Locus effects and sources of error in noninvasive genotyping

In spite of more than a decade of research on noninvasive genetic sampling, the low quality and quantity of DNA in noninvasive studies continue to plague researchers. Effects of locus size on error have been documented but are still poorly understood. Further, sources of error other than allelic dropout have been described but are often not well quantified. Here we analyse the effects of locus size on allelic dropout, amplification success and error rates in noninvasive genotyping studies of three species, and quantify error other than allelic dropout.     

Noninvasive Doppler-derived myocardial performance index in rats with myocardial infarction: validation and correlation by conductance catheter

The rodent model of myocardial infarction (MI) is extensively used in heart failure studies. However, long-term follow-up of echocardiographic left ventricular (LV) function parameters such as the myocardial performance index (MPI) and its ratio with the fractional shortening (LVFS/MPI) has not been validated in conjunction with invasive indexes, such as those derived from the conductance catheter (CC). Sprague-Dawley rats with left anterior descending coronary artery ligation (MI group, n = 9) were compared with a sham-operated control group (n = 10) without MI. Transthoracic echocardiography (TTE) was performed every 2 wk over an 8-wk period, after which classic TTE parameters, especially MPI and LVFS/MPI, were compared with invasive indexes obtained by using a CC. Serial TTE data showed significant alterations in the majority of the noninvasive functional and structural parameters (classic and novel) studied in the presence of MI. Both MPI and LVFS/MPI significantly (P < 0.05 for all reported values) correlated with body weight (r = -0.58 and 0.76 for MPI and LVFS/MPI, respectively), preload recruitable stroke work (r = -0.61 and 0.63), LV end-diastolic pressure (LVEDP) (r = 0.82 and -0.80), end-diastolic volume (r = 0.61 and -0.58), and end-systolic volume (r = 0.46 and -0.48). Forward stepwise linear regression analysis revealed that, of all variables tested, LVEDP was the only independent determinant of MPI (r = 0.84) and LVFS/MPI (r = 0.83). We conclude that MPI and LVFS/MPI correlate strongly and better than the classic noninvasive TTE parameters with established, invasively assessed indexes of contractility, preload, and volumetry. These findings support the use of these two new noninvasive indexes for long-term analysis of the post-MI LV remodeling.     

A genotyping‐in‐thousands by sequencing panel to inform invasive deer management using noninvasive fecal and hair samples

Studies in ecology, evolution, and conservation often rely on noninvasive samples, making it challenging to generate large amounts of high‐quality genetic data for many elusive and at‐risk species. We developed and optimized a Genotyping‐in‐Thousands by sequencing (GT‐seq) panel using noninvasive samples to inform the management of invasive Sitka black‐tailed deer (Odocoileus hemionus sitkensis) in Haida Gwaii (Canada). We validated our panel using paired high‐quality tissue and noninvasive fecal and hair samples to simultaneously distinguish individuals, identify sex, and reconstruct kinship among deer sampled across the archipelago, then provided a proof‐of‐concept application using field‐collected feces on SGang Gwaay, an island of high ecological and cultural value. Genotyping success across 244 loci was high (90.3%) and comparable to that of high‐quality tissue samples genotyped using restriction‐site associated DNA sequencing (92.4%), while genotyping discordance between paired high‐quality tissue and noninvasive samples was low (0.50%). The panel will be used to inform future invasive species operations in Haida Gwaii by providing individual and population information to inform management. More broadly, our GT‐seq workflow that includes quality control analyses for targeted SNP selection and a modified protocol may be of wider utility for other studies and systems where noninvasive genetic sampling is employed.     

^19FNMR在生物医学研究中的应用

核磁共振（ＮＭＲ）是一种无创伤的物理测试方法,它可以直接用于体内和体外的生物样品测定,提供分子水平的信息。正常体内含氟成分很少,测定进无本底信号干扰,因此在体内研究中引进氟代指示剂进行＾１９ＦＮＭＲ研究是目前普遍采用的方法。＾１９ＦＮＭＲ可可以用来测定药物在体内代谢过程、胸内游离的离子如Ｃａ＾２＋和Ｍｇ＾２＋、胞内ｐＨ、氧浓度或氧压力（ｐＯ２）、膜电位、组织温度、血液容积和细胞容积等多项生理生化指     

When can noninvasive samples provide sufficient information in conservation genetics studies?

Noninvasive sampling, of faeces and hair for example, has enabled many genetic studies of wildlife populations. However, two prevailing problems common to these studies are small sample sizes and high genotyping errors. The first problem stems from the difficulty in collecting noninvasive samples, particularly from populations of rare or elusive species, and the second is caused by the low quantity and quality of DNA extracted from a noninvasive sample. A common question is therefore whether noninvasive sampling provides sufficient information for the analyses commonly conducted in conservation genetics studies. Here, we conducted a simulation study to investigate the effect of small sample sizes and genotyping errors on the precision and accuracy of the most commonly estimated genetic parameters. Our results indicate that small sample sizes cause little bias in measures of expected heterozygosity, pairwise F_ST and population structure, but a large downward bias in estimates of allelic diversity. Allelic dropouts and false alleles had a much smaller effect than missing data, which effectively reduces sample size further. Overall, reasonable estimates of genetic variation and population subdivision are obtainable from noninvasive samples as long as error rates are kept below a frequency of 0.2. Similarly, unbiased estimates of population clustering can be made with genotyping error rates below 0.5 when the populations are highly differentiated. These results provide a useful guide for researchers faced with studying the conservation genetics of small, endangered populations from noninvasive samples.     

Noninvasive Saliva Collection for DNA Analyses From Free‐Ranging Tibetan Macaques (Macaca thibetana)

Cryptic and endangered fauna, including many primate taxa, pose challenges for noninvasive collection of biomaterials. As a result, application of noninvasive genotyping to primates has been limited to the use of samples such as feces and hair for the extraction of PCR‐amplifiable DNA. We present a method for noninvasive collection of saliva from habituated, free‐ranging monkeys. The method utilizes a low‐cost apparatus that controls for contamination and is usable with individual, free‐ranging primates. Saliva samples were collected from 18 individuals in a population of Tibetan macaques (Macaca thibetana) in the Valley of Wild Monkeys in Huangshan, People's Republic of China. DNA was extracted from these samples and PCR‐amplified for both mitochondrial and nuclear genes, Cytochrome B and MHC‐DR Beta 1, respectively. These results indicate this is an effective technique for the noninvasive collection of saliva across age and sex class, and dominance rank in a free‐ranging, terrestrial primate species. This device could have wide application for obtaining high‐quality saliva samples from free‐ranging primate populations for use in epidemiological studies, hormonal analyses of HPA axis function, pathogen screening, noninvasive genotyping, and behavioral genetics. Am. J. Primatol. 74:1064‐1070, 2012. © 2012 Wiley Periodicals, Inc.     

基于全场光学相干层析技术评估卵母细胞质量

卵胞质质量是影响卵母细胞发育能力的重要因素,然而目前仍缺少无损且客观高效地评估卵胞质成熟度的方法.已知胞质成熟度低是导致体外成熟卵母细胞发育能力远低于体内成熟卵母细胞的主要原因.全场光学相干层析技术(FF-OCT)具有无损非标记且三维高分辨的特点.经对比FF-OCT采集的体内成熟与体外成熟小鼠卵胞质图像发现,具有大面积高亮皮质区及胞质均匀分布的卵母细胞,其胞质成熟度更高.为验证这一点,对比了显微镜下筛选和FF-OCT筛选的体外成熟卵母细胞的体外受精率,发现显微镜筛选组的体外受精率远低于体内成熟的情况,FF-OCT筛选组的体外受精率与体内成熟组之间无显著差异.这说明FF-OCT筛选的体外成熟卵母细胞发育能力接近于体内成熟的卵母细胞.此外还利用FF-OCT成功观测到新鲜及老化卵母细胞之间的形态差别,即老化卵母细胞胞质均匀性更差及高亮皮质区更小.因此,FF-OCT可用于无损评估卵胞质质量,且大面积高亮皮质区的存在及胞质分布的均匀性是评估卵胞质质量的有效指标.     

Early comers occupy popular territories in a lycaenid butterfly,Chrysozephyrus smaragdinus (Lepidoptera: Lycaenidae)

Territorial activities of a lycaenid butterfly, Chrysozephyrus smaragdinus, were observed throughout its adult season to investigate the territorial dynamics. The territories occupied earlier in the season were abandoned later than later‐occupied territories, indicating that the site preference of male C. smaragdinus was largely maintained during the adult season. Three indexes of territory quality based on animal preference, occupation duration (OD), initial occupation date (IOD) and the number of contests (NOC), were highly correlated. Therefore, these preference‐based indexes are consistent and reliable, although they are indirect estimates of benefits obtained from territories. Males arriving at territory sites early in the season were more likely to hold territories of better quality when NOC was used as the index of territory quality. The positive relationship between territory quality and male arrival date is congruent with the result of a previous study showing that early comers tend to hold a territory whereas late comers do not. However, male arrival date was not related to territory quality when OD or IOD was used as an index of territory quality. One possible reason for this apparent discrepancy is that the coefficient of variation of NOC is larger than those of OD and IOD, and therefore NOC could evaluate territory quality more quantitatively than the other two indexes. To date, studies of territoriality in butterflies have not considered the quality of territories. More studies considering the quality of territories will provide new insights into butterfly territoriality.     

A factorial design experiment as a pilot study for noninvasive genetic sampling

Noninvasive genetic sampling has increasingly been used in ecological and conservation studies during the last decade. A major part of the noninvasive genetic literature is dedicated to the search for optimal protocols, by comparing different methods of collection, preservation and extraction of DNA from noninvasive materials. However, the lack of quantitative comparisons among these studies and the possibility that different methods are optimal for different systems make it difficult to decide which protocol to use. Moreover, most studies that have compared different methods focused on a single factor – collection, preservation or extraction – while there could be interactions between these factors. We designed a factorial experiment, as a pilot study, aimed at exploring the effect of several collection, preservation and extraction methods, and the interactions between them, on the quality and amplification success of DNA obtained from Asiatic wild ass (Equus hemionus) faeces in Israel. The amplification success rates of one mitochondrial DNA and four microsatellite markers differed substantially as a function of collection, preservation and extraction methods and their interactions. The most efficient combination for our system integrated the use of swabs as a collection method with preservation at ?20 °C and with the Qiagen DNA Stool Kit with modifications as the DNA extraction method. The significant interaction found between the collection, preservation methods and the extraction methods reinforces the importance of conducting a factorial design experiment, rather than examining each factor separately, as a pilot study before initiating a full‐scale noninvasive research project.     

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.     

Analysis of noninvasive macromolecular transport measurements in the lung

Several groups of investigators are measuring transcapillary protein flux in the lung using noninvasive methods. Results from these studies are reported using several different protein transport indexes, including pulmonary transvascular transfer coefficient, relative extravascular protein, pulmonary transcapillary escape rate, protein leak index, lung transferrin index, slope index, and lung-to-heart count ratios. The purpose of this study is to discover the relationships between these indexes by employing a two-compartment theory of protein transcapillary transport in the lung. We found that all the above indexes can be related to a single index, which we call the normalized slope index. This index is the time rate of change of radioactivity originating from protein in lung interstitium divided by radioactivity arising from protein in lung plasma, normalized by this ratio at time 0, and corrected for blood volume changes. In particular the normalized slope index is shown to be the same as pulmonary transcapillary escape rate under normal sampling conditions and is relatively unaffected by changes in interstitial volume. The response of the normalized slope index to changes in microvascular pressure and microvascular permeability is explored by applying a two-pore model of the microvascular barrier. Results indicate that the normalized slope index is relatively insensitive to changes in microvascular pressure but is greatly affected by changes in microvascular permeability (i.e., changes in large-pore size or number). Since all published leak indexes are related, we would encourage all investigators in the field to adopt a single leak index. We recommend that when a two-compartment model is applied to external detection data, the results be expressed as pulmonary transcapillary escape rate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Multiplex pre-amplification for noninvasive genetic sampling: is the extra effort worth it?

Microsatellite genotyping of hair and faeces using standard polymerase chain reaction (PCR) resulted in low success rates and high error rates in a 2003–2004 pilot study using noninvasive genetic sampling for the brown bear (Ursus arctos) in the Italian Alps. Thus, we evaluated the performance of multiplex pre-amplification for improving microsatellite genotyping results. Brown bear faecal DNA extracts of varying quality (n = 33) and hair DNA extracts of poor (n = 32) and good (n = 34) quality were used to compare standard PCR and pre-amplification. In contrast to previous studies, there was no significant difference between methods for individual locus amplification success, genotyping error and genotyping success rates for scat and hair samples. The use of pre-amplification requires an additional investment of time and resources, and our results raise questions about the universal value of pre-amplification approaches. We suggest that researchers carefully evaluate the performance of pre-amplification compared to standard PCR using field-collected samples from the study area of interest before engaging in large-scale noninvasive genetic analyses.     

食用菌产业化生产的质量安全保障措施

总结多年食用菌生产实践经验,并在多项科研项目中运用和充实,制定了《安全食用菌产品生产技术要求(企业标准)》。在食用菌产业化示范中对安全食用菌生产场地环境、制种和菌种经营、栽培工艺、产品质量等提出了专业和具体的要求,为食用菌生产技术的升级换代和规范化提供技术参数和质量指标,为提高食用菌产品质量、保障消费者安全与健康提供依据。     

Clinical application of near infrared fiber optic spectroscopy for noninvasive bone assessment

Approaches for noninvasive bone quality assessment are of great clinical need, particularly in individuals that require close monitoring of disease progression. X‐ray measurements are standard approaches to assess bone quality; however, they have several disadvantages. Here, a nonionizing approach for noninvasive assessment of the second metacarpal bone based on near infrared (NIR) spectroscopy was investigated. Transcutaneous bone signal detection was experimentally confirmed with cadaveric hand data, and Monte Carlo modeling further indicated that 50% of the measured signals arise from bone. Spectral data were collected via a NIR fiber optic from the bone of individuals with osteogenesis imperfecta, a disease marked by frequent bone fractures and fragility. Multiple significant correlations were found between spectral parameters related to water, protein and fat, and standard bone quality parameters obtained by X‐ray measurements. The results from this preliminary study highlight the potential application of NIR spectroscopy for the noninvasive assessment of bone quality.     

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.     

Noninvasive quality estimation of adherent mammalian cells for transplantation

The noninvasive quality estimation of adherent mammalian cells for transplantation is reviewed. The quality and heterogeneity of cells should be estimated before transplantation because cultured cells are not homogeneous but heterogeneous. The estimation of cell quality should be performed noninvasively because most protocols of regenerative medicine are autologous cell system. The differentiation level and contamination of other cell lineage could be estimated by two-dimensional cell morphology analysis and tracking using a conventional phase contrast microscope. The noninvasive determination of the laser phase shift of a cell using a phase-shifting laser microscope, which might be more noninvasive, and more useful than the atomic force microscope and digital holographic microscope, was carried out to determine the three-dimensional cell morphology, and the estimation of the cell cycle phase of each adhesive cell and the mean proliferation activity of a cell population. Chemical analysis of the culture supernatant by conventional analytical methods such as ELISA was also useful to estimate the differentiation level of a cell population. Chemical analysis of cell membrane and intracellular components using a probe beam, an infrared beam, and Raman spectroscopy was useful for diagnosing the viability, apoptosis, and differentiation of each adhesive cell.     

转录本组装与质量评价

转录本组装是基于第二代测序技术研究转录组的关键环节,其质量好坏直接影响到下游结果的可靠性,也是目前的研究热点与难点。转录本组装方法可以分为Genome-guided和de novo两类,它们在理论基础与算法实现方面各有优劣。转录本组装质量的高低依赖于PCR扩增错误率、第二代测序技术准确率、组装算法和参考基因组完整性等方面,而现有的算法还无法完全处理由这些因素带来的影响。本文从转录本组装方法与软件、影响组装质量的因素和对组装质量的评价指标等方面进行讨论,以期能指导纯生物学家对分析软件的选择。     

Noninvasive bovine oocyte quality assessment: possibilities of a single oocyte culture

Although bovine embryos are routinely produced in vitro for several decades, there still exists a critical need for techniques to accurately predict the oocyte's developmental competence in a noninvasive way, before the in vitro embryo production procedure. In this review, several noninvasive methods to evaluate oocyte quality are discussed, such as morphological assessment of the cumulus oocyte complex and the use of brilliant cresyl blue. Because an individual oocyte and embryo culture method can possibly generate additional insights into the factors that determine oocyte quality, the second part of this review summarizes the state of the art of bovine single oocyte culture. The optimization of individual in vitro embryo production can obviously accelerate the quest for better noninvasive oocyte quality markers, because more information about the oocyte's requirements and intrinsic quality will be revealed. Although each step of in vitro culture has to be re-examined in light of the hampered production of single embryos, the reward at the end will be substantial. Individual scored oocytes will be traceable along the in vitro embryo production procedure and the final blastocyst outcome can be linked to the original oocyte quality and follicular environment without the bias caused by simultaneously developing embryos.     

Applications of nonthermal plasma technology on safety and quality of dried food ingredients

Cold plasma technology is an efficient, environmental-friendly, economic and noninvasive technology; and in recent years these advantages placed this novel technology at the centre of diverse studies for food industry applications. Dried food ingredients including spices, herbs, powders and seeds are an important part of the human diet; and the growing demands of consumers for higher quality and safe food products have led to increased research into alternative decontamination methods. Numerous studies have investigated the effect of nonthermal plasma on dried food ingredients for food safety and quality purposes. This review provides critical review on potential of cold plasma for disinfection of dried food surfaces (spices, herbs and seeds), improvement of functional and rheological properties of dried ingredients (powders, proteins and starches). The review further highlights the benefits of plasma treatment for enhancement of seeds performance and germination yield which could be applied in agricultural sector in near future. Different studies applying plasma technology for control of pathogens and spoilage micro-organisms and modification of food quality and germination of dried food products followed by benefits and current challenges are presented. However, more systemic research needs to be addressed for successful adoption of this technology in food industry.     

Long-term, noninvasive imaging of regulated gene expression in living mice

We describe here an approach for monitoring regulated gene expression by noninvasive imaging in living mice. We have utilized the tetracycline inducible system to simultaneously coregulate the expression of two genes encoding the firefly luciferase and the Cre recombinase, respectively. Results from our model system demonstrate that luciferase can be used as a noninvasive imaging marker for the regulated expression of a second gene in living mice. The integration of noninvasive imaging and inducible gene expression into current approaches of functional genomics should greatly advance our capabilities of carrying out highly controlled long-term studies of gene function in individual mice.