Mechanisms of valence selectivity in biological ion channels

Transmembrane ion channels play a crucial role in the existence of all living organisms. They partition the exterior from the interior of the cell, maintain the proper ionic gradient across the cell membrane and facilitate signaling between cells. To perform these functions, ion channels must be highly selective, allowing some types of ions to pass while blocking the passage of others. Here we review a number of studies that have helped to elucidate the mechanisms by which ion channels discriminate between ions of differing charge, focusing on four channel families as examples: gramicidin, ClC chloride, voltage-gated calcium and potassium channels. The recent availability of high-resolution structural data has meant that the specific inter-atomic interactions responsible for valence selectivity can be pinpointed. Not surprisingly, electrostatic considerations have been shown to play an important role in ion specificity, although many details of the origins of this discrimination remain to be determined. Received 4 September 2005; received after revision 17 October 2005; accepted 2 November 2005     

Direct ink writing of polycaprolactone/polyethylene oxide based 3D constructs

There has been increasing interest over recent years in the application of three-dimensional(3 D) printing technologies in the biomedical field. One such method is Direct Ink Writing(DIW); this approach has the potential advantage of allowing room-temperature deposition of materials, presented as an ink, to build complex architectures. DIW offers the ability to process biomaterials containing temperature-sensitive components. Due to the fabrication principles of DIW, there are specific rheological requirements that the ink must exhibit for the 3 D construction. For this reason, hydrogel-based liquid feed stocks have been the focal point of ink development. As a consequence, studies based on inks comprising hydrophobic biomaterials, which are insoluble in water and hence unsuited to the hydrogel approach, have been limited.In this study, we investigate novel inks that utilize polycaprolactone(PCL), a hydrophobic polymer, as the primary constituent by dissolving the polymer in solvent systems based on dichloromethane(DCM) and acetone(ACE). Moreover, polyethylene oxide(PEO) was incorporated into the PCL systems in order to extend the range of hydrophilicity of the systems. The rheological properties of the inks were investigated as a function of polymer composition and solvent system. Woodpile constructs of PCL and PCL/PEO were fabricated using DIW method and were assessed by a series of material characterisation. The type of solvent system had a noticeable impact on the ink rheology, which ultimately affected the surface properties. The incorporation of PEO particularly enhanced the roughness and wettability of the constructs. Our results support the use of DIW as a new means to process hydrophobic polymers for biomedical applications.     

Detectable clonal mosaicism and its relationship to aging and cancer

In an analysis of 31,717 cancer cases and 26,136 cancer-free controls from 13 genome-wide association studies, we observed large chromosomal abnormalities in a subset of clones in DNA obtained from blood or buccal samples. We observed mosaic abnormalities, either aneuploidy or copy-neutral loss of heterozygosity, of >2 Mb in size in autosomes of 517 individuals (0.89%), with abnormal cell proportions of between 7% and 95%. In cancer-free individuals, frequency increased with age, from 0.23% under 50 years to 1.91% between 75 and 79 years (P = 4.8 × 10(-8)). Mosaic abnormalities were more frequent in individuals with solid tumors (0.97% versus 0.74% in cancer-free individuals; odds ratio (OR) = 1.25; P = 0.016), with stronger association with cases who had DNA collected before diagnosis or treatment (OR = 1.45; P = 0.0005). Detectable mosaicism was also more common in individuals for whom DNA was collected at least 1 year before diagnosis with leukemia compared to cancer-free individuals (OR = 35.4; P = 3.8 × 10(-11)). These findings underscore the time-dependent nature of somatic events in the etiology of cancer and potentially other late-onset diseases.     

Mutations in the gene encoding GlyT2 (SLC6A5) define a presynaptic component of human startle disease

Hyperekplexia is a human neurological disorder characterized by an excessive startle response and is typically caused by missense and nonsense mutations in the gene encoding the inhibitory glycine receptor (GlyR) alpha1 subunit (GLRA1). Genetic heterogeneity has been confirmed in rare sporadic cases, with mutations affecting other postsynaptic glycinergic proteins including the GlyR beta subunit (GLRB), gephyrin (GPHN) and RhoGEF collybistin (ARHGEF9). However, many individuals diagnosed with sporadic hyperekplexia do not carry mutations in these genes. Here we show that missense, nonsense and frameshift mutations in SLC6A5 (ref. 8), encoding the presynaptic glycine transporter 2 (GlyT2), also cause hyperekplexia. Individuals with mutations in SLC6A5 present with hypertonia, an exaggerated startle response to tactile or acoustic stimuli, and life-threatening neonatal apnea episodes. SLC6A5 mutations result in defective subcellular GlyT2 localization, decreased glycine uptake or both, with selected mutations affecting predicted glycine and Na+ binding sites.     

Conformational changes in the G protein Gs induced by the β2 adrenergic receptor

G protein-coupled receptors represent the largest family of membrane receptors that instigate signalling through nucleotide exchange on heterotrimeric G proteins. Nucleotide exchange, or more precisely, GDP dissociation from the G protein α-subunit, is the key step towards G protein activation and initiation of downstream signalling cascades. Despite a wealth of biochemical and biophysical studies on inactive and active conformations of several heterotrimeric G proteins, the molecular underpinnings of G protein activation remain elusive. To characterize this mechanism, we applied peptide amide hydrogen-deuterium exchange mass spectrometry to probe changes in the structure of the heterotrimeric bovine G protein, Gs (the stimulatory G protein for adenylyl cyclase) on formation of a complex with agonist-bound human β(2) adrenergic receptor (β(2)AR). Here we report structural links between the receptor-binding surface and the nucleotide-binding pocket of Gs that undergo higher levels of hydrogen-deuterium exchange than would be predicted from the crystal structure of the β(2)AR-Gs complex. Together with X-ray crystallographic and electron microscopic data of the β(2)AR-Gs complex (from refs 2, 3), we provide a rationale for a mechanism of nucleotide exchange, whereby the receptor perturbs the structure of the amino-terminal region of the α-subunit of Gs and consequently alters the 'P-loop' that binds the β-phosphate in GDP. As with the Ras family of small-molecular-weight G proteins, P-loop stabilization and β-phosphate coordination are key determinants of GDP (and GTP) binding affinity.     

Synaptic potentiation onto habenula neurons in the learned helplessness model of depression

The cellular basis of depressive disorders is poorly understood. Recent studies in monkeys indicate that neurons in the lateral habenula (LHb), a nucleus that mediates communication between forebrain and midbrain structures, can increase their activity when an animal fails to receive an expected positive reward or receives a stimulus that predicts aversive conditions (that is, disappointment or anticipation of a negative outcome). LHb neurons project to, and modulate, dopamine-rich regions, such as the ventral tegmental area (VTA), that control reward-seeking behaviour and participate in depressive disorders. Here we show that in two learned helplessness models of depression, excitatory synapses onto LHb neurons projecting to the VTA are potentiated. Synaptic potentiation correlates with an animal's helplessness behaviour and is due to an enhanced presynaptic release probability. Depleting transmitter release by repeated electrical stimulation of LHb afferents, using a protocol that can be effective for patients who are depressed, markedly suppresses synaptic drive onto VTA-projecting LHb neurons in brain slices and can significantly reduce learned helplessness behaviour in rats. Our results indicate that increased presynaptic action onto LHb neurons contributes to the rodent learned helplessness model of depression.     

韩国伞滑刃属线虫的形态和分子特征

以锥尾伞滑刃线虫（Bursaphelenchus conicaudatus）作为对照,在韩国不同地域和寄主中采集了15个松材线虫分离物、3 个拟松材线虫的分离物及5个未鉴定的该属的分离物,并对其进行了ITS 和 D2D3 rDNA序列分析。以单条雌线虫的DNA为模板,ITS 和 D2D3 区由PCR仪扩增并克隆其序列。结果表明,所有松材线虫的ITS 和 D2D3 区序列相同,没有种内变异。然而2个采自黑松和红松的拟松材线虫分离物的基因型分别是亚洲型和欧洲型。5个未鉴定线虫的数据表明,它们分别接近B. tusciae、B. lini、B. thailandae、B. doui和 B. hylobianum,该结果同时被形态学结果所支持。利用5种酶（Hinf I、Alu I、Msp I、Hae III、Rsa）,PCR产物克隆并测序的数据也可以区分不同种和基因型。     

Induction of deoxyribonucleic acid degradation in Escherichia coli by ozone.

Cell survival and deoxyribonucleic acid (DNA) degradation wave measured for wild-type Escherichia coli B251 cells after exposure to different concentrations of ozone. The results show that extensive breakdown of DNA occurs after ozonation and that the extent of ozone-induced DNA degradation generally correlates with the colony-forming ability of the cells.     

Forecasting time series with outliers

Time-series data are often contaminated with outliers due to the influence of unusual and non-repetitive events. Forecast accuracy in such situations is reduced due to (1) a carry-over effect of the outlier on the point forecast and (2) a bias in the estimates of model parameters. Hillmer (1984) and Ledolter (1989) studied the effect of additive outliers on forecasts. It was found that forecast intervals are quite sensitive to additive outliers, but that point forecasts are largely unaffected unless the outlier occurs near the forecast origin. In such a situation the carry-over effect of the outlier can be quite substantial. In this study, we investigate the issues of forecasting when outliers occur near or at the forecast origin. We propose a strategy which first estimates the model parameters and outlier effects using the procedure of Chen and Liu (1993) to reduce the bias in the parameter estimates, and then uses a lower critical value to detect outliers near the forecast origin in the forecasting stage. One aspect of this study is on the carry-over effects of outliers on forecasts. Four types of outliers are considered: innovational outlier, additive outlier, temporary change, and level shift. The effects due to a misidentification of an outlier type are examined. The performance of the outlier detection procedure is studied for cases where outliers are near the end of the series. In such cases, we demonstrate that statistical procedures may not be able to effectively determine the outlier types due to insufficient information. Some strategies are recommended to reduce potential difficulties caused by incorrectly detected outlier types. These findings may serve as a justification for forecasting in conjunction with judgment. Two real examples are employed to illustrate the issues discussed.