A Small‐Molecule AIE Chromosome Periphery Probe for Cytogenetic Studies

The chromosome periphery (CP) is a complex network that covers the outer surface of chromosomes. It acts as a carrier of nucleolar components, helps maintain chromosome structure, and plays an important role in mitosis. Current methods for fluorescence imaging of CP largely rely on immunostaining. We herein report a small‐molecule fluorescent probe, ID‐IQ , which possesses aggregation‐induced emission (AIE) property, for CP imaging. By labelling the CP, ID‐IQ sharply highlighted the chromosome boundaries, which enabled rapid segmentation of touching and overlapping chromosomes, direct identification of the centromere, and clear visualization of chromosome morphology. ID‐IQ staining was also compatible with fluorescence in situ hybridization and could assist the precise location of the gene in designated chromosome. Altogether, this study provides a versatile cytogenetic tool for improved chromosome analysis, which greatly benefits the clinical diagnostic testing and genomic research.     

Chromosome protein framework from proteome analysis of isolated human metaphase chromosomes

We have presented a structural model of the chromosome based on its constituent proteins. Development of a method of mass isolation for intact human metaphase chromosomes and proteome analysis by mass spectrometry of the isolated chromosomal proteins enabled us to develop a four-layer structural model of human metaphase chromosomes. The model consists of four layers, each with different chromosomal protein sets, i.e., chromosome coating proteins (CCPs), chromosome peripheral proteins (CPPs), chromosome structural proteins (CSPs), and chromosome fibrous proteins (CFPs). More than 200 identified proteins have been classified and assigned to the four layers with each layer occupying a distinct region of the chromosome. CCPs are localized at the most outer regions of the chromosomes and they attach to the regions tentatively and occasionally. CCPs include mostly mitochondrial and cytoplasmic proteins, e.g., 70 kDa heat shock protein 9B and Hsp60. CPPs are also localized at the peripheral regions of the chromosomes, but as the essential part of the chromosomes. CPPs include nucleolin, lamin A/C, fibrillarin, etc. CSPs are the primary chromosomal structure proteins, and include topoisomerase IIalpha, condensin subunits, histones, etc. CFPs have a fibrous nature, e.g., beta-actin, vimentin, myosin II, tublin, etc. A data set of these proteins, which we developed, contains essential chromosome proteins with classified information based on this four-layer model and presents useful leads for further studies on chromosomal structure and function.     

RNP STRUCTURE IN METAPHASE CHROMOSOMES OF Vicia faba

The results of studies on ultrastructural and cytochemical features of metaphase chromosomes of Vicia faba are reported. In conventionally stained specimens are observed many electron-lucent compartments, in which exists an internal fibrillar-granular structure. When stained with the Bernhard's technique, the internal structure is found to consist of ribonucleoprotein (RNP) fibrilsand granules that measure about 15 to 20 nmin diameter. In the transverse section of metaphase chromosomes, the RNP structures are distributed in both the central and the peripheral regions of chromatids. It is conceivable that this RNP structure constitutes a special component of chromosomes. The distribution of the RNP structure in a chromosome and some questions related to the chromosomal scaffold are discussed on the basis of these data.     

谷胱甘肽和凋亡酶-3响应的环肽分子荧光探针

设计、合成了一类新型谷胱甘肽(glutathione,GSH)和凋亡酶-3(Caspase-3)响应的环肽分子荧光探针.该类探针主要由能量共振转移(FRET)分子荧光对、Caspase-3特异性识别多肽序列和GSH响应双硫键组成,分为不含穿膜肽序列(CP)和包含穿膜肽序列(cp CP)的两种不同环肽分子荧光探针.2种环肽分子荧光探针均能实现在GSH和Caspase-3同时存在情况下的精确成像,同时具有良好的响应性、特异性和高信噪比.该类环肽分子荧光探针在细胞培养环境中具有良好的稳定性和生物相容性.利用该探针,可以实现对星形孢菌素(STS)诱发的细胞凋亡进行实时、原位的成像监测,并对抗肿瘤药物阿霉素(DOX)和顺铂(cisplatin)诱导的细胞凋亡进行成像.这种具有多重响应并能用于精确成像的分子荧光探针将极大地促进疾病的精确诊断.     

UV-A breakage sensitivity of human chromosomes as measured by COMET-FISH depends on gene density and not on the chromosome size

COMET-FISH, a single cell-based combination of COMET-assay (also known as single cell gel electrophoresis (SCGE)) with fluorescence in situ hybridization (FISH) allows region specific studies on DNA stability and damage. COMET-FISH can be used to investigate UV-A-induced DNA damage of selected whole chromosomes. In the present work, a modified COMET-FISH protocol with whole chromosome painting probes was used to study whether UV-A-induced DNA damage is distributed randomly over the whole genome or occurs at preferred sites. The study was performed with 12 different chromosome painting probes (for chromosomes 1, 2, 3, 8, 9, 11, 14, 18, 19, 21, X and Y). The results on human lymphocytes irradiated with 500 kJ/m2 at a wavelength of 365 nm indicate that the induced number of chromatin strand breaks does not correlate with the chromosome size. They therefore are distributed in a non-random manner. For example, fragments of the gene-rich chromosome chromosome 1 were found in the comet tail in only 3% of the examined cells, and thus chromosome 1 is rather stable, whereas fragmentation of the gene-poor chromosome 8 was observed in 25% of all comets. On the basis of all 12 chromosomes analyzed, an inverse correlation between the density of active genes and the sensitivity toward UV-A radiation is found.     

A device for extraction, manipulation and stretching of DNA from single human chromosomes

We describe the structure and operation of a micro/nanofluidic device in which individual metaphase chromosomes can be isolated and processed without being displaced during exchange of reagents. The change in chromosome morphology as a result of introducing protease into the device was observed by time-lapse imaging; pressure-driven flow was then used to shunt the chromosomal DNA package into a nanoslit. A long linear DNA strand (>1.3 Mbp) was seen to stretch out from the DNA package and along the length of the nanoslit. Delivery of DNA in its native metaphase chromosome package as well as the microfluidic environment prevented DNA from shearing and will be important for preparing ultra-long lengths of DNA for nanofluidic analysis.     

Multiplex genotyping assays for fine‐resolution subtyping of the major human Y‐chromosome haplogroups E,G, I,J, and R in anthropological,genealogical, and forensic investigations

Inherited DNA polymorphisms located within the nonrecombing portion of the human Y chromosome provide a powerful means of tracking the patrilineal ancestry of male individuals. Recently, we introduced an efficient genotyping method for the detection of the basal Y‐chromosome haplogroups A to T, as well as an additional method for the dissection of haplogroup O into its sublineages. To further extend the use of the Y chromosome as an evolutionary marker, we here introduce a set of genotyping assays for fine‐resolution subtyping of haplogroups E, G, I, J, and R, which make up the bulk of Western Eurasian and African Y chromosomes. The marker selection includes a total of 107 carefully selected bi‐allelic polymorphisms that were divided into eight hierarchically organized multiplex assays (two for haplogroup E, one for I, one for J, one for G, and three for R) based on the single‐base primer extension (SNaPshot) technology. Not only does our method allow for enhanced Y‐chromosome lineage discrimination, the more restricted geographic distribution of the subhaplogroups covered also enables more fine‐scaled estimations of patrilineal bio‐geographic origin. Supplementing our previous method for basal Y‐haplogroup detection, the currently introduced assays are thus expected to be of major relevance for future DNA studies targeting male‐specific ancestry for forensic, anthropological, and genealogical purposes.     

Localisation and accumulation of a new carotenoporphyrin in two primary tumour models

We have investigated the tumour-localising properties and in vivo fluorescence kinetics of a hexamethoxylated carotenqporphyrin (CP6) in two primary tumour models: UV-B-induced early skin cancer in hairless mice and chemically induced mucosal dysplasia in the rat palate. CP6 fluorescence kinetics are investigated by measuring in vivo fluorescence spectra and images of the mouse skin and the rat palate at different time points after injection. For the tumour-localising properties, microscopic phase-contrast and fluorescence images are recorded. The in vivo fluorescence kinetics in the mouse skin show localization of CP6 in the tumours. However, fluorescence microscopy images show that CP6 localises in the dermis and structures that are not related to the malignant transformation of the mouse skin. The fluorescence kinetics in the rat palate show a significant correlation between the degree of malignancy and the CP6 fluorescence build-up time in the palate. The microscopic images show that CP6 fluorescence localises in the connective tissue and not in the dysplastic epithelium. In conclusion, CP6 does not localise preferentially in (pre-) cancerous tissue in the two primary tumour models studied here, in contrast to reports about localisation of carotenoporphyrins in transplanted tumours. However, the CP6 build-up time in rat palates correlates with the degree of malignancy and this might possibly be a useful parameter in tumour detection.     

Heritability and linkage study on heart rates in a Mongolian population

Elevated heart rate has been proposed as an independent risk factor for cardiovascular diseases, but their interrelationships are not well understood. In this study, we performed a genome-wide linkage scan in 1,026 individuals (mean age 30.6 years, 54.5% women) from 73 extended families of Mongolia and determined quantitative trait loci that influence heart rate. The DNA samples were genotyped using deCODE 1,039 microsatellite markers for 3 cM density genome-wide linkage scan. Correlation analysis was carried out to evaluate the correlation of the covariates and the heart rate. T-tests of the heart rate were also performed on sex, smoking and alcohol intake. Consequently, this model was used in a nonparametric genome-wide linkage analysis using variance component model to create a multipoint logarithm of odds (LOD) score and a corresponding P value. In the adjusted model, the heritability of heart rate was estimated as 0.32 (P<.0001) and a maximum multipoint LOD score of 2.03 was observed in 77 cM region at chromosome 18. The second largest LOD score of 1.52 was seen on chromosome 5 at 216 cM. Genes located on the specified locations in chromosomes 5 and 18 may be involved in the regulation of heart rate.     

The application of an in situ karyotyping technique for mesenchymal stromal cells: a validation and comparison study with classical G-banding

The cytogenetic analysis of mesenchymal stromal cells (MSCs) is essential for verifying the safety and stability of MSCs. An in situ technique, which uses cells grown on coverslips for karyotyping and minimizes cell manipulation, is the standard protocol for the chromosome analysis of amniotic fluids. Therefore, we applied the in situ karyotyping technique in MSCs and compared the quality of metaphases and karyotyping results with classical G-banding and chromosomal abnormalities with fluorescence in situ hybridization (FISH). Human adipose- and umbilical cord-derived MSC cell lines (American Type Culture Collection PCS-500-011, PCS-500-010) were used for evaluation. The quality of metaphases was assessed by analyzing the chromosome numbers in each metaphase, the overlaps of chromosomes and the mean length of chromosome 1. FISH was performed in the interphase nuclei of MSCs for 6q, 7q and 17q abnormalities and for the enumeration of chromosomes via oligo-FISH in adipose-derived MSCs. The number of chromosomes in each metaphase was more variable in classical G-banding. The overlap of chromosomes and the mean length of chromosome 1 as observed via in situ karyotyping were comparable to those of classical G-banding (P=0.218 and 0.674, respectively). Classical G-banding and in situ karyotyping by two personnel showed normal karyotypes for both cell lines in five passages. No numerical or structural chromosomal abnormalities were found by the interphase-FISH. In situ karyotyping showed equivalent karyotype results, and the quality of the metaphases was not inferior to classical G-banding. Thus, in situ karyotyping with minimized cell manipulation and the use of less cells would be useful for karyotyping MSCs.     

A novel technique for observing the internal ultrastructure of human chromosomes with known karyotype

Observation of the internal ultrastructure of human chromosomes by transmission electron microscopy (TEM) has frequently been attempted in spite of the difficulties in detaching metaphase chromosome spreads from the glass slide for further processing. In this study we have used a method in which metaphase chromosome spreads were prepared on a flexible thermoplastic membrane (ACLAR) film. To assess chromosome identity, a diamidino-phenylindole staining and karyotying was first done using a conventional cytogenetic system. The chromosome spreads were then fixed with 1% osmium tetroxide, stained with freshly prepared 2% tannic acid, dehydrated, and flat-embedded in epoxy resin. The resin sheet was easily detachable and carried whole chromosome spreads. By this method, TEM observation of chromosomes from normal human lymphocytes allowed a thorough examination of the ultrastructure of centromeres, telomeres, fragile sites, and other chromosomal regions. Various ultrastructural patterns including thick electron dense boundaries, less dense internal regions, and extended chromatin loops at the periphery of the chromosomes were discernible. Application of the present method to chromosome research is expected to provide comprehensive information on the internal ultrastructure of different chromosomal regions in relation to function.     

含五苯基吡咯侧基聚丙烯酸酯的制备及其聚集诱导发光增强性能

设计合成了具有聚集诱导发光增强活性(AEE)的含五苯基吡咯的甲基丙烯酸酯单体M-PPP,并通过自由基聚合制备了系列均聚物及不同五苯基吡咯侧基含量的聚甲基丙烯酸酯共聚物.所制备的均聚物P与共聚物CP在THF/H_2O体系中均具有AEE特性,在水含量大于20%时荧光开始增加,大于80%时荧光快速增加,95%时相对荧光强度达到最大;单体M-PPP则在水含量低于70%时荧光强度略有降低,随后迅速增加,95%后荧光强度下降.五苯基吡咯侧基含量较高的共聚物表现出更好的AEE特性.进一步的研究发现,共聚物CP在THF/H_2O混合溶液中能够对赖氨酸产生荧光点亮型响应.     

MEIOTIC CHROMOSOME BEHAVIOR IN FEMALE INTERSEXES OF ARTIFICIAL TRIPLOID TRANSPARENT-COLORED CRUCIAN CARP

Chromosome behavior in meiosis was studied by ail-drying, C-banding and surface-spreading methods in female intersexes of artificial triploid transparent-colored crucian carp (Corassius auratus). Chromosome pairing and contraction were obviously asynchronous. The preferential pairing of two homologous chromosomes was the major pattern of chromosome pairing, and a few triple pairing, repeated pairing, telomere or centromere associating and multiple pairing were also observed in the pachytene cells. The metaphase I cells were main-ly composed of univalents, bivalents and trivalents, as well as a few of other multivalents, such as tetravalents, pentavalents, hexavalents and heptavalents, were also found in some metaphase I cells. The chromosome elements including uni-, bi-, tri- and other multivalents varied considerably among the metaphase I cells, and the associating patterns of multivalents were also diverse. Some 6 n and 12 n cells, in which premeiotic endomitosis occurred once or twice, were found at     

A TPA-caged precursor of (imino)coumarin for “turn-on” fluorogenic detection of Cu

We strategize to utilize the precursors of (imino)coumarin fluorophores to deliver novel reactive Cu⁺ probes, where tris[(2-pyridyl)-methyl] amine (TPA) works as a reactive receptor towards Cu⁺. To verify this strategy, CP1, a representative probe and relevant sensing behaviors towards Cu⁺ are presented here. CP1 features good solubility and fast response for monitoring labile copper in aqueous solution and live cells. The sensing mechanism of CP1 is determined by HPLC titration and mass spectrometric analysis. The probe CP1 exhibits a 60-fold fluorescence enhancement and a detection limitation of 10.8 nM upon the detection of Cu⁺. CP1 is further applied for imaging labile copper in live cells. This work provides a starting point for future development of Cu⁺ probes, based on in situ formation of (imino)coumarin scaffolds, as well as their further investigations of copper signaling and biological events.     

Synergistic enhancement of the emergency treatment effect of organophosphate poisoning by a supramolecular strategy

Poisoning by organophosphorus agents (OPs) is a serious public health issue across the world. These compounds irreversibly inhibit acetylcholinesterase (AChE), resulting in the accumulation of acetylcholine (ACh) and overstimulation of ACh receptors. A supramolecular detoxification system (SDS) has been designed with a view to deliver pyridine-2-aldoxime methochloride (PAM) with a synergistic inhibition effect on the ACh-induced hyperstimulation through host–guest encapsulation. NMR and fluorescence titration served to confirm the complexation between carboxylatopillar[6]arene (CP6A) and PAM as well as ACh with robust affinities. Patch-clamp studies proved that CP6A could exert an inhibition effect on the ACh-induced hyperstimulation of ACh receptors. Support for the feasibility of this strategy came from fluorescence imaging results. In vivo studies revealed that complexation by CP6A serves to increase the AChE reactivation efficiency of PAM. The formation of the PAM/CP6A complex contributed to enhance in a statistically significant way the ability of PAM not only to relieve symptoms of seizures but also to improve the survival ratio in paraoxon-poisoned model rats. These favorable findings are attributed to synergistic effects that PAM reactivates AChE to hydrolyze ACh and excess ACh is encapsulated in the cavity of CP6A to relieve cholinergic crisis symptoms.

This work provides a feasible supramolecular strategy for improving the emergency treatment effect of OPs poisoning through synergistic effect.     

The Hydroxypyridinone Iron Chelator CP94 Can Enhance PpIX-induced PDT of Cultured Human Glioma Cells

Photodynamic therapy (PDT) with the pro-drugs 5-aminolevulinic acid (ALA) or methyl aminolevulinate (MAL) utilizes the combined interaction of a photosensitizer, light and molecular oxygen to ablate tumor tissue. To potentially increase accumulation of the photosensitizer, protoporphyrin IX (PpIX), within tumor cells an iron chelator can be employed. This study analyzed the effects of ALA/MAL-induced PDT combined with the iron chelator 1, 2-diethyl-3-hydroxypyridin-4-one hydrochloride (CP94) on the accumulation of PpIX in human glioma cells in vitro. Cells were incubated for 0, 3 and 6 h with various concentrations of ALA/MAL with or without CP94 and the resulting accumulations of PpIX, which naturally fluoresces, were quantified prior to and following light irradiation. In addition, counts of viable cells were recorded. The use of CP94 in combination with ALA/MAL produced significant enhancements of PpIX fluorescence in human glioma cells. At the highest concentrations of each prodrug, CP94 enhanced PpIX fluorescence significantly at 3 h for ALA and by more than 50% at 6 h for MAL. Cells subsequently treated with ALA/MAL-induced PDT in combination with CP94 produced the greatest cytotoxicity. It is therefore concluded that with further study CP94 may be a useful adjuvant to photodiagnosis and/or PpIX-induced PDT treatment of glioma.     

Separation of polymerase chain reaction amplified bird genes by capillary electrophoresis

Female birds possess one Z and one W chromosome, whereas male birds possess two identical Z chromosomes. Thus, the presence of a W genetic marker is diagnostic of the female sex. Capillary electrophoresis with buffer containing an entangled solution of hydroxyethylcellulose was used to separate the polymerase chain reaction (PCR) amplified bird sexing genes CHD-Z and CHD-W. The relative standard deviations (RSD) were less than 0.6% for the male genes and less than 0.4% for the female genes for six runs and detection limits of 0.1 ng/microL were obtained with laser-induced fluorescence (LIF) detection. Using a DNA ladder and theoretical models for DNA separation in sieving media, the sizes of the two bird genes were determined.     

Synthesis and Characteristics of Self-Assembled Multifunctional Ln3+-DNA Hybrid Coordination Polymers

Owing to the unique catalytic, optical and magnetic properties, lanthanides (Ln) as multicomponent biomarkers, are widely used in the field of optical sensing, mass spectrometry and magnetic resonance imaging. As ligands, DNA molecules have good biocompatibility, high stability, cost efficiency, programmability and biodegradability. Based on the coordination-driven self-assembly between Ln ions (Ln³⁺) and DNA molecules, a multifunctional Ln³⁺-DNA hybrid coordination polymers (CPs) were synthesized. Not only a series of different Ln³⁺ (single Ln³⁺) and DNA hybrid CPs were synthesized, but one hybrid CP contains two kinds of Ln³⁺ was obtained. Besides, the synthetic CPs in cell fluorescence imaging and miRNA sensing also exhibited high performance. This work provides a novel idea for the synthesis of DNA based nanomaterials, which is promising for biologically-related applications.     

氯氮平与蛋白质相互作用的研究

用荧光光谱法和紫外-可见光谱法研究了在模拟人体生理条件下，氯氮平和牛血清蛋白（BSA）结合反应的特征，发现氯氮平对BSA有较强的荧光猝灭作用，且氯氮平的紫外吸收光谱和BSA的荧光光谱有一定程度的重叠，由此可得出其作用距离和结合过程的基本热力学参数。