构树种质资源遗传多样性SRAP分子标记研究

构树（Broussonetia papyrifera L.）为桑科（Moraceae）构树属（Broussonetia）落叶乔木，广泛分布于亚洲东部及太平洋岛屿。它是我国重要的经济林木，具有重要的经济价值，其树皮纤维品质优良，自古就是造纸的优良原料；叶片可用作饲料；果实具有重要的药用价值；环境适应性强，是迅速绿化荒山、荒滩和盐碱地的理想树种。因此对构树这些特性的深入研究和开发利用具有非常重要的实际应用价值。本研究以日本和国内构树主要分布区域的的10种生态型及杂交构树共23份材料，摸索并改进了构树DNA的提取方法，建立了稳定的SRAP分子标记体系，以杂交构树组培苗为材料从120对引物组合中筛选出条带信息较高的17对SRAP引物，以这些引物对23份构树材料进行PCR扩增和标记分析。 本研究取得的主要结果如下： （1）本实验首次将SRAP技术应用于构树的研究中，建立起构树稳定的SRAP-PCR反应体系；实验中对影响扩增的5个主要因素进行了优化，确定20 μL PCR反应体系中各因素最适浓度：模板DNA浓度80 ng/(20 μL)，Mg2+浓度2.0 mM，dNTP浓度0.6 mM，引物浓度0.8 mM，Taq 酶浓度1.5 U/(20 μL)。 （2）从120对引物组合中筛选出来的17对SRAP引物对21份不同生态型构树样本（21份材料指的是除两份杂交构树材料外的其它生态型构树，以下同）进行PCR扩增，共扩增出439条带，平均每对引物25.5条，其大小介于100～1,000 bp之间，其中多态性条带319条，占总数的72.67%。 （3）用Popgene1.32软件进行分析，计算出Shannon信息指数（I）值为0.2275（0.2042），物种水平的Nei基因多样性（H）值为0.1336（0.1436），表明各生态型构树之间的平均遗传多态性不高，中国大陆各生态型构树Shannon信息指数（I）值仅为0.1675（0.2271），物种水平的Nei基因多样性（H）值为0.1039（0.1540），群体遗传多样性较低，构树的遗传分化主要存在于中国和日本之间。 （4）用NTSYS-2.10e软件进行聚类分析，发现不同生态型构树按距离关系远近及分布区域可划分为不同类群。在遗传相似系数0.57附近，对21份材料进行聚类分析发现其可分为两个群体，一类为日本生态型，另一类为中国生态型，表明日本构树与中国野生种构树种源遗传相似性较小。中国各生态型构树在遗传相似系数0.91处可分为5类，总体而言，中国各地区之间的构树遗传相似度较高。对杂交构树分析表明，其亲缘关系与日本构树（母本）更接近。 （5）日本及杂交构树的SCAR标记。本研究找到两条日本及杂交构树的特异性条带，回收、测序，再根据序列往里重新设计引物，转变成稳定性更好，更直观的SCAR标记，这为挑选性状优良的日本及杂交构树提供重要的参考，对其育种有一定的指导意义。其中一条片段经与NCBI数据库比对发现与拟南芥磺基转移酶家族基因具有较高的同源性。

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.