Advancing ecological understandings through technological transformations in noninvasive genetics

Noninvasive genetic approaches continue to improve studies in molecular ecology, conservation genetics and related disciplines such as forensics and epidemiology. Noninvasive sampling allows genetic studies without disturbing or even seeing the target individuals. Although noninvasive genetic sampling has been used for wildlife studies since the 1990s, technological advances continue to make noninvasive approaches among the most used and rapidly advancing areas in genetics. Here, we review recent advances in noninvasive genetics and how they allow us to address important research and management questions thanks to improved techniques for DNA extraction, preservation, amplification and data analysis. We show that many advances come from the fields of forensics, human health and domestic animal health science, and suggest that molecular ecologists explore literature from these fields. Finally, we discuss how the combination of advances in each step of a noninvasive genetics study, along with fruitful areas for future research, will continually increase the power and role of noninvasive genetics in molecular ecology and conservation genetics.     

Relationship between changes in brachial artery flow-mediated dilation and basal release of nitric oxide in subjects with Type 2 diabetes

Assessment of flow-mediated dilation (FMD) after forearm ischemia is widely used as a noninvasive bioassay of stimulated nitric oxide (NO)-mediated conduit artery vasodilator function in vivo. Whether this stimulated endothelial NO function reflects basal endothelial NO function is unknown. To test this hypothesis, retrospective analysis of randomized crossover studies was undertaken in 17 subjects with Type 2 diabetes; 9 subjects undertook an exercise training or control period, whereas the remaining 8 subjects were administered an angiotensin II receptor blocker or placebo. FMD was assessed by using wall tracking of high-resolution brachial artery ultrasound images in response to reactive hyperemia. Resistance vessel basal endothelium-dependent NO function was assessed by using intrabrachial administration of NG-monomethyl-L-arginine (L-NMMA) and plethysmographic assessment of forearm blood flow (FBF). FMD was higher after intervention compared with control/placebo (6.15+/-0.53 vs. 3.81+/-0.72%, P<0.001). There were no significant changes in the FBF responses to L-NMMA. Regression analysis between FMD and L-NMMA responses at entry to the study revealed an insignificant correlation (r=-0.10, P=0.7), and improvements in FMD with the interventions were not associated with changes in the L-NMMA responses (r=-0.04, P=0.9). We conclude that conduit artery-stimulated endothelial NO function (FMD) does not reflect basal resistance vessel endothelial NO function in subjects with Type 2 diabetes.     

Current and future approaches using genetically modified mice in endocrine research

Genetically modified mouse models have been used widely to advance our knowledge in the field of endocrinology and metabolism. A number of different approaches to generate genetically modified mice are now available, which provide the power to analyze the role of individual proteins in vivo. However, there are a number of points to be considered in the use and interpretation of these models. This review discusses the advantages and disadvantages involved in the generation and use of different genetically modified mouse models in endocrine research, including conventional techniques (e.g., overexpression, knockout, and knock-in models), tissue- and/or time-specific deletion of target genes [e.g., Cre-loxP and short interfering (si)RNA transgenic approaches], and gene-trap approaches to undertake functional genomics. This review also highlights the many factors that should be considered when assessing the phenotype of these mouse models, many of which are relevant to all murine physiological studies. These approaches are a powerful means by which to dissect the function of genes and are revolutionizing our understanding of endocrine physiology and metabolism.     

Derivatization of carbohydrates for analysis by chromatography; electrophoresis and mass spectrometry

Carbohydrates display a large diversity of structures and their analysis presents many obstacles as the result of properties such as isomeric diversity, existence of branched structures and the lack of chromophores or fluorophores. Consequently, many analytical approaches depend on the application of chemical modifications such as hydrolysis or derivative formation. This review covers various aspects of derivatization that are used for such approaches as improving thermal stability and volatility for gas-phase analyses, introduction of fluorophores for optical detectors, introduction of charge for mass spectral analyses and attachment of bioaffinity tags for bioactivity studies. Reducing carbohydrates contain, in addition to multiple hydroxyl groups, several other sites for derivatization such as the single anomeric site that has been used in numerous methods for attaching various property-enhancing tags. Other sites are restricted to specific carbohydrates but include carboxy groups in sialic acids and amino groups in glycosylamines. All of these groups have been the targets of derivatization and this review attempts to summarise the main methods used for these various functional groups.     

An empirical exploration of data quality in DNA-based population inventories

I present data from 21 population inventory studies - 20 of them on bears - that relied on the noninvasive collection of hair, and review the methods that were used to prevent genetic errors in these studies. These methods were designed to simultaneously minimize errors (which can bias estimates of abundance) and per-sample analysis effort (which can reduce the precision of estimates by limiting sample size). A variety of approaches were used to probe the reliability of the empirical data, producing a mean, per-study estimate of no more than one undetected error in either direction (too few or too many individuals identified in the laboratory). For the type of samples considered here (plucked hair samples), the gain or loss of individuals in the laboratory can be reduced to a level that is inconsequential relative to the more universal sources of bias and imprecision that can affect mark-recapture studies, assuming that marker systems are selected according to stated guidelines, marginal samples are excluded at an early stage, similar pairs of genotypes are scrutinized, and laboratory work is performed with skill and care.     

Cryo-electron tomography of cells: connecting structure and function

Cryo-electron tomography (cryo-ET) allows the visualization of cellular structures under close-to-life conditions and at molecular resolution. While it is inherently a static approach, yielding structural information about supramolecular organization at a certain time point, it can nevertheless provide insights into function of the structures imaged, in particular, when supplemented by other approaches. Here, we review the use of experimental methods that supplement cryo-ET imaging of whole cells. These include genetic and pharmacological manipulations, as well as correlative light microscopy and cryo-ET. While these methods have mostly been used to detect and identify structures visualized in cryo-ET or to assist the search for a feature of interest, we expect that in the future they will play a more important role in the functional interpretation of cryo-tomograms.     

Targeted metagenomics: a high-resolution metagenomics approach for specific gene clusters in complex microbial communities

A major research goal in microbial ecology is to understand the relationship between gene organization and function involved in environmental processes of potential interest. Given that more than an estimated 99% of microorganisms in most environments are not amenable to culturing, methods for culture-independent studies of genes of interest have been developed. The wealth of metagenomic approaches allows environmental microbiologists to directly explore the enormous genetic diversity of microbial communities. However, it is extremely difficult to obtain the appropriate sequencing depth of any particular gene that can entirely represent the complexity of microbial metagenomes and be able to draw meaningful conclusions about these communities. This review presents a summary of the metagenomic approaches that have been useful for collecting more information about specific genes. Specific subsets of metagenomes that focus on sequence analysis were selected in each metagenomic studies. This 'targeted metagenomics' approach will provide extensive insight into the functional, ecological and evolutionary patterns of important genes found in microorganisms from various ecosystems.     

The significance of clinical assessment of brain tissue oxygenation in different pathological conditions: an overview.

The development of methods of measurement of brain oxygenation in man is reviewed and the possible clinical potential of some new and established laboratory techniques is evaluated. Advantages and problems associated with the various approaches are considered together with the difficulties that are encountered in interpretation of data obtained and the factors that may increase such difficulties. It is concluded that invasive techniques that can only be used intraoperatively are of limited value but may be helpful in confirming the edges of ischemic areas or the restoration of adequate local blood flow. Chronically implanted devices have been useful in detecting epileptic foci and in evaluation of anesthetic regimes in patients with brain lesions. Infrared spectroscopy may offer possibilities for noninvasive whole brain monitoring in patients, but the method lacks resolution.     

Advances in technologies for 3D genomics research

The spatial structure of the orderly organized chromatin in the nucleus has important roles in maintaining normal cell function and in regulation of gene expression, and the high-throughput Hi-C and Ch IA-PET methods have been widely used in various biological studies for determining potential spatial genome structures and their functions. However, there are still great difficulties and challenges in three-dimensional(3D) genomics research. More efficient, economical, and unbiased approaches to studying 3D genomics need to be developed for more widespread and easier applications. Here, we review the most recent studies on new 3D genomics research technologies, such as improvements of the traditional Hi-C and Ch IA-PET methods, new approaches based on non-proximal-ligation strategies, and imaging-based methods improved in recent years. Especially, we review the CRISPR-based methods for functional validations in 3D genomics, which could be the forthcoming directions. We hope this review can show some insights into the potential improvements for future 3D genomics.     

The clinical diagnosis of splenomegaly.

Assessing for the presence of splenomegaly is an important component of the physical examination. Although several methods of palpation and percussion of the spleen have been described, until recently they have not been validated by noninvasive imaging techniques such as ultrasonography, radionuclide scanning, and computed tomography that offer objective means to assess splenomegaly. We review the literature comparing various physical examination techniques with noninvasive imaging modalities and conclude that palpation and percussion of the spleen are complementary but frequently insensitive and that further studies are needed to evaluate the efficacy of specific diagnostic methods.     

Bacterial Sub-Species Typing Using Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry: What Is Promising?

Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) is routinely used for bacterial identification. It would be highly beneficial to also be able to use the technology as a fast way to detect clinically relevant clones of bacterial species. However, studies to this aim have often had limited success. The methods used for data acquisition, processing and data interpretation are highly diverse amongst studies on MALDI-TOF MS sub-species typing. In addition to this, feasibility may depend on the bacterial species and strains investigated, making it difficult to determine what methods may or may not work. In our paper, we have reviewed recent research on MALDI-TOF MS typing of bacterial strains. Although we found a lot of variation amongst the methods used, there were approaches shared by multiple research groups. Multiple spectra of the same isolate were often combined before further analysis for strain distinction. Many groups used a protein extraction step to increase resolution in their MALDI-TOF MS results. Peaks at a high mass range were often excluded for data interpretation. Three groups have found ways to determine feasibility of MALDI-TOF MS typing for their set of strains at an early stage of their project.     

Noninvasive reconstruction of cardiac electrical activity: update on current methods,applications and challenges

Electrical activity at the level of the heart muscle can be noninvasively reconstructed from body-surface electrocardiograms (ECGs) and patient-specific torso-heart geometry. This modality, coined electrocardiographic imaging, could fill the gap between the noninvasive (low-resolution) 12-lead ECG and invasive (high-resolution) electrophysiology studies. Much progress has been made to establish electrocardiographic imaging, and clinical studies appear with increasing frequency. However, many assumptions and model choices are involved in its execution, and only limited validation has been performed. In this article, we will discuss the technical details, clinical applications and current limitations of commonly used methods in electrocardiographic imaging. It is important for clinicians to realise the influence of certain assumptions and model choices for correct and careful interpretation of the results. This, in combination with more extensive validation, will allow for exploitation of the full potential of noninvasive electrocardiographic imaging as a powerful clinical tool to expedite diagnosis, guide therapy and improve risk stratification.     

Hair analysis for abused and therapeutic drugs

This review focuses on basic aspects and recent studies of hair analysis for abused and therapeutic drugs and is discussed with 164 references. Firstly, biology of hair and sampling of hair specimens have been commented for the sake of correct interpretation of the results from hair analysis. Then the usual washing methods of hair samples and the extraction methods for drugs in hair have been shown and commented on. Analytical methods for each drug have been discussed by the grouping of three analytical methods, namely immunoassay, HPLC–CE and GC–MS. The outcomes of hair analysis studies have been reviewed by dividing into six groups; morphine and related, cocaine and related, amphetamines, cannabinoids, the other abused drugs and therapeutic drugs. In addition, reports on stability of drugs in the living hair and studies on drug incorporation into hair and dose–hair concentration relationships have been reviewed. Applications of hair analysis to the estimation of drug history, discrimination between OTC drug use and illegal drug use, drug testing for acute poisoning, gestational drug exposure and drug compliance have also been reviewed. Finally, the promising prospects of hair analysis have been described.     

食线虫菌物胞外蛋白酶基因工程研究进展

在病原线虫的生物防治中 ,利用食线虫菌物在侵染过程中分泌的胞外蛋白酶 (重要毒力因子 )固定线虫并降解线虫体壁显示出巨大的潜力。综述了近年来食线虫菌物胞外蛋白酶的研究概况、目前开展蛋白酶基因工程研究存在的问题及解决策略;对真菌胞外蛋白酶应用于线虫生防和生物医药领域中的前景也进行了评述。     

Improved analysis of brachial artery ultrasound using a novel edge-detection software system.

Brachial artery ultrasound is commonly employed for noninvasive assessment of endothelial function. However, analysis is observer dependent and susceptible to errors. We describe studies on a computerized edge-detection and wall-tracking software program to allow more accurate and reproducible measurement. In study 1, three purpose-built Perspex phantom arteries, 3.00, 4.00, and 6.00 mm in diameter, were measured with the software. There was a mean bias of 11 microm (P < 0.001 at each level) between known and measured values; the mean resolving power of the software was estimated as 8.3 microm. In study 2, the mean intraobserver coefficient of variation of repeated measures of flow-mediated dilation (FMD) using the software (6.7%) was significantly lower than that for traditional manual measurements using the intima-lumen interfaces (24.8%, P < 0.05) and intima-media interfaces (32.5%, P < 0.05). In study 3, 24 healthy volunteers underwent repeat testing twice within 1 wk; the coefficients of variation for between-visit reproducibility of FMD and response to glyceryl trinitrate using the software were 14.7 and 17.6%, respectively. Assuming 80% power and an alpha of 0.05, eight subjects with matched controls would be required, in a parallel designed study, to detect an absolute 2.5% change in FMD. In summary, we have developed a semiautomated computerized vascular ultrasound analysis system that will improve the power of clinical intervention studies to detect small changes in arterial diameter.     

Target identification of bioactive compounds

To fully understand the regulation of cellular events, functional analysis of each protein involved in the regulatory systems is required. Among a variety of methods to uncover protein function, chemical genetics is a remarkable approach in which small molecular compounds are used as probes to elucidate protein functions within signaling pathways. However, identifying the target of small molecular bioactive compounds isolated by cell-based assays represents a crucial hurdle that must be overcome before chemical genetic studies can commence. A variety of methods and technologies for identifying target proteins have been reported. This review therefore aims to describe approaches for identifying these molecular targets.     

Network-assisted analysis to prioritize GWAS results: principles, methods and perspectives

Genome-wide association studies (GWAS) have rapidly become a powerful tool in genetic studies of complex diseases and traits. Traditionally, single marker-based tests have been used prevalently in GWAS and have uncovered tens of thousands of disease-associated SNPs. Network-assisted analysis (NAA) of GWAS data is an emerging area in which network-related approaches are developed and utilized to perform advanced analyses of GWAS data in order to study various human diseases or traits. Progress has been made in both methodology development and applications of NAA in GWAS data, and it has already been demonstrated that NAA results may enhance our interpretation and prioritization of candidate genes and markers. Inspired by the strong interest in and high demand for advanced GWAS data analysis, in this review article, we discuss the methodologies and strategies that have been reported for the NAA of GWAS data. Many NAA approaches search for subnetworks and assess the combined effects of multiple genes participating in the resultant subnetworks through a gene set analysis. With no restriction to pre-defined canonical pathways, NAA has the advantage of defining subnetworks with the guidance of the GWAS data under investigation. In addition, some NAA methods prioritize genes from GWAS data based on their interconnections in the reference network. Here, we summarize NAA applications to various diseases and discuss the available options and potential caveats related to their practical usage. Additionally, we provide perspectives regarding this rapidly growing research area.     

Genomic sequencing of single microbial cells from environmental samples

Recently developed techniques allow genomic DNA sequencing from single microbial cells [Lasken RS: Single-cell genomic sequencing using multiple displacement amplification. Curr Opin Microbiol 2007, 10:510-516]. Here, we focus on research strategies for putting these methods into practice in the laboratory setting. An immediate consequence of single-cell sequencing is that it provides an alternative to culturing organisms as a prerequisite for genomic sequencing. The microgram amounts of DNA required as template are amplified from a single bacterium by a method called multiple displacement amplification (MDA) avoiding the need to grow cells. The ability to sequence DNA from individual cells will likely have an immense impact on microbiology considering the vast numbers of novel organisms, which have been inaccessible unless culture-independent methods could be used. However, special approaches have been necessary to work with amplified DNA. MDA may not recover the entire genome from the single copy present in most bacteria. Also, some sequence rearrangements can occur during the DNA amplification reaction. Over the past two years many research groups have begun to use MDA, and some practical approaches to single-cell sequencing have been developed. We review the consensus that is emerging on optimum methods, reliability of amplified template, and the proper interpretation of 'composite' genomes which result from the necessity of combining data from several single-cell MDA reactions in order to complete the assembly. Preferred laboratory methods are considered on the basis of experience at several large sequencing centers where >70% of genomes are now often recovered from single cells. Methods are reviewed for preparation of bacterial fractions from environmental samples, single-cell isolation, DNA amplification by MDA, and DNA sequencing.     

Mitigation of stress: new treatment alternatives

Complaints of stress are common in modern life. Psychological stress is a major cause of lifestyle-related issues, contributing to poor quality of life. Chronic stress impedes brain function, causing impairment of many executive functions, including working memory, decision making and attentional control. The current study sought to describe newly developed stress mitigation techniques, and their influence on autonomic and endocrine functions. The literature search revealed that the most frequently studied technique for stress mitigation was biofeedback (BFB). However, evidence suggests that neurofeedback (NFB) and noninvasive brain stimulation (NIBS) could potentially provide appropriate approaches. We found that recent studies of BFB methods have typically used measures of heart rate variability, respiration and skin conductance. In contrast, studies of NFB methods have typically utilized neurocomputation techniques employing electroencephalography, functional magnetic resonance imaging and near infrared spectroscopy. NIBS studies have typically utilized transcranial direct current stimulation methods. Mitigation of stress is a challenging but important research target for improving quality of life.