There were no evident differences in how H. pluchei responded to tactile stimulation in the laboratory and the field.     

Effect of Dietary Magnesium on Post Mortem Phosphocreatine Utilization in Skeletal Muscle of Swine: A Non-Invasive Study Using 31P-NMR Spectroscopy

The effect of dietary magnesium on the post mortem PCr (phosphocreatine) decay in muscle of heterozygote malignant hyperthermia pigs was studied after in vivo exposure to a combination of halothane and succinylcholine. The pigs were anaesthetized with halothane and succinylcholine was injected in the ear vein. Immediately after initiation of the depolarizing neuromuscular blocking effect of succinylcholine the animals were captive-bolt stunned. The PCr decay, reflecting ATP turnover, was followed in situ by 31P-NMR spectroscopy in the biceps femoris muscle for the subsequent 40-70 min post mortem. In 3 of the 4 experiments, the Mg-fed pig had a significantly reduced rate of PCr hydrolysis compared to the control animal. The mechanism of this magnesium effect is unknown.     

An Epidemiologic and Economic Study of Respiratory Diseases in Two Conventional Danish Swine Herds. I: Prevalence of Respiratory Lesions at Slaughter and Their Effects on Growth

A total of 578 slaughter pigs from 2 Danish conventional farrow-to-finish operations (Herds A and B) were followed from an age of 14 days to slaughter. Pigs were weighed at 3 weeks intervals and at slaughter and an extended post mortem examination of the plucks was done. Comparison of growth rates in pigs with and without specific types of lesions by the t-test and those with multiple lesions with regression models demonstrated that Mycoplasma-like pneumonia, complicated pneumonia, anterio-ventral pleuritis, fissures and atrophic rhinitis significantly reduced mean daily gain and increased the time required to reach slaughter weight. The total impact of the lesions in Herd A was an estimated reduction in mean daily gain of 27 grams and a 2 day increase in the interval from 14 days of age until slaughter (MDG14). Decreases in MDG14 in Herd B were more substantial, 98 grams and 16.7 days. Reductions in mean daily gains during the interval from the fourth weighing until slaughter were 31 grams in Herd A and 137 grams in Herd B. Chronic dorso-caudal and parietal pleuritis, without other lesions present, had no significant adverse effects on growth rates in either herd. Interactions between lesions did not significantly alter the estimates. The R2 values obtained for the regression models showed that the presence, absence or extent of lesions at slaughter explained only 13-27% of the variations in growth rates in the 2 herds.     

THE NORMAL FINE STRUCTURE OF OPOSSUM TESTICULAR INTERSTITIAL CELLS

The interstitial tissue of the opossum testis includes interstitial or Leydig cells, macrophages, and small cells which morphologically resemble mesenchymal cells. The latter are thought to give rise to mature interstitial cells. The most prominent feature of the interstitial cell cytoplasm is an exceedingly abundant agranular endoplasmic reticulum. This reticulum is generally in the form of a meshwork of interconnected tubules about 300 to 450 A in diameter, but occasionally it assumes the form of flattened, fenestrated cisternae resembling those of pancreatic acinar cells, except for the lack of ribonucleoprotein particles on the surface of the membranes. The interstitial cells vary considerably in their cytoplasmic density. The majority are quite light, but some appear extremely dense, and in addition usually have a more irregular cell surface, with numerous small pseudopodia. These differences may well reflect variations in physiological state. Cytoplasmic structures previously interpreted as "crystalloids" consist of long bundles of minute parallel tubules, each about 180 A in diameter, which seem to be local differentiations of the endoplasmic reticulum. The mitochondria are rod-shaped, and contain a moderately complex internal membrane structure, and also occasional large inclusions that are spherical and homogeneous. The prominent juxtanuclear Golgi complex contains closely packed flattened sacs and small vesicles. The results of the present study, coupled with biochemical evidence from other laboratories, make it seem highly probable that the agranular endoplasmic reticulum is involved in the synthesis of the steroid hormones produced by the interstitial cell. This finding therefore constitutes one of the first functions of the agranular reticulum for which there is good morphological and biochemical evidence.     

Etlingera kenyalang (Zhgiberaceae) — a new species from Sarawak, Borneo, with notes on its ecology and Iban ethnobotany

Etlingera kenyalang is described from southern Sarawak. Ecological notes are given including a possible pollinator and seed dispersers. The Iban ethnobotany of the species is discussed; the shoots and fruits are edible and the leaves are used ritually during the Gawai Kenyalang (Hornbill Festival).     

Sex differences in heritability of BMI: a comparative study of results from twin studies in eight countries.

Body mass index (BMI), a simple anthropometric measure, is the most frequently used measure of adiposity and has been instrumental in documenting the worldwide increase in the prevalence of obesity witnessed during the last decades. Although this increase in overweight and obesity is thought to be mainly due to environmental changes, i.e., sedentary lifestyles and high caloric diets, consistent evidence from twin studies demonstrates high heritability and the importance of genetic differences for normal variation in BMI. We analysed self-reported data on BMI from approximately 37,000 complete twin pairs (including opposite sex pairs) aged 20-29 and 30-39 from eight different twin registries participating in the GenomEUtwin project. Quantitative genetic analyses were conducted and sex differences were explored. Variation in BMI was greater for women than for men, and in both sexes was primarily explained by additive genetic variance in all countries. Sex differences in the variance components were consistently significant. Results from analyses of opposite sex pairs also showed evidence of sex-specific genetic effects suggesting there may be some differences between men and women in the genetic factors that influence variation in BMI. These results encourage the continued search for genes of importance to the body composition and the development of obesity. Furthermore, they suggest that strategies to identify predisposing genes may benefit from taking into account potential sex specific effects.     

Stroke research in GenomEUtwin.

Stroke is one of the leading causes of severe disability and death in the world. In the present article we outline possibilities and limitations for future stroke research within the GenomEUtwin. The combined sample of twins born before 1958 from Denmark, Finland, and Sweden, and available for follow-up into the second millennium for non-fatal and fatal stroke events through national inpatient and death registers exceeds 70,000 twin pairs. This sample size will enable the study of genetic influences on stroke and major stroke subtypes. Large samples of twins in GenomEUtwin have been followed up repeatedly through interviews and questionnaires concerning a variety of exposures and potential risk factors for stroke. We briefly outline how this information can be combined with the health register information for epidemiologic and genetic epidemiologic studies of stroke. We also present the number of twin pairs concordant and discordant for stroke in Denmark, Finland and Sweden, and time lags between events for twins concordant for stroke. This information illustrates that the number of affected sib pairs for linkage studies is relatively limited, but the sample sizes are promising for association studies.