[Analysis of families with fetal congenital abnormalities but negative prenatal diagnosis by whole exome sequencing].

Objective: To evaluate the value of whole exome sequencing (WES) in prenatal clinical application. Methods: A total of 1 152 cases of congenital abnormal [including structural malformation, nuchal translucency (NT) thickening and intrauterine growth restriction] with traditional prenatal diagnosis [including G-band karyotype analysis and chromosome microarray analysis (CMA)] negative were analyzed. The congenital abnormal fetuses were divided into retrospective group and prospective group according to the time of WES detection, that is whether the pregnancy termination or not. According to the specific location of fetal malformation and their family history, the cohort was divided into subgroups. The clinical prognosis of all fetuses were followed up, and the effect of WES test results on pregnancy decision-making and clinical intervention were analyzed. According to the follow-up results, the data of fetuses with new phenotypes in the third trimester or after birth were re-analyzed. Results: Among 1 152 families who received WES, 5 families were excluded because of nonbiological parents. Among the remaining 1 147 families, 152 fetuses obtained positive diagnosis (13.3%,152/1 147), including 74 fetuses in the retrospective group (16.1%,74/460) and 78 fetuses in the prospective group (11.4%,78/687). In fetuses with negative CMA and G-band karyotype analysis results but new phenotypes in the third trimester or after birth, the positive rate by WES data re-analysis was 4.9% (8/163). A total of 34 (21.3%, 34/160) fetuses were directly affected by the corresponding positive molecular diagnosis. Among 68 cases of live births with diagnostic variation grade 4, 29 cases (42.7%, 29/68) received appropriate medical intervention through rapid review of WES results. Conclusions: WES could increase the detection rate of abnormal fetuses with negative G-banding karyotype analysis and CMA by 13.3%. Prenatal WES could guide pregnancy decision-making and early clinical intervention. It might be an effective strategy to pay attention to the special follow-up of the third trimester and postnatal fetus and to re-analyze the WES data.

Speciation and Potential Ecological Risk of Heavy Metals in Soils from Overlapped Areas of Farmland and Coal Resources in Northern Xuzhou, China.

Contamination caused by heavy metals (HMs) in soil of overlapped area of farmland and coal resources (OAFCR) has impact on crops. The concentrations and speciation of As, Cd, Cr, Cu, Pb and Zn were investigated in topsoil of an OAFCR in Xuzhou, China. The results showed that mean concentrations of all six metals were higher than the background values of Xuzhou city and Cd was moderate accumulated with the maximum Igeo equalled to 2.13. Cd showed moderate contamination level (IPi = 1.75) and potential ecological risk (Er = 44.06). Most of the total Cr, Cu, Ni, Pb, and Zn were presented in the residual forms (above 60%), and the percentages of reducible, oxidisable and residual forms of Pb were 23%, 21% and 43% respectively. Pb and Cd reflected a moderate degree of potential ecological risk and a considerable migration risk and ecotoxicity.

AXIN2 gene silencing reduces apoptosis through regulating mitochondria-associated apoptosis signaling pathway and enhances proliferation of ESCs by modulating Wnt/ß-catenin signaling pathway.

OBJECTIVE: Embryonic stem cells (ESCs) mainly originate from totipotent cells in early-stage of mammalian embryo and could proliferate in a manner of un-limitation. This study aimed to investigate roles of Axin2 in proliferation of ESCs and explore the associated mechanisms. MATERIALS AND METHODS: Axis inhibition protein 2 (AXIN2) over-expression (LV5-AXIN2) and AXIN2 RNA interfere (LV3-AXIN2-RNAi) vectors were structured and transfected into H9 cells. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) was used to evaluate cell proliferative activity. Flow cytometry analysis was employed to measure apoptosis of H9 cells. AXIN2, ß-catenin, transcription factor 4 (TCF4), c-myc, c-jun and Cyclin D mRNA levels and protein expressions were determined using quantitative real-time PCR (qRT-PCR) and Western blotting assay. RESULTS: LV5-AXIN2 and LV3-AXIN2-RNAi were successfully structured with higher transfecting efficacy. AXIN2 gene silencing remarkably increased proliferative activity and AXIN2 treatment significantly induced apoptosis of H9 cells, comparing with blank vector group (p<0.05). AXIN2 gene silencing significantly enhanced B-cell lymphoma-2 (Bcl-2) expression and remarkably inhibited cleaved caspase-3 expression comparing to that in blank vector group (p<0.05). AXIN2-RNAi treatment significantly enhanced and AXIN2 over-expression significantly reduced ß-catenin and TCF4 expression, comparing to that in blank vector group (p<0.05). AXIN2 gene silence activated down-stream molecules of Wnt/ß-catenin signaling pathway, including c-jun, c-myc, and Cyclin D1 (p<0.05). CONCLUSIONS: AXIN2 gene silencing reduced apoptosis by regulating mitochondria-associated apoptosis signaling pathway and enhanced proliferation by modulating molecules in Wnt/ß-catenin signaling pathway. Therefore, targeting of aberrant apoptosis and AXIN2 might be a novel clinical strategy to inhibit aging and enhance self-renewal of ESCs.

Discriminating two races of Liobagrus marginatoides by cytogenetic analysis.

Although two kinds of Liobagrus marginatoides should belong to the same species according to the taxonomy of fish morphology, the results of cytogenetic analysis showed they are two different races.

Spin structure transition in La(1.6-x)Nd(0.4)Sr(x)CuO(4) superconductors.

The field and temperature dependencies of the spin structures in the normal states of La(1.6-x)Nd(0.4)Sr(x)CuO(4) (x = 0.10, 0.12, and 0.15) single crystals have been studied by measuring the magnetoresistance and susceptibility. A negative magnetoresistance appears just below the spin-ordering temperature for the magnetic fields parallel to the CuO(2) plane, which can be attributed to the spin-flop transition of the special spin structure in the normal state of the system. The anisotropic variations of susceptibilities with temperature for all the three specimens can be described in the framework of the crystal-field theory. The well fitted broad peaks of the in-plane susceptibilities χ(ab) for the specimens suggest that the susceptibilities are dominated by Nd(3+), and thus the spin reorientation of Cu(2+) in the CuO(2) plane can not be observed from the study of the susceptibility.

The 5S rDNA related repetitive sequences in the sex chromosomes of the spiny eel (Mastacembelus aculeatus).

The karyotype of the spiny eel (Mastacembelus aculeatus) has highly evolved heteromorphic sex chromosomes. X and Y chromosomes differ from each other in the distribution of heterochromatin blocks. To characterize the repetitive sequences in these heterochromatic regions, we microdissected the X chromosome, constructed an X chromosome library, amplified the genomic DNA using PCR and isolated a repetitive sequence DNA family by screening the library. All family members were clusters of two simple repetitive monomers, MaSRS1 and MaSRS2. We detected a conserved 5S rDNA gene sequence within monomer MaSRS2; thus, tandem-arranged MaSRS1s and MaSRS2s may co-compose 5S rDNA multigenes and NTSs in M. aculeatus. FISH analysis revealed that MaSRS1 and MaSRS2were the main components of the heterochromatic regions of the X and Y chromosomes. This finding contributes additional data about differentiation of heteromorphic sex chromosomes in lower vertebrates.

Ag-staining pattern, FISH and ISH with rDNA probes in the rice field eel (Monopterus albus Zuiew) chromosomes.

The present paper reports the Results of rice field eel (Monopterus albus Zuiew) chromosomal analysis by Ag-staining techniques, FISH and ISH with 18S rDNA. Both the Ag-NORs and the hybridization signals were observed on the pericentromeric region of chromosome pairs 3 and 7. The Ag-NORs and the hybridization signals on homologous chromosomes 3 and 7 were corresponding to each other. And rDNA genes were mapped to bivalents 3q12-q24 and 7q14-q26. No heteromorphic sex chromosome pair was identified. The Results obtained were discussed with respect to the feature of Ag-NORs and the position of rDNA genes on chromosome pairs 3 and 7.

Molecular cytogenetic detection of paternal chromosome fragments in allogynogenetic gibel carp, Carassius auratus gibelio Bloch.

In gynogenesis, sperm from related species activates egg and embryonic development, but normally does not contribute genetically to the offspring. In gibel carp, Carassius auratus gibelio Bloch, however, gynogenetic offspring often show some phenotypes apparently derived from the heterologous sperm donor. This paternal effect of allogynogenesis is outstanding in an artificial clone F produced by cold treatment of clone E eggs after insemination with blunt-nose black bream (Megaloabrama amblycephala Yin) sperm. Karyotype analysis revealed 5-15 supernumerary microchromosomes in different individuals of clone F in addition to 156 normal chromosomes inherited from the maternal clone E. A painting probe was prepared from the microdissected microchromosomes, and used to investigate the origin of these microchromosomes. Strong positive signals were detected on each microchromosomes of clone F and on 4 pairs of chromosomes in blunt-nose black bream, whereas no signals were detected on the chromosomes of clone E. This result indicates that some paternal chromosome fragments of blunt-nose black bream have been incorporated into the artificial clone F. Therefore, the manipulation of allogynogenesis may provide a unique method to transfer DNA between diverse species for fish breeding.

Sex chromosomes in the spiny eel (Mastacembelus aculeatus) revealed by mitotic and meiotic analysis.

Lower vertebrates like fish exhibit tremendous diversity in sex determination. There are wide interplays between environment-dependent sex differentiation ranging from natural hermaphroditism to sex reversal and genetic sex determination. Diverse systems of male and female heterogamety coexist in fish and sex chromosomes are rarely distinguishable in morphology. Here we show that the spiny eel ((Mastacembelus aculeatus) of the Perciformes, has evolved highly heteromorphic X and Y chromosomes. The metacentric X and Y chromosomes are the largest among 24 homologous pairs, differ from each other in size and morphology, and become distinct after C-banding because of conspicuous heterochromatin blocks which exhibit alternate distribution around the centromeric region. Chromosome painting using probes from the microdissected X chromosome revealed sequence homology between X and Y. During the pachytene stage of meiosis the X and Y form a bivalent. However, their synapsis is delayed which is particularly evident in one terminus. Therefore, the X and Y have resulted from a pericentric inversion in the Y. We conclude that M. aculeatus represents an example of a highly advanced stage of sex chromosome evolution in fish.

[Painting the chromosomes of fishes with human sex chromosome-specific DNA probes].

Chromosome painting is one of the effective methods in studying the evolution of genomes. The chromosomes of the species of fishes--M. aculeatus, M. albus and D. rerio were painted respectively with human X and Y chromosome-specific DNA probes. The results showed that some homologous segments of human X chromosome were found in the genomes of all the three kind of fishes, all the segments were scattered on several pairs of homologous chromosomes in each kind of fish. But no homologous segment of the human Y chromosome was found.

[The chromosome mapping of PdSox8 and PdSox9 in two kinds of loaches].

With the Dig-labelled PdSox8 and PdSox9 as probes, the chromosome mapping of Sox8 and Sox9 in Paramisgurnus dabryanus and Misgurnus anguillicaudatus were analyzed by chromosome in situ hybridization technique. In P. dabryanus, the PdSox8 and PdSox9 were successfully localized in the No. 4 and No. 2 telocentric chromosome respectively, the relative distance to centromere is 40.2% and 67.5%; In M. anguillicaudatus, they were localized in the No. 9 and No. 6 telocentric chromosome respectively, the relative distance to centromere is 58.3% and 30.8%.

[The conservative region sequence analysis of PdSox9 in Paramisgurnus dabryanus].

Degenerate primers within HMG-box analyzed the Sox genes of P. dabryanus. Three amplification bands with the length 220, 550 and 1500 bp respectively were observed. A novel gene was obtained from the 550 bp band. The identity of possible amino acid sequence to Sox9 of human, mouse and chicken is 94%. The similarity to HSOX10, MSox10, HSRY, MSry is 87%, 55%, 40% and 47% respectively. So, it was named PdSox9. Northern blotting result showed that it overexposes in adult testis and brain.

[Px gene of rice-field eels primed in situ labeling].

Peroxidase (Px) gene of rice-field eels (Monopterus albus) was successfully localized to the segments of two bivalents, 8q15-q26 and 11q32-q37 with digoxigenin labeled primed in situ labeling (PRINS) technique, which is utilized for localization of single copy gene of fish for the first time. Our results demonstrated the possibility of mapping genes in fish by the DNA probes of other species and PRINS technique.

[Effects of NA and 5-HT on the spontaneous and evoked activities of Purkinje cells in cerebellar slice].

Effects of NA and 5-HT perfusion on the spontaneous activity and evoked responses of the Purkinje cells (PCs) to stimulation of white matter were studied in the rat cerebellar slices. The results were as follows: (1) Perfusion with NA could elicit inhibitory, excitatory or biphasic responses of the spontaneously discharging PCs, with inhibitory response in dominance (79.8%). 5-HT perfusion could also exert either excitatory or inhibitory effect on PC spontaneous activity. (2) When NA and 5-HT were perfused successively, NA tended to inhibit while 5-HT tended to excite in 53.8% of the tested cells. (3) Most of the complex spike (CS) and simple spike (SS) responses evoked by white matter stimulation could be potentiated by NA (68.2%, 57.1%) or depressed by 5-HT (60.0%, 68.2%). (4) On the same cell under successively perfusion with NA and 5-HT, the evoked CS and SS responses of most cells (60.0%, 52.9%) showed a tendency to be facilitated by NA and suppressed by 5-HT. These results suggest that aminergic afferent fibers, by releasing NA and 5-HT, may affect PC's excitability and responsiveness to synaptic inputs from mossy and climbing fibers, thus playing an important role in the cerebellar function.

[Effects of dorsal raphe stimulation on activities of cerebellar nuclear neurons in the rat].

The effects of stimulating dorsal raphe nucleus (DR) on activities of three deep cerebellar nuclei (DCN) (medial, interposed and lateral nuclei) were investigated. The results were as follows: (1) Stimulation of DR elicited three types of responses, inhibition, excitation, and biphasic response (excitation-inhibition or inhibition-excitation) from DCN cells with responsive latencies ranging from 10 to 84 ms. The majority of responsive cells showed an inhibitory response (76%-90%) with a latency less than 30 ms. (2) The spontaneous discharge frequencies of the DCN cells were 5 to 120 Hz. The cells with higher spontaneous discharge frequencies showed low responsive rates to the DR stimulation, as compared with lower firing frequency cells. (3) The depressive effect of DR stimulation could be blocked by injection of 5-HT2/1c antagonist methysergide (66.7%-83.3%). These results suggest that raphe-cerbellum serotonergic afferent fibers may be involved in the cerebellar sensorimotor integration process by exerting some modulatory effects on the DCN cell's activities.

Cerebellar interpositus nucleus modulates neuronal activity of lateral hypothalamic area.

Effects of stimulating the cerebellar interpositus nucleus (IN) on the neuronal activity of lateral hypothalamic area (LHA) were first observed in the cat. The results showed that (1) IN stimulation could elicit inhibitory, excitatory, inhibitory-excitatory and excitatory-inhibitory responses from LHA neurones, with a majority of inhibitory responses (46.9%); (2) the responsive latencies of LHA neurones to IN stimulation ranged from 5 to 45 ms, while most (83.6%) showed a short latency of < 15 ms; (3) of 67 LHA neurones which responded to the IN stimulation, 42 (62.7%) cells were identified to be glucose-sensitive neurones. These results suggest that IN may be involved in the role of LHA modulating food intake behaviour, as well as other non-somatic functions.

The microvasculature of human infant oral mucosa using vascular corrosion casts and india ink injection. II. Palate and lip.

The microvasculature of human hard and soft palate and lip originating from four infant males and six females, aged 6 months to 2 years was studied by scanning electron microscopy of vascular corrosion casts and light microscopy of India ink injected specimens. The capillary loops of the hard palate mucosa and vermilion border of the lips were found to be tall, numerous and consisted of primary, secondary and tertiary loops. Those of the soft palatal and labial mucosa were short, few in number and demonstrated a simple hair-pin shape originating directly from the subpapillary vascular network. It was concluded that the configuration of capillary loops is not only determined by the shape of the connective tissue papillae in the lamina propria but also influenced by the functional demands characteristic of the different areas of the oral mucosa.

[Effects of stimulating lateral hypothalamic area and ventromedial nucleus of hypothalamus on cerebellar cortical neuronal activity in the cat].

In pentobarbital anaesthetized cat, stimulating lateral hypothalamic area (LHA) and ventromedial nucleus (VMN) of the hypothalamus could elicit inhibitory, excitatory or inhibitory-excitatory responses of cerebellar Purkinje cells (PC) and non-Purkinje cells (NPC) in the loubles VI and VII. The majority of responses are inhibitory (80.4%) usually with a latency of 10-20 ms, or occassionaly as long as 90 ms. Usually stimulation of LHA was more effective and NPC was more responsive. The aforementioned inhibitory response could be blocked by iv. chlorpheniramine. The possible role of the hypothalamo-cerebellar activity was discussed.