CD146 as a new marker for an increased chondroprogenitor cell sub‐population in the later stages of osteoarthritis

Cartilage‐derived mesenchymal stem cells (MSCs) have been isolated with different methods. In this study lateral and medial femoral condyles were respectively collected from patients with late‐stage osteoarthritis during the total knee arthroplasty. After digestion of the cartilage tissues with type II collagenase and analysis by fluorescence‐activated cell sorting (FACS) with CD146, a chondroprogenitor cell sub‐population were isolated and purified. The expression of other MSC‐associated markers in the CD146⁺ chondroprogenitors was analyzed by flow cytometry. Multi‐lineage differentiation capacity of CD146⁺ chondroprogenitors was compared with that of unsorted chondrocytes and adipose‐derived MSCs (ADMSCs). Higher percentage of CD146⁺ chondroprogenitors isolated from the medial femoral condyles was observed than that from the lateral. CD146⁺ chondroprogenitors expressed high levels of MSC‐specific surface antigens, and showed higher chondrogenesis capacity than ADMSCs and unsorted chondrocytes in a 3D cell pellet culture model. Thus CD146 might be a new cell surface marker for cartilage progenitor cell population in the late‐stage osteoarthritis. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 33:84–91, 2015.     

Neuroprotective effect of interleukin-6 regulation of voltage-gated Na~+ channels of cortical neurons is time-and dose-dependent

Interleukin-6 has been shown to be involved in nerve injury and nerve regeneration, but the effects of long-term administration of high concentrations of interleukin-6 on neurons in the central nervous system is poorly understood. This study investigated the effects of 24 hour exposure of interleukin-6 on cortical neurons at various concentrations(0.1, 1, 5 and 10 ng/m L) and the effects of 10 ng/m L interleukin-6 exposure to cortical neurons for various durations(2, 4, 8, 24 and 48 hours) by studying voltage-gated Na+ channels using a patch-clamp technique. Voltage-clamp recording results demonstrated that interleukin-6 suppressed Na+ currents through its receptor in a time- and dose-dependent manner, but did not alter voltage-dependent activation and inactivation. Current-clamp recording results were consistent with voltage-clamp recording results. Interleukin-6 reduced the action potential amplitude of cortical neurons, but did not change the action potential threshold. The regulation of voltage-gated Na+ channels in rat cortical neurons by interleukin-6 is time- and dose-dependent.     

宫颈癌患者术后下肢淋巴水肿发生状况及危险因素分析

目的分析宫颈癌患者术后下肢淋巴水肿发生情况及相关危险因素。方法应用妇科癌症淋巴水肿问卷电话调查492例手术治疗的宫颈癌患者下肢淋巴水肿发生情况,并分析术后下肢淋巴水肿的危险因素。结果宫颈癌患者术后下肢淋巴水肿发生率为20.93%,严重程度以Ⅰ度为主(占86.41%),中位发生时间为5.5个月。logistic回归分析表明,年龄、淋巴结清扫数目、术后辅助放疗、持续站立时间是术后下肢淋巴水肿发生的危险因素,规律运动锻炼是其保护因素(P0.05,P0.01)。243例术后联合放疗患者下肢淋巴水肿发生的危险因素为淋巴结清扫数目20枚及术后放疗开始时间45d(P0.05,P0.01)。结论下肢淋巴水肿是宫颈癌术后常见的并发症,年龄大、淋巴结清扫数目多、辅助放疗、长时间站立的患者是下肢淋巴水肿发生的高危人群。医护人员应做好高危人群筛查,及时给予健康教育,并积极探寻有效的预防干预措施,降低术后下肢淋巴水肿的发生率。     

Value of handgrip strength in predicting the mortality of hemodialysis patients

Objective To analyze the risk factors of mortality among patients treated by maintenance hemodialysis (MHD), and identify whether handgrip strength (HGS) or other nutrient markers could predict the mortality independently. Methods One hundred and eight patients receiving regular MHD in Peking Union Medical College Hospital from July to September, 2008 were involved. Baseline data including clinical data, nutrient data such as subjective global assessment, anthropometrics and biochemical measurement were collected. After being followed for 72 months, the patients' mortality and morbidity of cardiovascular event were recorded. Cox regression model was used to estimate the risk factors of mortality. Results The average age of 108 MHD patients was (57.6±13.0) years. During the 6-years following up, 35 patients died (32.4%), of whom 62.9% died of cardiovascular events. Among variables, patients’ age, residual urine volume, serum creatinine level, prealbumin level and mean leg circumference were risk factors for all-cause mortality. The patient with lower HGS bore higher risk for all-cause mortality (HR=2.842, 95%CI 1.390-5.811) and cardiovascular death (HR=2.826, 95%CI 1.150-6.947). After adjusting gender, age, history of cardiovascular disease and diabetes, body mass index (BMI), dialysis vintage, Kt/V, nPCR and prealbumin, lower handgrip strength was still an independent risk factor of all-cause mortality (HR=2.505, 95%CI 1.112-5.642). In prediction for all-cause mortality by HGS, the area under the receiver operating characteristic curve(ROC) were 0.705 and 0.682 among men and women respectively. Conclusion Lower handgrip strength can predict mortality of maintenance hemodialysis patients independently.     

Analysis of risk factors for steroid resistance in children with primary nephritic syndrome and discussion on the prediction model of steroid resistance

Objective To seek the risk factors of steroid resistance in children with primary nephritic syndrome, and construct the predicting model of steroid resistance. Methods The clinical indicators of 185 patients with primary nephrotic syndrome (PNS) were collected, including clinical data, laboratory and imaging examination. The risk factors of steroid resistance were found using single factor analysis, receiver operator characteristic (ROC) and logistic regression test. The predicting model of steroid resistance was constructed based on integral method model. Results The results of single factor analysis, receiver operating characteristic curve (ROC) and logistic regression analysis showed that the age more than 6.5 years old, having microscopic hematuria and the 24 h urine protein content (24 hUP) more than 177.49 mg•kg-1•d-1 were the significant risk factors of steroid resistant nephrotic syndrome (SRNS). Logistic regression prediction model was Y=6.761-2.947X1-3.336X2-2.669X3. The result of receiver operator characteristic showed that when the score was 0.95, the sensitivity and specificity was 56.56%, 96.62% respectively and the area under ROC was 0.86, P＜0.05). Conclusions The age more than 6.5 years old, having microscopic hematuria and 24 h urine protein content more than 177.49 mg•kg-1•d-1 are the significant risk factors of SRNS.     

Synthetic heparan sulfate standards and machine learning facilitate the development of solid-state nanopore analysis

The application of solid-state (SS) nanopore devices to single-molecule nucleic acid sequencing has been challenging. Thus, the early successes in applying SS nanopore devices to the more difficult class of biopolymer, glycosaminoglycans (GAGs), have been surprising, motivating us to examine the potential use of an SS nanopore to analyze synthetic heparan sulfate GAG chains of controlled composition and sequence prepared through a promising, recently developed chemoenzymatic route. A minimal representation of the nanopore data, using only signal magnitude and duration, revealed, by eye and image recognition algorithms, clear differences between the signals generated by four synthetic GAGs. By subsequent machine learning, it was possible to determine disaccharide and even monosaccharide composition of these four synthetic GAGs using as few as 500 events, corresponding to a zeptomole of sample. These data suggest that ultrasensitive GAG analysis may be possible using SS nanopore detection and well-characterized molecular training sets.

Glycosaminoglycans (GAGs) are linear anionic polysaccharides found on cell surfaces and in the extracellular matrix in all animals. GAGs comprise an important class of biopolymers that are ubiquitous in nature and exhibit a number of critical functional roles including biological recognition and signaling (1–3). Such processes play critical roles in physiology, such as in development and wound healing, and pathophysiology, such as cancer and infectious disease. Sulfated GAGs result from template-independent synthesis in the Golgi of animal cells (4, 5) and are polydisperse, heteropolysaccharides comprising variable disaccharide repeating units that are classified by these repeating units. Like nucleic acids, sulfated GAGs are made up of repeating units that comprise a linear sequence (Fig. 1). Unlike the nucleic acids, GAGs have far more complicated structures and number of possible sequences and they present severe challenges to both synthesis and characterization. Thus, we undertook to chemoenzymatically synthesize defined GAGs and characterize these using solid-state nanopore analysis.Open in a separate windowFig. 1.Structures of four synthetic GAG samples. Polysaccharide NSH is made up of N-sulfoglucosamine (GlcNS) and glucuronic acid (GlcA),NS2S is made up with GlcNS and 2-O-sulfo-iduronic acid (IdoA2S), NS6S is made up with 6-O-sulfo-N-sulfoheparosan (GlcNS6S) and GlcA, and NS6S2S is made up with GlcNS6S and IdoA2S.Despite their structural complexities, sulfated GAGs often contain well-defined domain structures that are responsible for their diverse biological functions, yet even this level of structural complexity poses a significant general challenge to structural analysis and sequencing. The simple, short-chain, chondroitin sulfate GAG component of bikunin has been sequenced using liquid chromatography–tandem mass spectrometry (LC-MS/MS) (6). While LC-MS/MS is capable of sequencing such simple, short-chain GAGs, it is not yet able to distinguish all of the many isobaric isomers of the variably sulfated saccharide residues and uronic acid epimers commonly encountered in more structurally complex GAGs, such as heparan sulfate (HS) (7). NMR has been applied to determine GAG structures but often requires milligram amounts of samples. HS/heparin is made up of →4)-β-d-glucuronic acid (GlcA) [or α-l-iduronic acid (IdoA)] (1→4)-α-d-glucosamine (GlcN) [1→ repeating units with 2-O-sulfo (S) groups on selected uronic acid residues and 3- and/or 6-O-S and N-S or N-acetyl (Ac) group substitutions on the glucosamine residues] (Fig. 1). GAG structural analysis presents challenges beyond their chemical complexity. There are no amplification methods to detect small numbers of GAG chains, whereas nucleic acid analysis can rely on PCR. Similarly, there are few GAG-specific antibodies or aptamers (8), and no natural GAG chromophores or fluorophores (9), in contrast to the many used for protein sensing. Ultrasensitive (zeptomole) detection methods of modified GAGs, based on fluorescence resonance energy transfer (FRET) (10), DNA bar coding (11), and dye-based nanosensors (12) have been demonstrated, but their application to sequencing is particularly challenging because of the high level of structural complexity of sulfated GAGs.Nanopore single-molecule detection is now routinely applied to DNA (13, 14) and RNA (15–17) biopolymers, and is increasingly applied to protein characterization (18–22). In brief, a nanopore is a nanofluidic channel ∼10 nm long and <100 nm in diameter, serving as the sole fluid connection between two reservoirs of electrolyte separated by an otherwise impermeable membrane (Fig. 2A). On applying a voltage across this nanopore, the passage of supporting electrolyte ions results in a “baseline,” or open-pore current, i₀. The passage of a biopolymer analyte through this nanopore disrupts the flow of supporting electrolyte ions, often as a current blockage. This temporary reduction in ionic current is called an “event,” and its magnitude (mean blockage ratio over the dwell time, ⟨f_b⟩=⟨i⟩_Td/⟨i₀⟩) and its temporal features [dwell time (Td)] (Fig. 2 B and C) depend on the size and shape of the nanopore, the biopolymer analyte, and the applied voltage and interfacial charge distributions. Indeed, the passage of DNA through engineered protein nanopore devices produces current blockages that can be applied in sequencing, and the widespread use of these commercial protein nanopore DNA sequencing devices is increasing (23, 24). Despite this success with protein nanopores, the potential benefits of (abiotic) solid-state (SS) nanopores have continued to drive development efforts. Such a transition to the freely size-tunable SS platform (25, 26), however, is vital for the application of nanopores to the characterization of branched glycans (27). Yet the use of SS nanopores in even the better-established DNA sensing regime remains challenging. The application of nanopore sensing to glycans, while promising, remains profoundly exploratory using nanopores of any kind. The transition to the SS nanopores is accompanied by significant changes in pore geometry, chemistry, characteristics, and potential analyte–pore interactions and sensing modalities, so that there is a critical need for studies in the realm of nanopore glycomics (27, 28). For example, outcomes of early nanopore studies on a structurally simple unsulfated GAG, hyaluronan (HA, →4)- β -GlcA (1 → 3)- β -GlcNAc (1→), while providing some information on HA size does not provide definitive structural information (29, 30). SS nanopore analysis of two sulfated GAGs, heparin and a heparin contaminant, oversulfated chondroitin sulfate, using a silicon nitride SS nanopore was able to qualitatively identify these GAGs by either the magnitude or duration of characteristic current blockages (28). SS nanopore data on GAGs, analyzed using a machine-learning (ML) algorithm (i.e., a support vector machine [SVM]), distinguished heparin and chondroitin sulfate oligosaccharides and unfractionated heparin and low molecular weight heparin with >90% accuracy (31).Open in a separate windowFig. 2.Nanopore characteristics of four samples. (A) Schematic of the nanopore configuration. Anionic GAGs driven by electrophoresis to and through the pore with a negative applied voltage would be detected if they perturbed the open-pore current. (B) A representative current trace and events from polysaccharide NS6S2S test using an ∼6-nm-diameter nanopore. Measurements were collected using a −150-mV applied-voltage (details in Results and Discussion, and Materials and Methods) (C) Scatter plots of dwell time vs. current blockage ratio for four polysaccharides. To remove the bias of event numbers in human image recognition, all plots contain only the first 2,475 events. (D) PCA visualization of the embedded images from the four unique GAGs. The blue circles and region represent NSH, the red X and region represents NS2S, the green triangle and region represents NS6S, and the brown cross and region represents NS6S2S. The algorithm clusters signals from each GAG based on scatter plot images. Each insert shows one 500-events image from each sample class. All 500-events images are in SI Appendix, Fig. S12.Nanopore studies on GAGs, and glycans more broadly, have been severely limited by the lack of a library of structurally defined standards. The uniformity of sulfated GAGs prepared from animal sources is difficult to control and exhibits significant sequence heterogeneity and polydispersity (32). HS is particularly problematic as even for a small HS hexasaccharide, composed of an IdoA/GlcA:GlcNS/GlcNAc sequence with 12 available sites for random sulfation, there are 32,768 possible sequences. Recently, chemoenzymatic synthesis has made inroads in the preparation of high-purity sulfated HS GAGs from heparosan (→4)- β -GlcA (1→4)- β -GlcNAc (1→) (33). HS GAGs of approximately the same chain length and polydispersity and having a single repeating disaccharide unit (SI Appendix, Table S1) including, NSH (→4)- β -GlcA (1→4)- β -GlcNS (1→), NS2S (→4)-α α -IdoA2S (1 → 4)- β -GlcNS (1→), NS6S (→4)- β -GlcA (1→4)- β -GlcNS6S(1→)), NS6S2S (→4)- α -IdoA2S (1→4)- β -GlcNS6S (1→) have been prepared (see Materials and Methods and ref. 34) (Fig. 1). Here we use our recently developed synthetic technique, which has proven difficult to benchmark, in conjunction with a nanopore technique, which has only just begun to be applied to glycomics and has been severely challenged by the lack of available high-quality samples, to develop a fully integrated approach for the nanopore analysis of complex carbohydrates.     

局部晚期非小细胞肺癌后程加速超分割放射治疗临床分析

目的观察局部晚期非小细胞肺癌后程加速超分割放射治疗（LCAHRT）的毒性和疗效。方法从2000年8月至2002年3月56例经病理诊断或细胞学确诊不能手术Ⅲ期非小细胞肺癌患者随机进入研究，放疗第1阶段大野前后对穿垂直照射原发灶、转移淋巴结、同侧肺门和纵隔淋巴引流区，2．0Gy／次，1融d。总剂量40Gy。第2阶段缩野照射原发灶和转移淋巴结，1．5Gy／次，2次／d，总剂量26～30Gy。结果放疗结束1个月后增强CT评价疗效，56例有效率67．9％，其中完全缓解（cR）3例．部分缓解（PR）35例，稳定（SD）14例，进展（PD）4例。中位生存时间12个月，1、2、3、4年总生存率分别为53．6％、19．6％、5．4％、3．6％。Ⅲa、Ⅲb期中位生存期分别为13、9个月。急性放射反应有放射性食管炎2级20例，3级2例；放射性肺炎2级2例：晚期放射性肺损伤2级5例，3级1例。结论后程超分割放射治疗局部晚期非小细胞肺癌剂量能够得到一定的提高，有很好的近期疗效且副作用可以接受。     

Mitochondrial complex I inhibition is not required for dopaminergic neuron death induced by rotenone, MPP+, or paraquat

Inhibition of mitochondrial complex I is one of the leading hypotheses for dopaminergic neuron death associated with Parkinson's disease (PD). To test this hypothesis genetically, we used a mouse strain lacking functional Ndufs4, a gene encoding a subunit required for complete assembly and function of complex I. Deletion of the Ndufs4 gene abolished complex I activity in midbrain mesencephalic neurons cultured from embryonic day (E) 14 mice, but did not affect the survival of dopaminergic neurons in culture. Although dopaminergic neurons were more sensitive than other neurons in these cultures to cell death induced by rotenone, MPP⁺, or paraquat treatments, the absence of complex I activity did not protect the dopaminergic neurons, as would be expected if these compounds act by inhibiting complex 1. In fact, the dopaminergic neurons were more sensitive to rotenone. These data suggest that dopaminergic neuron death induced by treatment with rotenone, MPP⁺, or paraquat is independent of complex I inhibition.     

益肝康等活血化瘀中药抑制IL-1β刺激的HSC增殖及TIMP-1的表达

目的:探讨活血化瘀中药益肝康、丹参小复方、丹参对IL-1β刺激的活化的大鼠肝星状细胞(HSCs)增殖及基质金属蛋白酶抑制因子 mRNA(TIMP mRNA)表达的影响．方法:体外培养活化的大鼠HSC,随机分为8 组:对照组(A组)、IL-1β 10 μg/L(B组)、IL- 1β 10μg/L 益肝康2 g/L干预组(C组)、IL- 1β 10 μg/L 丹参小复方2g/L干预组(D组)、 IL-1β 10μg/L 丹参2g/L干预组(E组)、丹参 2 g／L(F组)、丹参小复方2 g/L(G组)、益肝康 2 g／L(H组)．加药后24 h．应用活细胞计数试剂盒-CCK-8检测各组HSC增殖,采用半定量 RT-PCR方法检测各组HSC TIMP-1 mRNA的表达．结果:A组HSC增殖和TIMP-1 mRNA表达强于F、G、H组(1．291±0．09 vs 1．055±0．105, 1±0．07,0．883±0．06,P<0．01:0．591±0．064 vs 0．493±0．088．0．458±0．076．0．356±0．046． P<0．05或P<0．01);H组HSC增殖和TIMP-1 mRNA表达低于F组和G组(P<0．05);B组HSC 增殖和TIMP-1 mRNA表达均明显强于A组 (1．575±0．017 vs 1.291±0,09,P<0．01;1．369± 0．097 vs 0．591±0．064,P<0．01)和C、D、E组 (1．575±0．017 vs 0．906±0．09,1．015±0．081, 1．097±0．038,P<0．01;1．369±0．097 vs 0．694 ±0．078,0．854±0．05,0．898±0．12,P<0．01);C 组HSC增殖和TIMP-1 mRNA表达低于D组和 E组(P<0．05)．结论:益肝康等活血化瘀中药能抑制IL-1β刺激的HSCs增殖及TIMP-1 mRNA表达,发挥其抗肝纤维化功效．益肝康抗肝纤维化作用强于丹参小复方和丹参单药．     

缺血性脑卒中后血管性认知功能障碍患者相关因素分析

目的探讨缺血性脑卒中后血管性认知功能障碍(VCI)发生的相关因素。方法收集2011年6月至2014年6月入住我院的208例缺血性脑卒中患者的临床资料,其中并发VCI患者82例,非VCI(NVCI)患者126例。分别采用Pearson单因素与多元Logistic回归分析的方法对缺血性脑卒中后并发VCI的相关因素加以确定。结果 (1)经Pearson单因素分析,VCI组与NVCI组在高血压、房颤、血脂异常、NIHSS评分、高糖化血红蛋白、同型半胱氨酸、超敏C反应蛋白及关键部位病灶差异具有统计学意义(P<0.05,P<0.01);(2)经多元Logistic回归分析,影响缺血性脑卒中后VCI的相关因素包括:高糖化血红蛋白(β=1.928,SE=0.382,Wald=6.928,P<0.05,OR值=2.559,95%CI为1.544~5.782)、高血压(β=2.024,SE=0.195,Wald=7.125,P<0.01,OR值=2.135,95%CI为1.672~3.225)、高同型半胱氨酸(β=2.359,SE=0.297,Wald=8.567,P<0.01,OR值=2.687,95%CI为1.938~4.253)、关键部位病灶(β=2.346,SE=0.597,Wald=7.259,P<0.05,OR值=2.187,95%CI为1.692~3.876)及高超敏C反应蛋白(β=1.856,SE=0.118,Wald=5.358,P<0.05,OR值=2.102,95%CI为1.432~3.870)。结论 影响缺血性脑卒中后并发VCI的因素较多,应对这些危险因素进行早期干预,降低缺血性脑卒中后VCI的发病率。