[Genetic diagnosis of microcephaly].

Objective: To explore the diagnostic value of chromosome karyotype analysis, chromosomal microarray analysis (CMA) and whole exome sequencing (WES) in microcephaly. Methods: A total of 9 cases of microcephaly fetuses diagnosed by prenatal ultrasound or children with microcephaly diagnosed after birth were selected from the Sixth Affiliated Hospital of Guangzhou Medical University from January 2014 to August 2022.Karyotype analysis and/or CMA were used to detect. The cases with negative karyotype analysis and CMA results were further sequenced by trio-based WES (Trio-WES). Then the coding genes contained in the pathogenic copy number variation (CNV) fragments were analyzed by gene ontology (GO) enrichment. The genes related to the development of the central nervous system contained in the pathogenic CNV and the pathogenic genes found by Trio-WES were combined for gene interaction network analysis. Results: In this study, 9 cases of microcephaly were recruited, with the time of diagnosis ranged from 23 weeks of gestation to 7 years after birth, and the head circumference of fetus or children ranged from 18.3 to 42.5 cm (-7SD to -2SD). Karyotype analysis was detected in all 9 cases and no abnormality result was found. Eight cases were detected by CMA, and one abnormal was found. Five cases were detected by Trio-WES, and two cases were detected with likely pathogenic genes. The GO enrichment analysis of the coding gene in the 4p16.3 microdeletion (pathogenic CNV) region showed that: in biological process, it was mainly concentrated in phototransduction, visible light; in terms of molecular function, it was mainly concentrated in fibroblast growth factor binding; in terms of cell components, it was mainly concentrated in rough endoplasmic reticulum. Gene interaction network analysis suggested that CDC42 gene could interact with CTBP1, HTT and ASPM gene. Conclusions: CMA could be used as a first-line detection technique for microcephaly. When the results of chromosome karyotype analysis and/or CMA are negative, Trio-WES could improve the detection rate of pathogenicity of microcephaly.

[NEK2 gene expression in mouse cryptorchidism model and its mechanism involved in apoptosis].

Objective: To observe the expression of NEK2 mRNA and protein in the cryptorchidism mice model, and to explore its role in apoptosis of testicular tissue. Methods: A mouse cryptorchid model was constructed, and the spermatids in the spermatic tubules were observed by HE staining. Apoptosis was detected by Tunel test, and expression of NEK2 mRNA and protein was detected by RT-PCR and immunohistochemistry, respectively. Results: After the mouse cryptorchidism model was successfully constructed, the HE staining results showed that the damage of spermatogonia cells, primary spermatocytes and sperm cells in the seminiferous tubules became more severe with time. The results of Tunel test showed that the number of apoptotic cells first increased and then decreased, 1, 3, 6, 9 and 15 d apoptotic cells were 3.67±2.08 (t=2, P=0.0412), 7.67±1.53 (t=6.325, P=0.003), 17.67±3.51 (t=7.906, P=0.001), 30.67±3.51 (t=14.072, P<0.001) and 14.33±3.21 (t=6.860, P=0.002). The results of immunohistochemistry showed that NEK2 protein was expressed in the nucleus and cytoplasm in normal testis and cryptorchidism. RT-PCR and immunohistochemistry showed that expression of NEK2 mRNA and protein gradually increased after modeling. After reaching the peak, the expression gradually decreased with time, and was significantly lower than the normal control group. Conclusion: The trend of NEK2 expression in cryptorchidism tissue is consistent with the trend of cell apoptosis in cryptorchidism tissue, suggesting that abnormal expression of NEK2 may affect the damage of sperm cells in the seminiferous tubules through apoptosis, leading to infertility in patients with cryptorchidism.

[The expression of VHL/HIF signaling pathway in the erythroid progenitor cells with chronic mountain sickness].

Objective: To investigate the expression and interaction of VHL/HIF-α pathways including HIF-1α, HIF-2α as well as VHL in erythroid progenitor cells of bone marrow from chronic mountain sickness (CMS) patients. Methods: A total of 25 patients with CMS and 21 healthy controls were recruited for this study. The CD71(+)CD235a(+) cells in bone marrow mononuclear cells, marked as erythroid progenitor cells, were isolated using MACS separation technology. The expression levels of HIF-1α, HIF-2α and VHL in erythroid progenitor cells were detected by Western blotting and real-time fluorescence quantitative PCR. Results: The mRNA levels of HIF-2α were higher in erythroid progenitor cells of CMS than in healthy controls [1.68 (0.81, 2.22) vs 0.98 (0.60, 1.19), P<0.05], while HIF-1α and VHL mRNA levels were similar between the two groups (P>0.05). Spearman analyses indicated that HIF-2α mRNA was positively associated with hemoglobin (Hb) levels in the erythroid progenitor cells of CMS (ρ=0.504, P<0.05). Furthermore, the mRNA level of HIF-2α was correlated with the mRNA level of VHL in the erythroid progenitor cells of CMS (ρ=0.647, P<0.05).The protein levels of HIF-2α in the erythroid progenitor cells of CMS were higher than that of healthy controls [0.94(0.68, 3.30) vs 0.59(0.30, 0.88), P<0.05], but the protein levels of HIF-1α and VHL were similar between the two groups (P>0.05). Conclusions: The abnormal increased expression of HIF-2α in the erythroid progenitor cells of CMS patients leads to the abnormal expression of hypoxia sensitive genes downstream, participating in the occurrence and development of CMS.

[The expressions of VEGF and VEGFR signaling pathway in the bone marrow mononuclear cells with chronic mountain sickness].

Objective: This study was aimed at investigating the levels and relationships of vascular endothelial growth factor (VEGF) and its receptor(VEGFR) in the bone marrow mononuclear cells (MNC) of chronic mountain sickness (CMS). Methods: A total of 34 patients with CMS and 30 controls residing at altitudes of 3 000-4 500 m were recruited for this study. The levels of VEGF, VEGFR1 and VEGFR2 in bone marrow MNC were detected by flow cytometry technique and RT-qPCR. Results: The percentage of VEGFR2 positive cells in the bone marrow MNC of CMS were higher than that of the controls[20.7% (8.1%, 67.6%) vs 8.1% (2.2%, 14.9%), P<0.05], but that of VEGFR1-positive and VEGF-positive were similar in CMS and controls. The mRNA levels of VEGFR2 were higher in the bone marrow MNC of CMS than in the controls[1.7(1.0, 5.1) vs 1.0(0.4, 2.7), P<0.05], while VEGF and VEGFR1 mRNA levels were similar between the two groups. The percentage of VEGFR2 positive cells in CMS were significantly correlated with hemoglobin (r=0.453, P=0.007) and the percentage of VEGF-positive cells (r=0.373, P=0.030). Conclusions: Bone marrow MNC of CMS may show enhanced activity of the VEGF-VEGFR2 pathway, and it appears to be involved in the pathogenesis of CMS.

[Study on apoptosis, cytochrome C and mitochondrial membrane potential in CD71(+) nucleated erythrocytes in patients with chronic mountain sickness].

Objective: To investigate the changes of CD71(+) nucleated erythrocyte apoptosis, cytochrome C (Cyt-C) and mitochondrial membrane potential (MMP) in bone marrow of chronic mountain sickness (CMS). Methods: 14 patients with CMS and 15 patients with simple old fracture were divided into CMS group and control group, respectively.Bone marrow mononuclear cells (BMMNC) were separated, marked with CD71 monoclonal antibody and stained with Annexin V-FITC/PI.Then the apoptotic index of CD71(+) nucleated erythrocytes was determined by flow cytometry.CD71(+) nucleated erythrocytes were sorted out by magnetic column separation, and Cyt-C mRNA was detected by RT-qPCR, MMP was detected by JC-1 staining flow cytometry. Results: The apoptotic index of CD71(+) nucleated erythrocytes was (1.9±1.4)% in the CMS group, and was (3.2±1.5)% in the control group, with significant difference between the two groups (P<0.05). The expression of Cyt-C mRNA was (0.72±0.14) in the CMS group, and was (1.00±0.15) in the control group, with significant difference between the two groups (P<0.01). The MMP was (5.0±2.2) in the CMS group, and was (3.3±0.9) in the control group, with significant difference between the two groups (P<0.05). The apoptotic index of CD71(+) nucleated erythrocytes was negatively correlated with hemoglobin in CMS group (r=-0.569, P=0.034). But there was no significant correlation among apoptosis index, MMP and Cyt-C mRNA. Conclusions: The apoptosis index of CD71(+) nucleated erythrocytes decreased in CMS patients, which was negatively correlated with the level of hemoglobin, indicating that the decline of apoptosis index of CD71(+) nucleated erythrocytes may be related to the accumulation of red blood cells in CMS.The MMP increased and Cyt-C mRNA expression decreased in CD71(+) nucleated erythrocytes of CMS patients, which suggests that the change of mitochondrial pathway of apoptosis might be involved in the down-regulation of CD71(+) nucleated erythrocytes apoptosis in CMS patients.But there was no significant correlation among CD71(+) nucleated erythrocyte apoptosis index, MMP and Cyt-C mRNA levels, which indicates that the mechanism of CD71(+) nucleated erythrocytes apoptosis is complex in CMS.

[Influence of HIF- 2α on the expression of GATA- 1 in bone marrow CD71(+) cell of high altitude polycythemia rat model].

OBJECTIVE: To explore the influence of hypoxia-inducible factor-2 αlpha (HIF-2α) on the expression of erythroid-specific transcription factor GATA-1 in bone marrow CD71(+) cells of rat model with high altitude polycythemia (HAPC). METHODS: A total of 48 male SD rats were selected and randomly divided into normal control group and HAPC group. HAPC model was established at an altitude of 4 300 meters in the natural environment and verified by bone marrow cell classification and counting, hematologic parameters and serum EPO detection. Bone marrow CD71 (+) cells were separated by a combination of methods with density gradient centrifugation and magnetic activated cell sorting. The changes of expression level of HIF-2α, GATA-1 mRNA and proteins were detected by Q-PCR and Western blot. CD71 (+) cells were cultured under hypoxia condition and transfected with selected optimal HIF- 2α shRNAi3 for 96 h. And the expression level of HIF-2α and GATA-1 mRNA and proteins were detected by Q- PCR and Western blot. RESULTS: The results of bone marrow cell counts, the hematologic parameters and the serum EPO content showed that the HAPC rat model was successfully established. The expression of HIF-2α and GATA-1 mRNA and protein in bone marrow CD71(+) cells of HAPC group was higher than that in control group (P<0.05). And HIF-2α and GATA-1 of HAPC group were positively correlated at the expression levels of mRNA and protein, respectively (r=0.923, P<0.01; r=0.838, P<0.01). However, the expression of HIF-2α and GATA-1 mRNA and protein in HAPC group was significantly lower than that in control groups after interfered by HIF-2α shRNAi3 for 96 h (P<0.05). CONCLUSION: The effect of HIF-2α on GATA-1 expression may be correlated with the pathogenesis of HAPC.

The cultivation of bone marrow mesenchymal stem cells derived from patients with high altitude polycythemia.

The aim of this study was to isolate and culture bone marrow mesenchymal stem cells (MSCs) from patients with high altitude polycythemia (HAPC) in order to provide a foundation for further exploration of their biological characteristics. MSCs were isolated and cultured from 10 HAPC patients and 10 healthy controls by using a density gradient centrifugation and an adherent screening method. The morphous of MSCs were observed under an inverted microscope, and its surface antigens were determined using flow cytometry. The growth of the MSCs was also detected to evaluate its proliferation. Bone marrow mononuclear cells were isolated from the bone marrow using a density gradient centrifugation, and they were cultured in vitro. The bone marrow MSCs were successfully isolated and cultured, which presented as fusiform and adherent cells. The MSCs in both groups expressed CD90,CD44,CD29,CD105, CD106, CD146, CD166,Stro—1 and CD13, but they did not express CD45, CD4,CD8,CD19,CD20,CD80,CD14,CD3,CD34 or HLA—DR (P>0.05). The bone marrow MSCs from HAPC patients had a higher proliferation than the bone marrow MSCs from the healthy controls (P<0.01). The bone marrow MSCs from HAPC patients can be effectively cultured in vitro.

The suppressor of transgene RNA silencing encoded by Cucumber mosaic virus interferes with salicylic acid-mediated virus resistance.

The Cucumber mosaic virus (CMV)-encoded 2b protein (Cmv2b) is a nuclear protein that suppresses transgene RNA silencing in Nicotiana benthamiana. Cmv2b is an important virulence determinant but nonessential for systemic spread in N. glutinosa, in contrast to its indispensable role for systemic infections in cucumber. Here, we report that Cmv2b became essential for systemic infections in older N. glutinosa plants or in young seedlings pretreated with salicylic acid (SA). Expression of Cmv2b from the genome of either CMV or Tobacco mosaic virus significantly reduced the inhibitory effect of SA on virus accumulation in inoculated leaves and systemic leaves. A close correlation is demonstrated between Cmv2b expression and a reduced SA-dependent induction of the alternative oxidase gene, a component of the recently proposed SA-regulated antiviral defense. These results collectively reveal a novel activity of Cmv2b in the inhibition of SA-mediated virus resistance. We used a N. tabacum line expressing a bacterial nahG transgene that degrades SA to provide evidence for a Cmv2b-sensitive antiviral defense mechanism in tobacco in which SA acts as a positive modifier but not as an essential component. We propose that SA induces virus resistance by potentiating a RNA-silencing antiviral defense that is targeted by Cmv2b.

Strong host resistance targeted against a viral suppressor of the plant gene silencing defence mechanism.

The 2b protein encoded by cucumber mosaic cucumovirus (Cmv2b) acts as an important virulence determinant by suppressing post-transcriptional gene silencing (PTGS), a natural plant defence mechanism against viruses. We report here that the tomato aspermy cucumovirus 2b protein (Tav2b), when expressed from the unrelated tobacco mosaic tobamovirus (TMV) RNA genome, activates strong host resistance responses to TMV in tobacco which are typical of the gene-for-gene disease resistance mechanism. Domain swapping between Cmv2b, which does not elicit these responses, and Tav2b, revealed functional domains in Tav2b critical for triggering virus resistance and hypersensitive cell death. Furthermore, substitution of two amino acids from Tav2b by those found at the same positions in Cmv2b, Lys21-->Val and Arg28-->Ser, abolished the ability to induce hypersensitive cell death and virus resistance. However, in Nicotiana benthamiana, a species related to tobacco, Tav2b functions as a virulence determinant and suppresses PTGS. Thus, a viral suppressor of the host gene silencing defence mechanism is the target of another independent host resistance mechanism. Our results provide new insights into the complex molecular strategies employed by viruses and their hosts for defence, counter-defence and counter counter-defence.

Viral pathogenicity determinants are suppressors of transgene silencing in Nicotiana benthamiana.

Post-transcriptional gene silencing (PTGS) of a green fluorescent protein (GFP) transgene is suppressed in Nicotiana benthamiana plants infected with potato virus Y (PVY) or with cucumber mosaic virus (CMV), but not in plants infected with potato virus X (PVX). By expressing PVY and CMV-encoded proteins in a PVX vector we have shown that the viral suppressors of gene silencing are the HCPro of PVY and the 2b protein of CMV. The HCPro acts by blocking the maintenance of PTGS in tissues where silencing had already been set, whereas the 2b protein prevents initiation of gene silencing at the growing points of the plants. Combined with previous findings that viruses are both activators and targets of PTGS, these data provide compelling evidence that PTGS represents a natural mechanism for plant protection against viruses.

Ilarviruses encode a Cucumovirus-like 2b gene that is absent in other genera within the Bromoviridae.

We found that RNA 2 of the four ilarviruses sequenced to date encodes an additional conserved open reading frame (ORF), 2b, that overlaps the 3' end of the previously known ORF, 2a. A novel RNA species of 851 nucleotides was found to accumulate to high levels in plants infected with spinach latent virus (SpLV). Further analysis showed that RNA 4A is a subgenomic RNA of RNA 2 and encodes all of ORF 2b. Moreover, a protein species of the size expected for SpLV ORF 2b was translated in vitro from the RNA 4A-containing virion RNAs. The data support the suggestion that the SpLV 2b protein is translated in vivo. The 2b gene of ilarviruses, which is not encoded by alfamoviruses and bromoviruses, shares several features with the previously reported cucumovirus 2b gene; however, their encoded proteins share no detectable sequence similarities. The evolutionary origin of the 2b gene is discussed.

An early meiosis cDNA clone from wheat.

Meiosis occupies only a very short period of the life cycle of higher plants but it is a crucial process ensuring the correct passage and maintenance of genetic information from parent to offspring. A clone (designated pAWJL3) has been isolated from a cDNA library generated from RNA prepared from young wheat florets at early meiosis. The clone was identified through cross-hybridisation to a cDNA clone from maize that, in turn, had been isolated by hybridisation to a Lilium meiosis-specific cDNA clone. The genes encoding the sequence represented in the wheat cDNA clone have been assigned to chromosomes in wheat. The clone, pAWJL3, represents a small family of genes with about 20 members located on the short arms of group 3 and 5 chromosomes. The chromosomal regions harbour genes known to control chromosomal pairing in wheat. DNA prepared from a deletion mutation affecting one of the major genes controlling pairing, Ph2 located on the short arm of 3DS, lacks the 3DS-specific members of the pAWJL3 family bands. The genes are shown to be expressed only after leptotene and predominantly at zygotene and pachytene of meiosis I. The deduced amino acid sequence encoded by the cDNA clone shows two domains, one with three leucine-rich, 24-amino acid repeats and the other with four leucine heptad repeats that resemble those found in basic leucine zipper proteins.

Toward a free-space parallel optoelectronic computer: a 300-MHz optoelectronic counter using holographic interconnects.

We built and tested one of the key components of a free-space holographically interconnected stored-program optoelectronic computer: a counter. The counter is constructed with 1-ns-latency optoelectronic NOR gates and is interconnected with holographic optical elements. Two synchronization methods were also demonstrated: the gate-and-strobe method and the time-of-flight method. These counters represent prototypical optoelectronic finite-state controllers. They were developed to demonstrate the feasibility of providing optoelectronic controllers for optoelectronic processors.