Cells Derived from the Coelomic Epithelium Contribute to Multiple Gastrointestinal Tissues in Mouse Embryos

Gut mesodermal tissues originate from the splanchnopleural mesenchyme. However, the embryonic gastrointestinal coelomic epithelium gives rise to mesenchymal cells, whose significance and fate are little known. Our aim was to investigate the contribution of coelomic epithelium-derived cells to the intestinal development. We have used the transgenic mouse model mWt1/IRES/GFP-Cre (Wt1^cre) crossed with the Rosa26R-EYFP reporter mouse. In the gastrointestinal duct Wt1, the Wilms’ tumor suppressor gene, is specific and dynamically expressed in the coelomic epithelium. In the embryos obtained from the crossbreeding, the Wt1-expressing cell lineage produces the yellow fluorescent protein (YFP) allowing for colocalization with differentiation markers through confocal microscopy and flow cytometry. Wt1^cre-YFP cells were very abundant throughout the intestine during midgestation, declining in neonates. Wt1^cre-YFP cells were also transiently observed within the mucosa, being apparently released into the intestinal lumen. YFP was detected in cells contributing to intestinal vascularization (endothelium, pericytes and smooth muscle), visceral musculature (circular, longitudinal and submucosal) as well as in Cajal and Cajal-like interstitial cells. Wt1^cre-YFP mesenchymal cells expressed FGF9, a critical growth factor for intestinal development, as well as PDGFRα, mainly within developing villi. Thus, a cell population derived from the coelomic epithelium incorporates to the gut mesenchyme and contribute to a variety of intestinal tissues, probably playing also a signaling role. Our results support the origin of interstitial cells of Cajal and visceral circular muscle from a common progenitor expressing anoctamin-1 and SMCα-actin. Coelomic-derived cells contribute to the differentiation of at least a part of the interstitial cells of Cajal.     

不同光强对越南抱茎茶光合及荧光特性日变化的影响

为研究不同光强对越南抱茎茶光合作用机理的影响,设置3种处理方式,即全光照(L1)、光强度45%(L2)、光强度15%(L3),分别在遮阴3个月后测定其光合荧光参数的变化。结果表明,不同光强处理下,净光合速率均呈单峰曲线,峰值出现在11:00左右;光合速率下降主要是气孔限制因素引起的;叶绿素荧光参数表明,强光条件下电子传递受阻,热耗散增加,PSII反应中心受到破坏,从而降低了茶花的光合效率;过度遮阴条件下受到光抑制,从而导致光合机构损伤;45%光照条件下光合效率较高。相关性分析表明,净光合速率(Pn)与空气温度(Ta)、相对湿度(RH)、气孔导度(Gs)、蒸腾速率(Tr)、水分利用效率(WUE)、表观电子传递速率(ETR)呈极显著相关,与RH呈极显著负相关,与胞间CO2浓度(Ci)呈显著负相关。影响越南抱茎茶净光合速率的主要环境因子是空气温度和相对湿度。     

Porcine Pluripotent Stem Cells Derived from IVF Embryos Contribute to Chimeric Development In Vivo

Although the pig is considered an important model of human disease and an ideal animal for the preclinical testing of cell transplantation, the utility of this model has been hampered by a lack of genuine porcine embryonic stem cells. Here, we derived a porcine pluripotent stem cell (pPSC) line from day 5.5 blastocysts in a newly developed culture system based on MXV medium and a 5% oxygen atmosphere. The pPSCs had been passaged more than 75 times over two years, and the morphology of the colony was similar to that of human embryonic stem cells. Characterization and assessment showed that the pPSCs were alkaline phosphatase (AKP) positive, possessed normal karyotypes and expressed classic pluripotent markers, including OCT4, SOX2 and NANOG. In vitro differentiation through embryonic body formation and in vivo differentiation via teratoma formation in nude mice demonstrated that the pPSCs could differentiate into cells of the three germ layers. The pPSCs transfected with fuw-DsRed (pPSC-FDs) could be passaged with a stable expression of both DsRed and pluripotent markers. Notably, when pPSC-FDs were used as donor cells for somatic nuclear transfer, 11.52% of the reconstructed embryos developed into blastocysts, which was not significantly different from that of the reconstructed embryos derived from porcine embryonic fibroblasts. When pPSC-FDs were injected into day 4.5 blastocysts, they became involved in the in vitro embryonic development and contributed to the viscera of foetuses at day 50 of pregnancy as well as the developed placenta after the chimeric blastocysts were transferred into recipients. These findings indicated that the pPSCs were porcine pluripotent cells; that this would be a useful cell line for porcine genetic engineering and a valuable cell line for clarifying the molecular mechanism of pluripotency regulation in pigs.     

Genetic Variations in the Serotoninergic System Contribute to Body-Mass Index in Chinese Adolescents

Objective

Obesity has become a worldwide health problem in the past decades. Human and animal studies have implicated serotonin in appetite regulation, and behavior genetic studies have shown that body mass index (BMI) has a strong genetic component. However, the roles of genes related to the serotoninergic (5-hydroxytryptamine,5-HT) system in obesity/BMI are not well understood, especially in Chinese subjects.

Subjects and Design

With a sample of 478 healthy Chinese volunteers, this study investigated the relation between BMI and genetic variations of the serotoninergic system as characterized by 136 representative polymorphisms. We used a system-level approach to identify SNPs associated with BMI, then estimated their overall contribution to BMI by multiple regression and verified it by permutation.

Results

We identified 12 SNPs that made statistically significant contributions to BMI. After controlling for gender and age, four of these SNPs accounted for 7.7% additional variance of BMI. Permutation analysis showed that the probability of obtaining these findings by chance was low (p = 0.015, permuted for 1000 times).

Conclusion

These results showed that genetic variations in the serotoninergic system made a moderate contribution to individual differences in BMI among a healthy Chinese sample, suggesting that a similar approach can be used to study obesity.     

用随机扩增多态性DNA产物做探针产生鸡的DNA指纹图

我们用12个随机扩增多态性DNA(RAPD)引物对来自不同品系的4只鸡进行了RAPD分析,在扩增出的共99条带中,表现多态性的带为38条,占总带数的38％.回收了4个表现个体特异性的RAPD产物,当用鸡的基因组总DNA探针与它们杂交时,其中3个表现阳性,说明RAPD方法扩增出的高变异产物含有重复序列.用含重复序列的个体特异性RAPD产物作探针,与无关个体鸡基因组DNA的HaeⅢ酶切产物进行DNA印迹,获得了变异性较高的DNA指纹图谱.因此,高变异的RAPD产物可以有效地用作DNA指纹探针.     

Quantification of Different Eubacterium spp. in Human Fecal Samples with Species-Specific 16S rRNA-Targeted Oligonucleotide Probes

Species-specific 16S rRNA-targeted, Cy3 (indocarbocyanine)-labeled oligonucleotide probes were designed and validated to quantify different Eubacterium species in human fecal samples. Probes were directed at Eubacterium barkeri, E. biforme, E. contortum, E. cylindroides (two probes), E. dolichum, E. hadrum, E. lentum, E. limosum, E. moniliforme, and E. ventriosum. The specificity of the probes was tested with the type strains and a range of common intestinal bacteria. With one exception, none of the probes showed cross-hybridization under stringent conditions. The species-specific probes were applied to fecal samples obtained from 12 healthy volunteers. E. biforme, E. cylindroides, E. hadrum, E. lentum, and E. ventriosum could be determined. All other Eubacterium species for which probes had been designed were under the detection limit of 10⁷ cells g (dry weight) of feces⁻¹. The cell counts obtained are essentially in accordance with the literature data, which are based on colony counts. This shows that whole-cell in situ hybridization with species-specific probes is a valuable tool for the enumeration of Eubacterium species in feces.     

Fluorescent Protein Voltage Probes Derived from ArcLight that Respond to Membrane Voltage Changes with Fast Kinetics

We previously reported the discovery of a fluorescent protein voltage probe, ArcLight, and its derivatives that exhibit large changes in fluorescence intensity in response to changes of plasma membrane voltage. ArcLight allows the reliable detection of single action potentials and sub-threshold activities in individual neurons and dendrites. The response kinetics of ArcLight (τ1-on ~10 ms, τ2-on ~ 50 ms) are comparable with most published genetically-encoded voltage probes. However, probes using voltage-sensing domains other than that from the Ciona intestinalis voltage sensitive phosphatase exhibit faster kinetics. Here we report new versions of ArcLight, in which the Ciona voltage-sensing domain was replaced with those from chicken, zebrafish, frog, mouse or human. We found that the chicken and zebrafish-based ArcLight exhibit faster kinetics, with a time constant (τ) less than 6ms for a 100 mV depolarization. Although the response amplitude of these two probes (8-9%) is not as large as the Ciona-based ArcLight (~35%), they are better at reporting action potentials from cultured neurons at higher frequency. In contrast, probes based on frog, mouse and human voltage sensing domains were either slower than the Ciona-based ArcLight or had very small signals.     

两种标记方法对60mer寡核苷酸芯片背景信号影响的初步分析

研究两种不同的样本标记方法对人全基因组高密度60mer寡核苷酸芯片背景信号的影响。收集5对患病与健康人外周血单个核细胞,分别提取总RNA后,采用限制性显示技术（restriction display,RD）进行样本双色（Cy3/Cy5）荧光标记,与5张Agilent 60mer高密度（22K）Human 1B寡核苷酸芯片进行杂交。芯片全部杂交点分3组：基因探针组、阳性对照组和阴性对照组。阳性对照采用荧光标记寡核苷酸直接掺入法进行标记。对全部杂交信号点的Cy3和Cy5背景信号值,用SPSS软件进行数据转换、正态性检验、方差齐性检验、变异系数分析和重复数据的方差分析。数据分析结果显示,Cy3 标记的背景信号值均高于 Cy5标记的背景信号值。重复测量数据的方差分析表明,在Cy3 和Cy5标记中,两种不同标记方法间的背景信号值的差异极显著（PCy3＜0.01, PCy5＜0.01）,且RD标记点的背景信号平均值低于荧光标记寡核苷酸直接掺入标记法标记的阳性对照点。RD标记方法是一种有用的低背景信号的高密度长链寡核苷酸芯片样本标记方法。     

鼎湖山国家自然保护区林下土壤微生物新结构生物活性代谢产物的研究进展

鼎湖山国家自然保护区特殊的地理位置和气候条件造就了独特的生态环境,孕育了丰富的植物和微生物资源。为了挖掘其中的微生物资源,对近年来鼎湖山林下土壤来源的真菌和放线菌次生代谢物研究进行了综述。     

Correlated Conductance Parameters in Leech Heart Motor Neurons Contribute to Motor Pattern Formation

Neurons can have widely differing intrinsic membrane properties, in particular the density of specific conductances, but how these contribute to characteristic neuronal activity or pattern formation is not well understood. To explore the relationship between conductances, and in particular how they influence the activity of motor neurons in the well characterized leech heartbeat system, we developed a new multi-compartmental Hodgkin-Huxley style leech heart motor neuron model. To do so, we evolved a population of model instances, which differed in the density of specific conductances, capable of achieving specific output activity targets given an associated input pattern. We then examined the sensitivity of measures of output activity to conductances and how the model instances responded to hyperpolarizing current injections. We found that the strengths of many conductances, including those with differing dynamics, had strong partial correlations and that these relationships appeared to be linked by their influence on heart motor neuron activity. Conductances that had positive correlations opposed one another and had the opposite effects on activity metrics when perturbed whereas conductances that had negative correlations could compensate for one another and had similar effects on activity metrics.     

Exosomes Derived from Human Primary and Metastatic Colorectal Cancer Cells Contribute to Functional Heterogeneity of Activated Fibroblasts by Reprogramming Their Proteome

Cancer‐associated fibroblasts (CAFs) are a heterogeneous population of activated fibroblasts that constitute a dominant cellular component of the tumor microenvironment (TME) performing distinct functions. Here, the role of tumor‐derived exosomes (Exos) in activating quiescent fibroblasts into distinct functional subtypes is investigated. Proteomic profiling and functional dissection reveal that early‐ (SW480) and late‐stage (SW620) colorectal cancer (CRC) cell‐derived Exos both activated normal quiescent fibroblasts (α‐SMA^?, CAV⁺, FAP⁺, VIM⁺) into CAF‐like fibroblasts (α‐SMA⁺, CAV^?, FAP⁺, VIM⁺). Fibroblasts activated by early‐stage cancer‐exosomes (SW480‐Exos) are highly pro‐proliferative and pro‐angiogenic and display elevated expression of pro‐angiogenic (IL8, RAB10, NDRG1) and pro‐proliferative (SA1008, FFPS) proteins. In contrast, fibroblasts activated by late‐stage cancer‐exosomes (SW620‐Exos) display a striking ability to invade through extracellular matrix through upregulation of pro‐invasive regulators of membrane protrusion (PDLIM1, MYO1B) and matrix‐remodeling proteins (MMP11, EMMPRIN, ADAM10). Conserved features of Exos‐mediated fibroblast activation include enhanced ECM secretion (COL1A1, Tenascin‐C/X), oncogenic transformation, and metabolic reprogramming (downregulation of CAV‐1, upregulation of glycogen metabolism (GAA), amino acid biosynthesis (SHMT2, IDH2) and membrane transporters of glucose (GLUT1), lactate (MCT4), and amino acids (SLC1A5/3A5)). This study highlights the role of primary and metastatic CRC tumor‐derived Exos in generating phenotypically and functionally distinct subsets of CAFs that may facilitate tumor progression.     

Head Tilt and Fertility Contribute to Different Aspects of Female Facial Attractiveness

Subjective attractiveness ratings of facial portraits of women taken at the fertile phase of the menstrual cycle are higher than those of portraits of the same women taken during non‐fertile periods. As female faces tilted downward are rated as more attractive and female courtship behaviours change across the menstrual cycle, we investigated whether systematic downward tilt of women's faces during the fertile phase might be responsible for increased attractiveness ratings. In the original study (Proc. R. Soc. Lond. B, 271, 2004, S272), the fertile‐phase portrait of each woman was deemed more attractive in 56–62% of cases. When the portraits were reclassified by head pitch, the more downward‐tilted portrait was preferred in 64–73% of cases. The fertile‐phase portrait was no more likely to be the downward‐tilted one, however, suggesting that effects of fertility on attractiveness are not simply due to changes in head position. We also had these portraits rated (N = 130) for physical attractiveness and behavioural allure. Fertile‐phase portraits were rated as more physically attractive than non‐fertile portraits, while more downward‐tilted portraits were rated as more behaviourally alluring than less downward‐tilted ones. These data not only confirm reported effects of head tilt and fertility on perceived female attractiveness, but also suggest that these factors influence different components of the attractiveness percept.     

Quantification of Mature MicroRNAs Using Pincer Probes and Real-Time PCR Amplification

The robust and reliable detection of small microRNAs (miRNAs) is important to understand the functional significance of miRNAs. Several methods can be used to quantify miRNAs. Selectively quantifying mature miRNAs among miRNA precursors, pri-miRNAs, and other miRNA-like sequences is challenging because of the short length of miRNAs. In this study, we developed a two-step miRNA quantification system based on pincer probe capture and real-time PCR amplification. The performance of the method was tested using synthetic mature miRNAs and clinical RNA samples. Results showed that the method demonstrated dynamic range of seven orders of magnitude and sensitivity of detection of hundreds of copies of miRNA molecules. The use of pincer probes allowed excellent discrimination of mature miRNAs from their precursors with five Cq (quantification cycle) values difference. The developed method also showed good discrimination of highly homologous family members with cross reaction less than 5%. The pincer probe-based approach is a potential alternative to currently used methods for mature miRNA quantification.     

Quantification of DNA structure from NMR data: conformation of d-ACATCGATGT

Resonance assignments of nonexchangeable base and sugar protons have been obtained in double-helical d-ACATCGATGT by using two-dimensional correlated spectroscopy (COSY) and nuclear Overhauser enhancement spectroscopy (NOESY). The exchangeable imino protons have been assigned on the basis of their chemical shifts. The characteristic phase-sensitive multiplet patterns of the intrasugar cross-peaks in the omega 1-scaled COSY spectrum have been used to estimate several scalar coupling constants (J). The information on the J values combined with the intranucleotide COSY cross-peak intensities has been used to identify sugar puckers of individual nucleotide units. In most cases, the deoxyribofuranose rings are found to adopt a conformation close to O4'-endo. Spin diffusion has been monitored from the buildup of the normalized volumes of NOE cross-peaks in NOESY spectra as a function of mixing time. A set of 52 intranucleotide and internucleotide proton-proton distances have been estimated by using low mixing time NOESY spectra (tau m = 40 and 80 ms). The estimated intrasugar proton-proton distances rule out possibilities of existence of a fast equilibrium between C2'-endo and C3'-endo conformations. Intranucleotide proton-proton distances combined with the knowledge of sugar puckers have been used to fix the glycosidic bond torsion angle (chi). For this purpose, simulated distance contours depicting the dependence of intranucleotide proton-proton distances on pseudorotational phase angle (P) and glycosidic bond torsion angle (chi) have been used. Further, the proton homonuclear (J, delta) spectrum has been used to monitor the 31P-1H heteronuclear couplings, which are preserved in the omega 2 projection.(ABSTRACT TRUNCATED AT 250 WORDS)     

Two Different Conductances Contribute to the Anion Currents in Coffea arabica Protoplasts

The anion conductance of the plasma membrane of Coffea arabica protoplasts was isolated and characterized using the whole-cell patch clamp technique. Voltage pulse protocols revealed two components: a voltage-gated conductance (G _s ) and a voltage-independent one (G _l ). G _s is activated upon depolarization (e-fold activation every +36 mV) with time constants of 1 sec and 5 sec at all potentials. G _l and G _s also differ by their kinetic and biophysical properties. In bi-ionic conditions the current associated with G _s shows strong outward rectification and its permeability sequence is F⁻ > NO₃ ⁻ > Cl⁻. In the same conditions the current associated with G _l does not rectify and its permeability sequence is F⁻≫ NO₃ ⁻= Cl⁻. Furthermore, at potentials over +50 mV G _s , but not G _l , increases with a time constant of several minutes. Finally the gating of G _s is affected by stretch of the membrane, which leads to an increased activation and a reduced voltage sensitivity. Anion conductances similar to the ones described here have been found in many plant preparations but G _l -type components have been generally interpreted as the background activation of the slow voltage-gated channels (corresponding to G _s ). We show that in coffee protoplasts G _l and G _s are kinetically and biophysically distinct, suggesting that they correspond to two different molecular entities. Received: 25 November 1996/Revised: 9 April 1997     

Quantification of Methanosaeta Species in Anaerobic Bioreactors Using Genus- and Species-Specific Hybridization Probes

A BSTRACTTo evaluate the role of Methanosaeta spp. in a variety of anaerobic environments, small-subunit rRNA targeted oligonucleotide hybridization probes were developed and experimentally characterized. The probes were designed to be genus specific for Methanosaeta and species specific for Methanosaeta concilii and Methanosaeta thermophila. The temperature of dissociation was determined for each probe. Probe specificities were determined using a diverse collection of Archaea and through an evaluation of probe nesting using samples from a variety of anaerobic bioreactors. Cell fixation and hybridization conditions for fluorescence in situ hybridizations were also evaluated. Although permeability of methanogens was variable, M. concilii cells could be permeabilized using a range of paraformaldehyde and ethanol based fixation conditions. Using the newly designed probes together with previously designed probes for methanogens, it was determined that Methanosaeta spp. were the dominant aceticlastic methanogens in a variety of anaerobic bioreactors when acetate concentrations were low. Their levels were higher in bioreactors with granular sludge than in those with flocculent sludge. In lab-scale upflow anaerobic sludge blanket reactors, the levels of M. concilii rRNA were as high as 30% of the total rRNA.     

Three-dimensional Quantification of Dendritic Spines from Pyramidal Neurons Derived from Human Induced Pluripotent Stem Cells

Dendritic spines are small protrusions that correspond to the post-synaptic compartments of excitatory synapses in the central nervous system. They are distributed along the dendrites. Their morphology is largely dependent on neuronal activity, and they are dynamic. Dendritic spines express glutamatergic receptors (AMPA and NMDA receptors) on their surface and at the levels of postsynaptic densities. Each spine allows the neuron to control its state and local activity independently. Spine morphologies have been extensively studied in glutamatergic pyramidal cells of the brain cortex, using both in vivo approaches and neuronal cultures obtained from rodent tissues. Neuropathological conditions can be associated to altered spine induction and maturation, as shown in rodent cultured neurons and one-dimensional quantitative analysis ¹. The present study describes a protocol for the 3D quantitative analysis of spine morphologies using human cortical neurons derived from neural stem cells (late cortical progenitors). These cells were initially obtained from induced pluripotent stem cells. This protocol allows the analysis of spine morphologies at different culture periods, and with possible comparison between induced pluripotent stem cells obtained from control individuals with those obtained from patients with psychiatric diseases.     

Exchangeable Colloidal AFM Probes for the Quantification of Irreversible and Long-Term Interactions

An original method is presented to study single-colloid interaction with a substrate in liquid environment. Colloids, either in solution or adsorbed on a surface, are fixed by suction against the aperture of a microchanneled atomic force microscopy cantilever. Their adhesion to the substrate is measured, followed by their release via a short overpressure surge. Such colloid exchange procedure allows for 1), the quick variation of differently functionalized colloids within the same experiment; 2), the investigation of long-term interactions by leaving the colloids on a surface for a defined time before detaching them; and 3), the inspection of irreversible interactions. After validation of the method by reproducing literature results obtained with traditional colloidal atomic force microscopy, the serial use of colloids with different surface functionalization was shown on a micropatterned surface. Finally, concanavalin A-coated colloids were allowed to adsorb on human embryonic kidney cells and then detached one by one. The adhesion between cells and colloids was up to 60 nN, whereas individual cells adhered with 20 nN to the glass substrate. A cellular elastic modulus of 0.8 kPa was determined using the attached colloid as indenter.