青藏高原东缘青杨（Populus cathayana Rehd.）遗传多样性研究

青杨（Populus cathayana Rehd.）是青杨派杨树的主要树种之一，为我国特有乡土树种，其主要分布区之一是我国的青藏高原，集中分布地带在甘肃省中部及青海省东部，四川省西北部岷江上游和松潘等地区。本研究以青藏高原东缘青杨天然分布区的6个群体143个个体为材料，用AFLP、SSR和叶绿体SSR分子标记分析青杨天然群体的遗传多样性，分析其遗传结构和分化，比较6个群体间遗传多样性的高低和群体间的遗传关系。旨在为青杨基因资源评价、保护与保存、遗传改良策略制定等提供科学理论依据。通过以上研究，得出如下主要研究结果： 1 AFLP分子标记研究结果 采用4对选择性引物对6个青杨天然群体143个个体进行分析，扩增谱带分析共检测到175个位点，其中173个位点表现为多态，多态位点百分率高达98.9%。从整体上表现出较高的遗传多样性，Nei’s基因多样度(h)水平为0.306。从青杨天然群体位点分布来看，有高达20%的位点（32位点）为群体所特有，仅有9.14%的位点（16位点）在所有群体中存在。群体间的遗传分化极大，所有遗传变异中，有48.9%的遗传变异存在于群体间。在个体群丛（Individuals cluster）和主坐标（PCO analysis）分析中，青杨各群体未呈现任何地理模式，Mantel检测也显示各群体间遗传距离与地理距离无明显相关。研究认为，由于地理和空间上大尺度的隔离和地形地貌复杂使得群体间无法进行基因交流，导致群体间遗传分化极大，另外各群体在不同的选择压力下，经历各自独立的进化历程，这些都可能导致群体间遗传距离与地理距离的不相关。 2 SSR分子标记研究结果 在SSR分析中，7个位点在6个青杨天然群体143个个体中共检测到79个等位基因，每位点检测到的等位基因数在5-16之间，平均11.3个，总体上多态位点百分率达100%。平均观察杂合度和期望杂合度分别为0.792和0.802。Hardy-Weinberg平衡检验表明青杨大部分群体都处于非平衡状态，群体大部分位点都是偏离哈迪-温伯格平衡（76.3%），只有23.7%的测验满足哈迪-温伯格平衡。分析青杨天然群体内和群体间的遗传变异，基因分化系数（GST）为0.373，即有62.7%的遗传变异存在群体内，37.3%的遗传变异存在群体间。群体内的遗传变异高于群体间水平。根据各群体遗传距离UPGMA聚类分析，有来自相临分布区、近似气候类型的群体聚在一起的趋势，但Mantel检测反映遗传距离与地理距离间并无明显相关性。 3 cpSSR分子标记研究结果 分析来自青藏高原东缘6个青杨天然群体，所用cpSSR引物中有5对cpSSR引物（CCMP2、CCMP5、SCUO01、SCU03、SCU07）都表现较高的多态性，单个引物检测的片段数都在4以上。5对cpSSR引物共检测片段数26个，组成了12种叶绿体DNA单倍型。各群体的单倍型分布和频率有较大差异，群体单倍型多样性范围为0-0.4926，TS、JZ、PW和SHY群体单倍型多样性高于QHY和LED群体水平。本研究发现，分布在青藏高原东缘的青杨天然群体，群体间不存在共享的单倍型，各群体间存在极大的遗传分化（GST=0.9223）。从青藏高原东缘地区经历的地质历史事件来看，第四纪的冰期气候变迁可能是造成青杨现今遗传结构模式的主要因素之一。根据单倍型在各群体的分布情况，进行青杨群体聚类分析结果，各群体无明显的分组现象，青杨各群体也未呈现任何清晰地理模式。 由于不同分子标记在对群体遗传多样性检测能力与效率上存在差异，所以三种标记检测的青藏高原东缘青杨天然群体遗传多性水平也不尽一致，但在与用同种方法检测其它物种或同一物种不同种源群体比较，三种分子标记方法都揭示了青藏高原东缘青杨天然群体具有中等偏上的遗传多样性水平。结果分析表明，群体间遗传分化极大，这是由于青杨天然群体分布于青藏高原东缘，既有高原又有高山峡谷，由于地理和空间上大尺度的隔离和地形地貌复杂导致了基因流物理上的阻隔。三种分子标记研究结果经Mantel分析检测，遗传距离与地理距离之间都无明显相关性。较为一致的解释是，青杨分布区域地理和空间上大尺度的隔离和和地形地貌复杂导致群体之间不存在均匀扩散现象，另外各群体在不同的选择压力下，经历各自独立的进化历程，这些都可能导致群体间遗传距离与地理距离的不相关。 The wide geographical and climatic distribution of P. cathayana Rehd. indicates that there is a large amount of genetic diversity available, which can be exploited for conservation, breeding programs and afforestation schemes. The results are as follows: 1 Research results of AFLP genetic diversity In present study, genetic diversity was evaluated in the natural populations of P. cathayana originating from southern and eastern edge of the Qinghai-Tibetan Plateau of China by means of AFLP markers. For four primer combinations, a total of 175 bands were obtained, of which 173 (98.9%) were polymorphic. Six natural populations of P. cathayana possessed different levels of genetic diversity, high level of genetic differentiation existed among populations (GST=0.489) of P. cathayana. Individuals cluster and PCO analysis based on Jaccard’s similarity coefficient also showed evident population genetic structure with high level population genetic differentiation. The long evolutionary process coupled with genetic drift within populations, rather than contemporary gene flow, are the major forces shaping genetic structure of P. cathayana populations. Moreover, there is no correspondence between geographical and genetic distances in the populations of P. cathayana, seldom gene exchange among populations and different selection pressures may be the causes. Our finding of different levels of genetic diversity within population and high level of genetic differentiation among populations provided promising condition for further breeding or conservation programs. 2 Research results of SSR genetic diversity In this study, the genetic diversity of P. cathayana was investigated using microsatellite markers. In a total of 150 individuals collected from six natural populations in the southeastern part of the Qinghai-Tibetan Plateau in China, a high level of microsatellite polymorphism was detected. At the seven investigated microsatellite loci, the number of alleles per locus ranged from 5 to 16, with a mean of 11.3, the observed heterozygosities across populations ranged from 0.408 to 0.986, with a mean of 0.792, and the expected heterozygosities across populations ranged from 0.511 to 0.891, with a mean of 0.802. The proportion of genetic differentiation among populations accounted for 37.3% of the whole genetic diversity. The presence of such a high level of genetic diversity could be attributed to the features of the species and the habitats where the sampled populations occur: The southeastern part of the Qinghai-Tibetan Plateau is regarded as the natural distribution and variation center of the genus Populus in China. Variation in environmental conditions and selection pressures in different populations, and topographic dispersal barriers could be factors associated with the high level of genetic differentiation found among populations. The populations possessed significant heterozygosity excesses, which may be due to extensive population mixing at the local scale. The cluster analysis showed that the populations are not strictly grouped according to their geographic distances but the habitat characteristics also influence the divergence pattern. In addition, we suggest that population SHY should be regarded as an ecologically divergent species of P. cathayana. 3 Research results of cpSSR genetic diversity Genetic diversity of six natural populations of P. cathayana originating from the southeastern part of the Qinghai-Tibetan Plateau in China was studied by use of cpSSR markers. Based on 5 pairs of polymorphic primers screened from 12 pairs of primers, twenty-six different length fragments and twelve different kinds of haplotypes were reduced in 143 samples. There were significant variant haplotypes among the populations．There were no shared haplotypes found among populations, analysis of molecular variance indicated that a high proportion of the total genetic variance was attributable to variations among populations (92.23%). The pattern of genetic structure which is associated with spatial separation, variation in environmental conditions and selection pressures in different populations, is also the result of geological historical factor. A molecular phylogenetic tree based on the 12 haplotypes showed that the populations are not strictly grouped according to their geographic distances.

Spectrometric and voltammetric studies of the interaction between quercetin and bovine serum albumin using warfarin as site marker with the aid of chemometrics

The interaction of quercetin, which is a bioflavonoid, with bovine serum albumin (BSA) was investigated under pseudo-physiological conditions by the application of UV–vis spectrometry, spectrofluorimetry and cyclic voltammetry (CV). These studies indicated a cooperative interaction between the quercetin–BSA complex and warfarin, which produced a ternary complex, quercetin–BSA–warfarin. It was found that both quercetin and warfarin were located in site I. However, the spectra of these three components overlapped and the chemometrics method – multivariate curve resolution-alternating least squares (MCR-ALS) was applied to resolve the spectra. The resolved spectra of quercetin–BSA and warfarin agreed well with their measured spectra, and importantly, the spectrum of the quercetin–BSA–warfarin complex was extracted. These results allowed the rationalization of the behaviour of the overlapping spectra. At lower concentrations ([warfarin] < 1 × 10−5 mol L−1), most of the site marker reacted with the quercetin–BSA, but free warfarin was present at higher concentrations. Interestingly, the ratio between quercetin–BSA and warfarin was found to be 1:2, suggesting a quercetin–BSA–(warfarin)2 complex, and the estimated equilibrium constant was 1.4 × 1011 M−2. The results suggest that at low concentrations, warfarin binds at the high-affinity sites (HAS), while low-affinity binding sites (LAS) are occupied at higher concentrations.

Corneal sensitivity as an ophthalmic marker of diabetic neuropathy

Purpose. The objective of this study was to explore the discriminative capacity of non-contact corneal esthesiometry (NCCE) when compared with the neuropathy disability score (NDS) score—a validated, standard method of diagnosing clinically significant diabetic neuropathy. Methods. Eighty-one participants with type 2 diabetes, no history of ocular disease, trauma, or surgery and no history of systemic disease that may affect the cornea were enrolled. Participants were ineligible if there was history of neuropathy due to non-diabetic cause or current diabetic foot ulcer or infection. Corneal sensitivity threshold was measured on the eye of dominant hand side at a distance of 10 mm from the center of the cornea using a stimulus duration of 0.9 s. The NDS was measured producing a score ranging from 0 to 10. To determine the optimal cutoff point of corneal sensitivity that identified the presence of neuropathy (diagnosed by NDS), the Youden index and “closest-to-(0,1)” criteria were used. Results. The receiver-operator characteristic curve for NCCE for the presence of neuropathy (NDS ≥3) had an area under the curve of 0.73 (p = 0.001) and, for the presence of moderate neuropathy (NDS ≥6), area of 0.71 (p = 0.003). By using the Youden index, for an NDS ≥3, the sensitivity of NCCE was 70% and specificity was 75%, and a corneal sensitivity threshold of 0.66 mbar or higher indicated the presence of neuropathy. When NDS ≥6 (indicating risk of foot ulceration) was applied, the sensitivity was 52% with a specificity of 85%. Conclusions. NCCE is a sensitive test for the diagnosis of minimal and more advanced diabetic neuropathy and may serve as a useful surrogate marker for diabetic and perhaps other neuropathies.

Identification of circulating tumour cells in early stage breast cancer patients using multi marker immunobead RT-PCR

Introduction The ability to screen blood of early stage operable breast cancer patients for circulating tumour cells is of potential importance for identifying patients at risk of developing distant relapse. We present the results of a study of the efficacy of the immunobead RT-PCR method in identifying patients with circulating tumour cells. Results Immunomagnetic enrichment of circulating tumour cells followed by RT-PCR (immunobead RT-PCR) with a panel of five epithelial specific markers (ELF3, EPHB4, EGFR, MGB1 and TACSTD1) was used to screen for circulating tumour cells in the peripheral blood of 56 breast cancer patients. Twenty patients were positive for two or more RT-PCR markers, including seven patients who were node negative by conventional techniques. Significant increases in the frequency of marker positivity was seen in lymph node positive patients, in patients with high grade tumours and in patients with lymphovascular invasion. A strong trend towards improved disease free survival was seen for marker negative patients although it did not reach significance (p = 0.08). Conclusion Multi-marker immunobead RT-PCR analysis of peripheral blood is a robust assay that is capable of detecting circulating tumour cells in early stage breast cancer patients.

Maximum recovery after knee replacement - the MARKER study rationale and protocol

Background There is little scientific evidence to support the usual practice of providing outpatient rehabilitation to patients undergoing total knee replacement surgery (TKR) immediately after discharge from the orthopaedic ward. It is hypothesised that the lack of clinical benefit is due to the low exercise intensity tolerated at this time, with patients still recovering from the effects of major orthopaedic surgery. The aim of the proposed clinical trial is to investigate the clinical and cost effectiveness of a novel rehabilitation strategy, consisting of an initial home exercise programme followed, approximately six weeks later, by higher intensity outpatient exercise classes. Methods/Design In this multicentre randomised controlled trial, 600 patients undergoing primary TKR will be recruited at the orthopaedic pre-admission clinic of 10 large public and private hospitals in Australia. There will be no change to the medical or rehabilitative care usually provided while the participant is admitted to the orthopaedic ward. After TKR, but prior to discharge from the orthopaedic ward, participants will be randomised to either the novel rehabilitation strategy or usual rehabilitative care as provided by the hospital or recommended by the orthopaedic surgeon. Outcomes assessments will be conducted at baseline (pre-admission clinic) and at 6 weeks, 6 months and 12 months following randomisation. The primary outcomes will be self-reported knee pain and physical function. Secondary outcomes include quality of life and objective measures of physical performance. Health economic data (health sector and community service utilisation, loss of productivity) will be recorded prospectively by participants in a patient diary. This patient cohort will also be followed-up annually for five years for knee pain, physical function and the need or actual incidence of further joint replacement surgery. Discussion The results of this pragmatic clinical trial can be directly implemented into clinical practice. If beneficial, the novel rehabilitation strategy of utilising outpatient exercise classes during a later rehabilitation phase would provide a feasible and potentially cost-effective intervention to optimise the physical well-being of the large number of people undergoing TKR.

Serum bone formation marker correlation with improved osseointegration in osteoporotic rats treated with simvastatin

Objective: Simvastatin has been shown to enhance osseointegration of pure titanium implants in osteoporotic rats. This study aimed to evaluate the relationship between the serum level of bone formation markers and the osseointegration of pure titanium implants in osteoporotic rats treated with simvastatin. Materials and methods: Fifty-four female Sprague Dawley rats, aged 3 months old, were randomly divided into three groups: Sham-operated group (SHAM; n=18), ovariectomized group (OVX; n=18), and ovariectomized with Simvastatin treatment group (OVX+SIM; n=18). Fifty-six days after ovariectomy, screw-shaped titanium implants were inserted into the tibiae. Simvastatin was administered orally at 5mg/kg each day after the placement of the implant in the OVX+SIM group. The animals were sacrificed at either 28 or 84 days after implantation and the undecalcified tissue sections were processed for histological analysis. Total alkaline phosphatase (ALP), bone specific alkaline phosphatase (BALP) and bone Gla protein (BGP) were measured in all animal sera collected at the time of euthanasia and correlated with the histological assessment of osseointegration. Results: The level of ALP in the OVX group was higher than the SHAM group at day 28, with no differences between the three groups at day 84. The level of BALP in the OVX+SIM group was significantly higher than both OVX and SHAM groups at days 28 and 84. Compared with day 28, the BALP level of all three groups showed a significant decrease at day 84. There were no significant differences in BGP levels between the three groups at day 28, but at day 84 the OVX+SIM group showed significantly higher levels than both the OVX and SHAM groups. There was a significant increase in BGP levels between days 28 and 84 in the OVX+SIM group. The serum bone marker levels correlated with the histological assessment showing reduced osseointegration in the OVX compared to the SHAM group which is subsequently reversed in the OVX+SIM group.

Neural differentiation potential of human bone marrow-derived mesenchymal stromal cells: misleading marker gene expression

Background In contrast to pluripotent embryonic stem cells, adult stem cells have been considered to be multipotent, being somewhat more restricted in their differentiation capacity and only giving rise to cell types related to their tissue of origin. Several studies, however, have reported that bone marrow-derived mesenchymal stromal cells (MSCs) are capable of transdifferentiating to neural cell types, effectively crossing normal lineage restriction boundaries. Such reports have been based on the detection of neural-related proteins by the differentiated MSCs. In order to assess the potential of human adult MSCs to undergo true differentiation to a neural lineage and to determine the degree of homogeneity between donor samples, we have used RT-PCR and immunocytochemistry to investigate the basal expression of a range of neural related mRNAs and proteins in populations of non-differentiated MSCs obtained from 4 donors. Results The expression analysis revealed that several of the commonly used marker genes from other studies like nestin, Enolase2 and microtubule associated protein 1b (MAP1b) are already expressed by undifferentiated human MSCs. Furthermore, mRNA for some of the neural-related transcription factors, e.g. Engrailed-1 and Nurr1 were also strongly expressed. However, several other neural-related mRNAs (e.g. DRD2, enolase2, NFL and MBP) could be identified, but not in all donor samples. Similarly, synaptic vesicle-related mRNA, STX1A could only be detected in 2 of the 4 undifferentiated donor hMSC samples. More significantly, each donor sample revealed a unique expression pattern, demonstrating a significant variation of marker expression. Conclusion The present study highlights the existence of an inter-donor variability of expression of neural-related markers in human MSC samples that has not previously been described. This donor-related heterogeneity might influence the reproducibility of transdifferentiation protocols as well as contributing to the ongoing controversy about differentiation capacities of MSCs. Therefore, further studies need to consider the differences between donor samples prior to any treatment as well as the possibility of harvesting donor cells that may be inappropriate for transplantation strategies.

A fragment of the LG3 peptide of endorepellin is present in the urine of physically active mining workers : a potential marker of physical activity

Biomarker analysis has been implemented in sports research in an attempt to monitor the effects of exertion and fatigue in athletes. This study proposed that while such biomarkers may be useful for monitoring injury risk in workers, proteomic approaches might also be utilised to identify novel exertion or injury markers. We found that urinary urea and cortisol levels were significantly elevated in mining workers following a 12 hour overnight shift. These levels failed to return to baseline over 24h in the more active maintenance crew compared to truck drivers (operators) suggesting a lack of recovery between shifts. Use of a SELDI-TOF MS approach to detect novel exertion or injury markers revealed a spectral feature which was associated with workers in both work categories who were engaged in higher levels of physical activity. This feature was identified as the LG3 peptide, a C-terminal fragment of the anti-angiogenic / anti-tumourigenic protein endorepellin. This finding suggests that urinary LG3 peptide may be a biomarker of physical activity. It is also possible that the activity mediated release of LG3 / endorepellin into the circulation may represent a biological mechanism for the known inverse association between physical activity and cancer risk / survival.

A radiological method to determine the accuracy of motion capture marker placement on palpable anatomical landmarks through a shoe

The accuracy of marker placement on palpable surface anatomical landmarks is an important consideration in biomechanics. Although marker placement reliability has been studied in some depth, it remains unclear whether or not the markers are accurately positioned over the intended landmark in order to define the static position and orientation of the segment. A novel method using commonly available X-ray imaging was developed to identify the accuracy of markers placed on the shoe surface by palpating landmarks through the shoe. An anterior–posterior and lateral–medial X-ray was taken on 24 participants with a newly developed marker set applied to both the skin and shoe. The vector magnitude of both skin- and shoe-mounted markers from the anatomical landmark was calculated, as well as the mean marker offset between skin- and shoe-mounted markers. The accuracy of placing markers on the shoe relative to the skin-mounted markers, accounting for shoe thickness, was less than 5mm for all markers studied. Further, when using the developed guidelines provided in this study, the method was deemed reliable (Intra-rater ICCs¼0.50–0.92). In conclusion, the method proposed here can reliably assess marker placement accuracy on the shoe surface relative to chosen anatomical landmarks beneath the skin.

Effects of oxygen on zonal marker expression in human articular chondrocytes

Articular cartilage is organized in depth zones with phenotypically distinct subpopulations of chondrocytes that are exposed to different oxygen tensions. Despite growing evidence of the critical role for oxygen in chondrogenesis, little is known about its effect on chondrocytes from different zones. This study evaluates zonal marker expression of human articular chondrocytes from different zones under various oxygen tensions. Chondrocytes isolated from full-thickness, superficial, and middle/deep cartilage from knee replacement surgeries were expanded and redifferentiated under hypoxic (5% O 2) or normoxic (20% O 2) conditions. Differentiation under hypoxia increased expression of hypoxia-inducible factors 1alpha and 2alpha and accumulation of extracellular matrix, particularly in middle/deep chondrocytes, and favored re-expression of proteoglycan 4 by superficial chondrocytes compared with middle/deep cells. Zone-dependent expression of clusterin varied with culture duration. These results demonstrate that zonal chondrocytes retain important phenotypic differences during in vitro cultivation, and that these characteristics can be improved by altering the oxygen environment. However, transcript levels for pleiotrophin, cartilage intermediate layer protein, and collagen type X were similar between zones, challenging their reliability as zonal markers for tissue-engineered cartilage from osteoarthritis patients. Key factors including oxygen tension and cell source should be considered to prescribe zone-specific properties to tissue-engineered cartilage. © 2012, Mary Ann Liebert, Inc.