整合化学物质组学的整体系统生物学——中药复方配伍和作用机理研究的整体方法论

本文以中医药学和系统生物学为基础,探讨建立一个整合化学物质组学的整体系统生物学体系,用于研究外部干预系统(中药复方)与生物应答系统(人体复杂系统)之间“系统-系统”的相互作用。整合化学物质组学的整体系统生物学提供了一个中医药学与现代科学交流融合的平台,有助于全面、系统、深刻的揭示中医方剂的药效物质基础和作用机理,阐明中药复方的配伍规律,指导复方新药研发,更好地传承和发展中医药理论。

中国精准医学的发展

精准医学是依据患者内在的生物学与临床症状的特征数据,对患者实施量身定制的临床医疗策略。其核心技术是测序技术与组学研究、大规模人群队列研究、生物大数据技术等。

从临床出发,以信号通路为靶标的复方新药研发策略、途径与实践——六论创建新医药学

本文对中医药现代化之路朝向何方之问,提出了新药研发重点将从新化学实体(NCE)转向复方新药(innovative compound drug,ICD)的观点。在分析新化学实体研究开发模式的缺陷——从动物模型和从靶点出发的新药研究开发的局限性基础上,指出"病"(西医)和"证"(中医)具有统一的生物学意义和生物物质基础(基因、蛋白质、代谢物等)。本文提出了基于"系统-系统"(人体系统-药物系统)模式的复方药物的定义和特点,给出了从临床出发,以信号通路为靶标的复方新药(包括中药复方、西药复方和中西药复方)研发策略。以中药方剂糖肾方治疗糖尿病肾病(DN)的临床研究为例,阐述了研究途径和实例。最后,再次提出设立中国"新医药学发展计划"的倡议。

中国精准医学的发展

综述我国精准医学发展情况,阐述精准医学的生物学基础、核心技术,介绍测序技术与组学研究、大规模人群队列研究、生物大数据技术研发、个性化治疗技术开发状况,在此基础上对未来发展进行展望。

复方药物研发创新体系展望

本文通过分析创新药物研发的机遇和挑战,提出了复方药物的定义:即指为了实现整体最佳的疗效目标,综合多种治疗原则和多种作用机理导向下所开发的由多个化合物或化合物群配伍组成的治疗药物。复方药物既包括中药复方药物,也包括西药复方药物(化学药复方药物),甚至还可能开发中西药结合的复方药物。复方药物研发创新体系的主要特点在于:强调治疗的对象是生病(或亚健康状态)的人(而不只是病);充分体现"医生参与、医药结合"的特色;研发途径应体现"临床-动物-临床"的特点;应具有独特的临床疗效综合评价体系;应具有体现其作用模式(机理)的药物综合筛选模式;应具有体现其化合物群的整体表征和局部特征的综合表达形式。当前创制中药复方药物的关键在于:亟待发展能体现其临床疗效的综合评价体系;亟待发展能体现其作用模式(机理)的复方药物综合筛选体系;亟须将中药复方药物综合表达形式规范化。作为中药复方药物的研究示范,作者领导的研究组采用所建立的整合化学物质组学的整体系统生物学方法,成功的实现了从清开灵复方开发出治疗缺血性脑中风的复方药物,从双龙方开发出治疗缺血性心脏病的复方药物。当前创制化学药复方新药的关键在于:亟须转变观念,确立化学药复方药物也是一种新的普适模式;亟须由临床医生从头开始参与新药研发,建立"临床-动物-临床"研发模式;加强中西药合用的复方药物的研究。

Cytological and Molecular Identification of Alien Chromatin in Giant Spike Wheat Germplasm

Alien chromosomes of twelve giant spike wheat germplasm lines were identified by C-banding, genomic in situ hybridization (GISH), sequence characterized amplified region (SCAR), and random amplified polymorphic DNA (RAPD). All lines showed a chromosome number of 2n = 42, five of them carried both a pair of wheat-rye (Triticum aestivum-Secale cereal) 1BL/1RS translocation chromosomes and a pair of Agropyron intermedium (Ai) chromosomes, three carried a pair of Ai chromosomes only, three others carried a pair of 1BL/1RS chromosomes only, and one carried neither 1BL/1BS nor Ai chromosome. Further identification revealed that the identical Ai chromosome in these germplasm lines substituted the chromosome 2D of common wheat (T aestivum L.), designated as 2Ai. The genetic implication and further utilization of 2Ai in wheat improvement were also discussed.

Development of Studies on RNA Interference

RNA interference （RNAi）, caused by endogenous or exogenous double- stranded RNA （dsRNA） homologous with target genes, refers to gene silencing widely existing in animals and plants. It was first found in plants, and now it has developed into a kind of biotechnology as well as an important approach in post- genome era. This paper is to summarize the achievements of studies on RNAi tech- nology in basic biology, medicine, pharmacy, botany and other fields.

Glutamine depletion induces murine neonatal melena with increased apoptosis of the intestinal epithelium

AIM:To investigate the possible biological outcome and effect of glutamine depletion in neonatal mice and rodent intestinal epithelial cells.METHODS:We developed three kinds of artificial milk with different amounts of glutamine;Complete amino acid milk (CAM),which is based on maternal mouse milk,glutamine-depleted milk (GDM),and glutaminerich milk (GRM).GRM contains three-fold more glutamine than CAM.Eighty-seven newborn mice were divided into three groups and were fed with either of CAM,GDM,or GRM via a recently improved nipple-bottle system for seven days.After the feeding period,the mice were subjected to macroscopic and microscopic observations by immunohistochemistry for 5-bromo-2'deoxyuridine (BrdU) and Ki-67 as markers of cell proliferation,and for cleaved-caspase-3 as a marker of apoptosis.Moreover,IEC6 rat intestinal epithelial cells were cultured in different concentrations of glutamine and were subject to a 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)2H-5-tetrazolio]-1,3-benzene disulfonate cell proliferation assay,flow cytometry,and western blotting to examine the biological effect of glutamine on cell growth and apoptosis.RESULTS:During the feeding period,we found colonic hemorrhage in six of 28 GDM-fed mice (21.4%),but not in the GRM-fed mice,with no differences in body weight gain between each group.Microscopic examination showed destruction of microvilli and the disappearance of glycocalyx of the intestinal wall in the colon epithelial tissues taken from GDM-fed mice.Intake of GDM reduced BrdU incorporation (the average percentage of BrdU-positive staining;GRM:13.8%,CAM:10.7%,GDM:1.14%,GRM vs GDM:P < 0.001,CAM vs GDM:P < 0.001) and Ki-67 labeling index (the average percentage of Ki67-positive staining;GRM:24.5%,CAM:22.4% GDM:19.4%,GRM vs GDM:P=0.001,CAM vs GDM:P =0.049),suggesting that glutamine depletion inhibited cell proliferation of intestinal epithelial cells.Glutamine deprivation further caused the deformation of the nuclear membrane and the plasma membrane,accompanied by chromatin degeneration and an absence of fat droplets from the colonic epithelia,indicating that the cells underwent apoptosis.Moreover,immunohistochemical analysis revealed the appearance of cleaved caspase-3 in colonic epithelial cells of GDM-fed mice.Finally,when IEC6 rat intestinal epithelial cells were cultured without glutamine,cell proliferation was significantly suppressed after 24 h (relative cell growth;4 mmol/L:100.0% ± 36.1%,0 mmol/L:25.3% ± 25.0%,P < 0.05),with severe cellular damage.The cells underwent apoptosis,accompanied by increased cell population in sub-G0 phase (4 mmol/L:1.68%,0.4 mmol/L:1.35%,0 mmol/L:5.21%),where dying cells are supposed to accumulate.CONCLUSION:Glutamine is an important alimentary component for the maintenance of intestinal mucosa.Glutamine deprivation can cause instability of the intestinal epithelial alignment by increased apoptosis.

Peripheral nerve regeneration and intraneural revascularization

Peripheral nerves are particularly vulnerable to injuries and are involved in numerous pathologies for which specific treatments are lacking. This review summarizes the pathophysiological features of the most common traumatic nerve injury in humans and the different animal models used in nerve regeneration studies. ~Ihe current knowledge concerning Wallerian degeneration and nerve regrowth is then described. Finally, the involvement of intraneural vascularization in these processes is addressed. As intraneural vascularization has been poorly studied, histological experiments were carried out from rat sciatic nerves damaged by a glycerol injection. The results, taken together with the data from literature, suggest that revascularization plays an important role in peripheral nerve regeneration and must therefore be studied more carefully.

SPARCL1, Shp2, MSH2, E-cadherin, p53, ADCY-2 and MAPK are prognosis-related in colorectal cancer

AIM: To investigate the expression of markers that are correlated with the prognosis of colorectal cancer (CRC) patients. METHODS: One hundred and fifty-six CRC patientswere followed up for more than 3 years after radical surgery. Immunohistochemical (IHC) analysis was performed to detect the expression of 14 pathway-related markers (p53, APC, p21ras, E-cadherin, endothelin-B receptor, Shp2, ADCY-2, SPARCL1, neuroligin1, hsp27, mmp-9, MAPK, MSH2 and rho) in specimens from these patients. Bioinformatics analysis involving a Support Vector Machine (SVM) was used to determine the best prognostic model from combinations of these markers. RESULTS: Seven markers (SPARCL1, Shp2, MSH2, E-cadherin, p53, ADCY-2 and MAPK) were significantly related to the prognosis and clinical pathological features of the CRC patients (P < 0.05). Prognostic models were established through SVM from combinations of these 7 markers and proved able to differentiate patients with dissimilar survival, especially in stage Ⅱ/Ⅲ patients. According to the best prognostic model, the p53/SPARCL1 model, patients having high p53 and low SPARCL1 expression had about 50% lower 3-year survival than others (P < 0.001). CONCLUSION: SPARCL1, Shp2, MSH2, E-cadherin, p53, ADCY-2 and MAPK are potential prognostic markers in CRC. A p53/SPARCL1 bioinformatics model may be used as a supplement to tumor-nodes-metastasis staging.

Effects of TaN nanoparticles on microstructure,mechanical properties and tribological performance of PEEK coating prepared by electrophoretic deposition

In order to solve the friction,wear and lubrication problems of titanium,a series of TaN/ployether−ether−ketone(PEEK)coatings were developed by electrophoretic deposition,and the effects of TaN nanoparticles on the microstructure,mechanical properties and tribological performance of coatings were explored.Results manifest that the introduction of TaN nanoparticles into PEEK coatings could improve the deposition efficiency,enhance the resistant deform capacity,increase the hardness,elastic modulus and adhesive bonding strength.Compared with the pure PEEK coating,the friction coefficient of P-TN-3 was greatly reduced by 31.25%.The wear resistance of P-TN-3 was also improved in huge boost,and its specific wear rate was decreased from 9.42×10^(−5) to 1.62×10^(−5) mm^(3)·N^(−1)·m^(−1).The homogeneous composite TaN/PEEK coatings prepared by electrophoretic deposition were well-adhered to the titanium alloy substrate,TaN nanoparticles could improve the strength of PEEK coating,and provide wear-resistance protection for titanium alloys.

Effect of thermo-mechanical processing on microstructure and electrochemical behavior of Ti-Nb-Zr-V new metastable β titanium biomedical alloy

The influence of thermo-mechanical processing (TMP) on the microstructure and the electrochemical behavior of new metastableβ alloy Ti?20.6Nb?13.6Zr?0.5V (TNZV) was investigated. The TMP included hot working in belowβ transus, solution heat treatments at the same temperature and different cooling rates in addition to aging. Depending upon the TMP conditions, a wide range of microstructures with varying spatial distributions and morphologies of equiaxed/elongatedα andβ phases were attained, allowing for a wide range of electrochemical properties to be achieved. The corrosion behavior of the studied alloy was evaluated in a Ringer’s solution at 37 °C via open circuit potential?time and potentiodynamic polarization measurements.

Expression and significance of cyclin E in gastric carcinoma

Objective:The aim of the study was to investigate the expression and significance of cyclin E in gastric carcinoma.Methods:We detected the expression of cyclin E in three different pathologic types gastric carcinoma samples by immuno-histochemical staining technique (SP method).Results:In 59 gastric carcinoma samples the positive rate of cyclin E expression in gastric carcinoma was 55.93% (33/59), and it was significantly higher than that of normal gastric mucosa (10.53%, 2/19).The positive rates of cyclin E expression in poor differentiation group and mucoid carcinoma group were 68.75% (11/16) and 66.67% (16/24), respectively, and these were significantly higher than that in well-middle differentiation group (31.58%, 6/19), but there was no significant difference between the fronted two groups (P>0.05).Conclusion:The high expression of cyclin E is associated with the progression of gastric carcinoma and probably related to the behavior of cellular biology.

A de novo germline MLH1 mutation in a Lynch syndrome patient with discordant immunohistochemical and molecular biology test results

We describe a patient with a Homo sapiens mutL homolog 1 (MLH1)-associated Lynch syndrome with previous diagnoses of two distinct primary cancers:a sigmoid colon cancer at the age of 39 years, and a right colon cancer at the age of 50 years. The mutation identified in his blood and buccal cells, c.1771delG, p.Asp591Ilefs*25, appears to be a de novo event, as it was not transmitted by either of his parents. This type of de novo event is rare in MLH1 as only three cases have been reported in the literature so far. Further-more, the discordant results observed between repli-cation error phenotyping and immunohistochemistry highlight the importance of the systematic use of both pre-screening tests in the molecular diagnosis of Lynch syndrome.

Comparative genomics of Helicobacter pylori

Genomic sequences have been determined for a number of strains of Helicobacter pylori （H pylori） and related bacteria. With the development of microarray analysis and the wide use of subtractive hybridization techniques, comparative studies have been carried out with respect to the interstrain differences between H pylori and inter-species differences in the genome of related bacteria. It was found that the core genome of H pylori constitutes 1111 genes that are determinants of the species properties. A great pool of auxiliary genes are mainly from the categories of cag pathogenicity islands, outer membrane proteins, restriction-modification system and hypothetical proteins of unknown function. Persistence of H pylori in the human stomach leads to the diversification of the genome. Comparative genomics suggest that a host jump has occurs from humans to felines. Candidate genes specific for the development of the gastric diseases were identified. With the aid of proteomics, population genetics and other molecular methods, future comparative genomic studies would dramatically promote our understanding of the evolution, pathogenesis and microbiology of Hpylori.

Clinicopathological characterization of gastroenteropancreatic neu-roendocrine neoplasms： a retrospective study of 48 cases

Objective Gastroenteropancreatic neuroendocrine neoplasms （GEP-NENs） constitute a rare and heterogeneous group of tumors with varied biology and still constitute a diagnostic and therapeutic challenge for physicians of all specialties. In the present study, we aimed to review and study the clinicopathological characteristics of GEP-NENs applying the World Health Organization （WHO） 2010 grading criterion. Methods A total of 48 patients were enrolled in the study. The study included patients diagnosed with GEP-NENs who were treated and followed up at our Hospital from January 2013 to December 2017. Data regarding clinicopathological features of the patients were retrospectively evaluated. The expression of neuroendocrine markers was measured using the immunohistochemical Ultra SensitiveTM S-P method of staining in 48 cases of primary GEP-NENs; and serum levels of neuron-specific enolase, carbohydrate an-tigen 19-9, and carcinoembryonic antigen in 36 GEP-NEN patients were measured using the electrochemiluminescence method. Results The median age at presentation was 59.3 （range 48-82） years, and 39 cases （81.3%） were seen between the 5th and 6th decades. There was a male predilection （male： female=3：1）. In 79.2% cases （38/48）, tumors were hormonally nonfunctional. The most common presentation was abdominal pain, and the most frequent primary site of the tumor was the rectum, followed by the stomach （n = 15, 31.3%）, colon （n = 5, 10.4%）, and so on. Of the 48 tumors, 16 （33.3%） were G1,6 （12.5%） cases were G2, 16 （33.3%） were neuroendocrine carcinoma （NEC）, and 10 （20.8%） were mixed adenoneuroendocrine carcinoma （MANEC）. According to the AJCC/UICC classification, 45.8% （n = 22） were diagnosed at low stage （stage Ⅰ or Ⅱ） while 54.2% （n = 26） were diagnosed at high stage （stage Ⅲ or Ⅳ） （the majority of NEC, G3, and MANEC）. A male preponderance was noted for all tumors except for G2 neoplasms, which showed no gender predilection. Thirty-nine patients underwent endoscopic biopsy. The lesions in 18.8% （n = 9） of the patients were indentified only radiologically. After the surgical procedures, 36 had at least one follow-up visit with a median follow-up duration of 5 months; the mean follow-up period was 28 ± 16 months. The one- year and three-year survival rates were 72.2% （26/36） and 61.1% （22/36）, respectively. This study did not find an effect of grade 3 （G3） of tumor on the short-term clinical outcome of these patients. In the survival analysis, NEN G3, higher stage （stage Ⅲ or Ⅳ） according to the AJCC/UICC classification （P 〈 0.05）, and metastases at diagnosis （P 〈 0.05） were associated with poorer prognosis. Conclusion Most GEP-NENs are nonfunctional and nonspecific in presentation. The most frequent primary site of the tumor was the rectum and the commonest ages at diagnosis were the 5th and 6th decades. Endoscopic biopsy is the main diagnostic and histological grading method for GEP-NEN. In the survival analysis, NEN G3, a higher stage according to the AJCC/UICC classification, and metastases at diagnosis are associated with poorer prognosis.

Lasertherapy as A Strategy for Treatment Healing under Caloric Restriction – Study in Rats

Complications in the healing process are challenging, especially in clinical situations of caloric restriction （CR）. The lasertherapy becomes an important strategy that aids the repair, especially in CR. Thus, it is important to investigate the InGaAlP-660 nm laser as an strategy to repair cutaneous wounds in rats submitted to 30% of CR and to understand the tissue repair in clinical situations of CR. Thirty-six male Wistar rats were used, of which half were fed with 30% less ration, and half with ad libitum diet, for 21 days. Then, punch lesions of 1.5 cm in diameter were made on the animals backs, which were divided into： NR （no-restricted）, R （restricted）-both before lesion; C （control）, RC （restricted-control）, L （laser）, RL （restricted-laser）-after lesion. Samples of the skin/lesion/scar were collected on the 2nd, 7th and 14th days post-injury for histological, biochemical and molecular analyses. The R group showed reduction of body mass, epidermal/dermal thickness, inflammation, angiogenesis, fibroplasia and collagenesis. The RL group showed control of inflammation, oxidative damage and increase of antioxidants than RC, which probably favored angiogenesis, collagenesis and reepithelialization, similar to C and L. Thus, 30% of CR impaired the skin （before lesion）. In the lesion, lasertherapy has shown to be effective in tissue repair mainly in CR status, being thus, the laser clinically important strategy to tissue repair in critical situations of caloric restriction.

Sacro-anterior haemangiopericytoma: a case report

Haemangiopericytoma(HPC) is a rare vascular tumor with borderline malignancy, considerable histological variability, and unpredictable clinical and biological behavior. HPC can present a diagnostic challenge because of its indeterminate clinical, radiological, and pathological features. HPC generally presents in adulthood and is equally frequent in both sexes. HPC can arise in any site in the body as a slowly growing and painless mass. The precise cell type origin of HPC is uncertain. One third of HPCs occur in the head and neck areas. Exceptional cases of hemangioblastoma arising outside the head and neck areas have been reported, but little is known about their clinicopathologic and immunohistochemical features. This study reports on a case of a large sacro-anterior HPC in a 65-year-old male.

Histological,enzymohistochemical and biomechanical observation of skeletal muscle injury in rabbits

Objective： To explore the pathophysiological and biomechanical features of skeletal muscular injury for providing a rational basis for its treatment, prevention and rehabilitation. Methods ： In 70 adult rabbits, the left tibialis anterior （TA） muscle was stretched to injury, while the right TA muscle served as control. Histological, enzymohistochemical and biomechanical changes were observed on days 0, 1, 2, 3, and 7 after injury. Cytochrome oxidase （ CCO ）, acid phosphatase （ ACP ）, ATPase, succinate dehydrogenase （ SDH ）, malate dehydrogenase （ MDH ）, NADH-diaphorase （ NADHD ）, giutamatedehydrogenase （ GDH ）, α-giycerophosphate dehydrogenase （ α-GPD ） and lactate dehydrogenase （LDH） were measured. The examined biomechanical parameters included maximal contractile force, ultimate load, length, energy absorption, tangent stiffness, and rupture site. Results： Partial or complete rupture of TA muscle occurred near the muscle-tendon junction. There was an intense inflammatory reaction on day 1 and 2 after injury. Endomysium fibrosis and myotube formation were observed on day 3, and developed further on day 7. The activity of cell oxidases （ CCO, ATPase, MDH, α-GPD, SDH,NADHD and GDH） showed a significant drop from day 0 to 2, and resumed with different levels on day 3. The increment of enzymatic activities continued on day 7 and the levels of NADHD and α-GPD reached to the levels of control muscle. Maximal contractile force was 70.17 % ± 3.82% of controls immediately after injury, 54.82% ±3.09% at 1 day, 66.41% ±4.36% at 2 days, 78.39%± 4.90 % at 3 days and 93.64 % ±5.02 % at 7 days. Ultimate load was 85.78 %±7.54 % of controls at the moment of injury, 61.44 % ± 5.91% at 1 day, 49. 17 %± 4.26 % at 2 days, 64.43% ±5.02% at 3 days, and 76.71% ±6.46% at 7 days. Conclusions ： Endomysium fibrosis and scar formation at the injured site are responsible for frequent recurrence of skeletal muscle injury. Recovery of tensile load slower than that of maximal contractile force may be another cause. Whether the injured muscle returns to normal exercise is mainly determined by the teusility on which the muscle-tendon can bear rather than the maximal contractile force.

Characterizing and annotating the genome using RNA-seq data

Bioinformatics methods for various RNA-seq data analyses are in fast evolution with the improvement of sequencing technologies. However, many challenges still exist in how to efficiently process the RNA-seq data to obtain accurate and comprehensive results. Here we reviewed the strategies for improving diverse transcriptomic studies and the annotation of genetic variants based on RNA-seq data. Mapping RNA-seq reads to the genome and transcriptome represent two distinct methods for quantifying the expression of genes/transcripts. Besides the known genes annotated in current databases, many novel genes/transcripts(especially those long noncoding RNAs) still can be identified on the reference genome using RNA-seq. Moreover, owing to the incompleteness of current reference genomes, some novel genes are missing from them. Genome-guided and de novo transcriptome reconstruction are two effective and complementary strategies for identifying those novel genes/transcripts on or beyond the reference genome. In addition, integrating the genes of distinct databases to conduct transcriptomics and genetics studies can improve the results of corresponding analyses.