Spastin interacts with CRMP5 to promote spindle organization in mouse oocytes by severing microtubules.

Microtubule-severing protein (MTSP) is critical for the survival of both mitotic and postmitotic cells. However, the study of MTSP during meiosis of mammalian oocytes has not been reported. We found that spastin, a member of the MTSP family, was highly expressed in oocytes and aggregated in spindle microtubules. After knocking down spastin by specific siRNA, the spindle microtubule density of meiotic oocytes decreased significantly. When the oocytes were cultured in vitro, the oocytes lacking spastin showed an obvious maturation disorder. Considering the microtubule-severing activity of spastin, we speculate that spastin on spindles may increase the number of microtubule broken ends by severing the microtubules, therefore playing a nucleating role, promoting spindle assembly and ensuring normal meiosis. In addition, we found the colocalization and interaction of collapsin response mediator protein 5 (CRMP5) and spastin in oocytes. CRMP5 can provide structural support and promote microtubule aggregation, creating transportation routes, and can interact with spastin in the microtubule activity of nerve cells (30). Knocking down CRMP5 may lead to spindle abnormalities and developmental disorders in oocytes. Overexpression of spastin may reverse the abnormal phenotype caused by the deletion of CRMP5. In summary, our data support a model in which the interaction between spastin and CRMP5 promotes the assembly of spindle microtubules in oocytes by controlling microtubule dynamics, therefore ensuring normal meiosis.

Placenta-specific 1 regulates oocyte meiosis and fertilization through furin.

Placenta-specific 1 (Plac1) has been found to be essential for placentation, and abnormal Plac1 expression and distribution is highly correlated with preeclampsia and implantation failure; however, its function in mammalian oocytes has not been elucidated. Here, we report that Plac1 was more prominent in mouse oocytes and enriched at the membrane region throughout meiosis. On the one hand, Plac1 knockdown severely disrupted microvillus organization; however, on the other hand, Plac1 significantly decreased oocyte maturation and increased aneuploidy, consequently disrupting normal fertilization. On the basis of immunoprecipitate matrix-assisted laser desorption/ionization, we established a working model, then verified and suggested that, at the germinal vesicle stage, Plac1 enriches the membrane to activate furin, and active furin subsequently activates IGF-1 receptor to maintain regular microvillus organization. Upon meiosis onset, active furin/IGF-1 receptor relocates into the cytoplasm to activate (phosphorylate) Akt to promote meiosis. In summary, our finding suggests that Plac1, a protein that is crucial for placentation, is also essential for oocyte meiosis and fertilization.-Shi, L.-Y., Ma, Y., Zhu, G.-Y., Liu, J.-W., Zhou, C.-X., Chen, L.-J., Wang, Y., Li, R.-C., Yang, Z.-X., Zhang, D. Placenta-specific 1 regulates oocyte meiosis and fertilization through furin.

Molecular Characterization of Organics Removed by a Covalently Bound Inorganic-Organic Hybrid Coagulant for Advanced Treatment of Municipal Sewage.

Coagulation is an important process to remove organics from water. The molecular composition and structure of organic matter influence water quality in many ways, and the lack of information regarding the organics removed by different coagulants makes it challenging to optimize coagulation processes and ensure reclaimed water safety. In this paper, we investigated coagulation of secondary biological effluent from a municipal sewage treatment plant with different coagulants. We emphasized investigation of organics removal characteristics at the molecular level using Fourier transform-ion cyclotron resonance mass spectrometry (FT-ICR MS) coupled with electrospray ionization (ESI). We found that conventional coagulants can only partially remove condensed polycyclic aromatics and polyphenols with low H/C (H/C < 0.7) and highly unsaturated and phenolic compounds and aliphatic compounds with high O/C (O/C > 0.6). A new coagulant, CBHyC, had better removal efficiencies for all organics with different element compositions and molecular structures, especially organics that are resistant to conventional coagulants such as highly unsaturated and phenolic compounds and aliphatic compounds located in 0.3 < O/C < 0.8 and 1.0 < H/C < 2.0 regions and sulfur-containing compounds with higher O/C (e.g., anionic surfactants and their metabolites or coproducts). This study provides molecular insights into the organics removed by different coagulants and provides data supporting the possible optimization of advanced wastewater treatment processes.

Isolation and Characterization of Rat Mesenchymal Stem Cells Derived from Granulocyte Colony-Stimulating Factor-Mobilized Peripheral Blood.

Mesenchymal stem cells (MSCs) have been isolated from many tissues and organs. However, there is much dispute as to whether MSCs exist in peripheral blood. This may be due to the limited identification methods of MSCs, especially the lack of detection markers for phenotypic characteristics. In this study, as many as 10 surface markers of MSCs derived from rat peripheral blood (rPBMSCs) were analyzed after granulocyte colony-stimulating factor mobilization. Our results suggest that mobilized rPBMSCs overexpress mesenchymal markers, including CD90, CD44, CD29, CD73 and CD105, but do not express CD45, CD11b, CD79a, CD34 or HLA-DR. This is in conformity with the standard definition of MSCs by the International Society for Cellular Therapy. In addition, the colony-forming efficiency of the mobilized rat peripheral blood was 15.83 ± 1.61/106, significantly outnumbering that of the nonmobilized group, which was 0.28 ± 0.1/106 (p < 0.01). Combining the growth characteristics with the differential capacities of mobilized rPBMSCs towards forming osteocytes, chondrocytes and adipocytes, we further confirmed the existence of rPBMSCs. Additionally, this treatment could improve locomotive function after spinal cord injury (SCI) in rats. Due to their convenient collection, fewer complications, cost effectiveness and suitability for autograft, PBMSCs might be a substitute for MSCs derived from bone marrow and provide promising prospects for the cell-based therapy of SCI.

Overlapping infections of Mycobacterium canariasense and Nocardia farcinica in an immunocompetent patient: A case report.

BACKGROUND: Infections by non-tuberculous mycobacteria (NTM) have become more common in recent years. Mycobacterium canariasense (M. canariasense) was first reported as an opportunistic pathogen in 2004, but there have been very few case reports since then. Nocardia is a genus of aerobic and Gram-positive bacilli, and these species are also opportunistic pathogens and in the Mycobacteriales order. Conventional methods for diagnosis of NTM are inefficient. Metagenomic next-generation sequencing (mNGS) can rapidly detect many pathogenic microorganisms, even rare species. Most NTM and Nocardia infections occur in immunocompromised patients with atypical clinical symptoms. There are no previous reports of infection by M. canariasense and Nocardia farcinica (N. farcinica), especially in immunocompetent patients. This case report describes an immunocompetent 52-year-old woman who had overlapping infections of M. canariasense, N. farcinica, and Candida parapsilosis (C. parapsilosis) based on mNGS. CASE SUMMARY: A 52-year-old woman presented with a productive cough and chest pain for 2 wk, and recurrent episodes of moderate-grade fever for 1 wk. She received antibiotics for 1 wk at a local hospital, and experienced defervescence, but the productive cough and chest pain persisted. We collected samples of a lung lesion and alveolar lavage fluid for mNGS. The lung tissue was positive for M. canariasense, N. farcinica, and C. parapsilosis, and the alveolar lavage fluid was positive for M. canariasense. The diagnosis was pneumonia, and application of appropriate antibiotic therapy cured the patient. CONCLUSION: Etiological diagnosis is critical for patients with infectious diseases. mNGS can identify rare and novel pathogens, and does not require a priori knowledge.

[Study on effect and mechanism of volatile oil of schizonepetae herba and its essential components against influenza virus].

OBJECTIVE: To observe the effect of volatile oil of Schizonepetae Herba (VOSH), and its essential components-menthone and pulegone against anti-influenza virus A/PR/8/34 (H1N1) in vivo and in vitro, as well as the signaling mechanism of its toll-like receptor/interferon (TLR/IFN). METHOD: The lung-adapted PR-8 virus model was prepared in mice. They were administered with preventive and therapeutic drugs, and the hemagglutination titer of model animals was determined to evaluate in vivo effect against H1N1. ELISA test was conducted to observe the effect on IFN-alpha, IFN-beta, IL-2, IL-6 and TNF-alpha in serum, as well as IFN-beta secretion in H1N1 infected MDCK supernatant. Real-time RT-PCR was employed to observe the expression levels of IRAK4 and TLR3 mRNA. RESULT: The in vivo experiment shows that the hemagglutination titer was significantly decreased when the mice were treated with VOSH (0.266 mg x kg(-1)), menthone(0.5 mg x kg(-1)) and pulegone (0.19 mg x kg(-1)) in therapeutic way; VOSH (0.226 mg x kg(-1)) had a significant effect on increasing serum levels of IFN-alpha, IL-2; Methone (0.5 mg x kg(-1)) had a significant effect on increasing serum levels of IFN-beta; Methone (0.5 mg x kg(-1)) and pulegone (0.19 mg x kg(-1)) had a significant effect on decreasing serum levels of IL-6; VOSH (0.452, 0.226 mg x kg(-1)) and pulegone (0.19 mg x kg(-1)) had a significant effect on decreasing serum levels TNF-alpha. The in vitro experiment showed that the expression levels of IRAK4 mRNA and IFN-beta were significantly increased in VOHS (0.1 g x L(-1)) and pulegone (0.1 g x L(-1)) groups; and the menthone (0.25 g x L(-1)) group showed a significant rise in the expression levels of IRAK4 mRNA, but a notable decline in TLR3 mRNA. CONCLUSION: The administration with VOSH, methone and pulegone in therapeutic way can significantly decrease the hemagglutination titer, which demonstrates the anti-virus effect of the administration in therapeutic way, but no notable efficacy of the administration in preventive way. The in vivo anti-virus mechanism is related to regulation of IFN-alpha, IFN-beta and IL-2.

Risk assessment, fitness cost, cross-resistance, and mechanism of tetraniliprole resistance in the rice stem borer, Chilo suppressalis.

The rice stem borer (RSB), Chilo suppressalis, a notorious rice pest in China, has evolved a high resistance level to commonly used insecticides. Tetraniliprole, a new anthranilic diamide insecticide, effectively controls multiple pests, including RSB. However, the potential resistance risk of RSB to tetraniliprole is still unknown. In this study, the tetraniliprole-selection (Tet-R) strain was obtained through 10 continuous generations of selection with tetraniliprole 30% lethal concentration (LC30 ). The realized heritability (h2 ) of the Tet-R strain was 0.387, indicating that resistance of RSB to tetraniliprole developed rapidly under the continuous selection of tetraniliprole. The Tet-R strain had a high fitness cost (relative fitness = 0.53). We established the susceptibility baseline of RSB to tetraniliprole (lethal concentration at LC50  = 0.727 mg/L) and investigated the resistance level of 6 field populations to tetraniliprole. All tested strains that had resistance to chlorantraniliprole exhibited moderate- to high-level resistance to tetraniliprole (resistance ratio = 27.7-806.8). Detection of ryanodine receptor (RyR) mutations showed that the Y4667C, Y4667D, I4758M, and Y4891F mutations were present in tested RSB field populations. RyR mutations were responsible for the cross-resistance between tetraniliprole and chlorantraniliprole. Further, the clustered regularly interspaced palindromic repeats (CRISPR) / CRISPR-associated protein 9-mediated genome-modified flies were used to study the contribution of RyR mutations to tetraniliprole resistance. The order of contribution of a single RyR mutation to tetraniliprole resistance was Y4667D > G4915E > Y4667C ≈ I4758M > Y4891F. In addition, the I4758M and Y4667C double mutations conferred higher tetraniliprole resistance than single Y4667C mutations. These results can guide resistance management practices for diamides in RSB and other arthropods.

Pen-grip kinetics in children with and without handwriting difficulties.

INTRODUCTION: Handwriting difficulty (HD) is a widely discussed issue. Previous researchers have revealed many valuable kinematics related to the handwriting performance. However, a clear understanding of the kinetics of handwriting performance in children with HD is still lacking. Therefore, this study investigated the writing performance of children with HD via a force acquisition pen (FAP), which detects the force applied from the digits and pen tip. METHODS: Data from 64 school-age children were divided into control (36 children without HD; mean age: 7.97 years) and HD (28 children with HD; mean age: 8.67 years) groups. The participants were asked to perform a tracing task using the FAP at their usual writing pace. RESULTS: Compared with the control group, the HD group had significantly less pen-tip force, an average amount of force (in-air) from all three digits, higher force variations (whole task) in the index finger, less force fluctuations with the index and middle fingers and a smaller force ratio. CONCLUSION: The findings of this study suggest that an understanding of the handwriting kinetics and the role of digits in handwriting may be crucial for further planning strategies for handwriting training for children with HD.

Serum levels of MMP-11 correlate with clinical outcome in Chinese patients with advanced gastric adenocarcinoma.

BACKGROUND: The aim of the present study was to investigate the association between serum levels of matrix metalloproteinase 11 (MMP-11) and responses to front-line chemotherapy and prognosis in advanced unresectable gastric adenocarcinoma. METHODS: Clinical data concerning 86 patients with advanced gastric adenocarcinoma (stages III c to IV), treated in Beijing Chao-Yang Hospital from 2005 to 2009, were reviewed retrospectively. Adenocarcinoma was confirmed by pathology and patients received 5-fluorouracil-based front-line combination chemotherapy with third generation chemotherapeutic agents including paclitaxel, docetaxel and oxaliplatin. The regimen was repeated every two to three weeks, and the first evaluation was carried out after three cycles. The median cycle of chemotherapy was 6 (ranging from three to twelve cycles). Serum MMP-11 protein from the 86 patients was examined using enzyme-linked-immunosorbent-assay (ELISA) prior to chemotherapy and after three cycles of chemotherapy. Serum samples from healthy individuals were used as controls. RESULTS: The response rate (RR, complete response plus partial response) to chemotherapy in the 86 patients was 44.2% (38/86). The median TTP (time to progression) and overall survival (OS) in patients who responded to chemotherapy were 6.0 and 10.0 months, respectively. The response rate to chemotherapy in patients with high levels of serum MMP-11 (42.9%; 9/21) was similar to that in patients with low levels (44.6%; 29/65) (P = 0.935). Patients with low serum levels of MMP-11 had a higher median survival time and 1-year survival rate than those with high levels (11 months vs. 8 months, 50.2% vs. 21.7%, P = 0.017), although the TTP was comparable in all patients, irrespective of serum MMP-11 level (P = 0.178). Serum MMP-11 levels were correlated with lymph node metastasis (P = 0.006). Cox multivariate regression analysis demonstrated that the serum level of MMP-11 was an independent prognostic factor for patients presenting with advanced gastric carcinoma. CONCLUSIONS: Serum levels of MMP-11 in Chinese patients with advanced gastric carcinoma were not associated with the response to front-line chemotherapy, but could play an important role in lymph node metastasis and prognosis.

[Detecting and identifying 3 novel single nucleotide polymorphisms in DNA sequence of epidermal growth factor precursor from Buyi and Han individuals].

OBJECTIVE: To examine the single nucleotide polymorphism (SNP) distributed in exon 20, 21 and intron 20 of epidermal growth factor precursor gene (preproEGF) of Buyi and Han individuals. METHODS: Eleven primer sets were designed and synthesized for PCR, that genomic DNA of Buyi or Han individual was used as the template, to amplify and sequence respectively the large fragment DNA from preproEGF gene. BLAST programs were applied to compare and identify the SNPs from the sequenced PCR products or amplified DNA fragments. RESULTS: 4.5 kb DNA fragments long over 20th, 21st exon and 20th intron structures of preproEGF gene were got by PCR respectively from genomic DNAs of Buyi and Han individuals. Results of DNA sequencing showed two SNPs in 4. 5 kb fragment of Han individual, of which one was sited at C86380T of preproEGF gene and another positioned at 84580 bp (T/-), while one SNP was observed in Buyi individual, which was located at T84329C of preproEGF gene. GenBank dbSNP database showed that C86380T SNP in 20th intron of preproEGF gene has not been reported yet from Han group and other cohorts except it has been reported from European and Sub-Saharan groups; and also that T84329C SNP has not been reported yet from Buyi group although it has been reported from Han group. CONCLUSIONS: Our study demonstrates that the Han individual is with C86380T SNP located in 20th intron of human preproEGF gene; and that the Buyi individual has the T84329C SNP sited in 20th intron of human preproEGF gene. However, another Han SNP (T/-) positioned at 84580 bp need to be further confirmed.

[Tumor cells lysates pulsed dendritic cells for immunotherapy of ovarian cancer in rats: in-vitro study].

OBJECTIVE: To study the feasibility of ovarian tumor cells lysates pulsed dendritic cells (DCs) in the immunotherapy of tumor prevention via cytotoxic test in-vitro. METHODS: Forty rats were randomly divided into 2 groups: experimental and control groups. Ten rats from each group were killed and DCs were isolated and cultured. Frozen-thawed antigen of in the study group, rat's Ovarian cancer cells of the line NuTu-19 were cultured, freeze-melt antigens were added at day 3. Two groups co-cultured with T lymphocytes from the other rat's spleen on the 14th day. Then observed the shape of DCs, contrasted the proliferation and killing rate. RESULTS: In the study group, DCs could significantly stimulate T lymphocytes multiplication and produce tumor specific cytotoxic T lymphocytes (CTL). When the ratios of stimulating cells (SC) to reaction cells (RC) were 1:3, 1:10, 1:30 and 1:100, the stimulating index (SI) in the study group were much higher than those in control group (P<0.01). When the ratios of SC is the same with RC (SC:RC=3:1, 10:1, 30:1): the killing and wounding rate to tumor cells of study group [(40.9+/-1.2)%, (69.8+/-1.8)%, (89.0+/-0.4%)] is significantly higher than those of control group [(30.9+/-1.9)%, (34.4+/-2.1)%, (51.0+/-1.5)%] (P < 0.01). CONCLUSION: DC could activate T lymphocytes proliferation and produce CTL to kill and wound tumor cells in-vitro.

[Separation and preconcentration by extraction and anti-extraction-determination of trace Te in complex geological samples by hydride generation atomic fluorescence spectrometry].

A method for the determination of trace Te in the complex geological samples by hydride generation atomic fluorescence spectrometry using MIBK as extraction reagent was developed. The extraction ability of Te (IV) in the HCl-NaBr-MIBK system and the anti-extraction behavior of Te (IV) in the HCl-KMnO4-MIBK system were studied. Under the optimum extraction condition of 3.6 mol x L(-1) HCl-100 g x L(-1) NaBr, Te (IV) was extracted completely by MIBK, Te (IV) in the MIBK phase was oxidized to Te (VI) with HCl-KMnO4, Te (VI) in the MIBK phase was anti-extracted using water, then the interference elements such as Au, Ag, Pt, Pd, Cu, Pb, Co, Ni, Cd, As, Sb, Bi, Hg, Tl and Se for the determination of Te by hydride generation atomic fluorescence spectrometry were eliminated successfully. The detection limit of Te was 1.14 x 10(-4) microg(-1), and the relative standard deviations of Te was 6.84%. The method was applied to complicated geological samples.

Microtubule­severing protein Katanin p60 ATPase­containing subunit A­like 1 is involved in pole­based spindle organization during mouse oocyte meiosis.

Microtubule­severing proteins (MTSPs) are a group of microtubule­associated proteins essential for multiple microtubule­related processes, including mitosis and meiosis. Katanin p60 ATPase­containing subunit A­like 1 (p60 katanin­like 1) is an MTSP that maintains the density of spindle microtubules at the poles in mitotic cells; however, to date, there have been no studies about its role in female meiosis. Using in vitro­matured (IVM) oocytes as a model, it was first revealed that p60 katanin­like 1 was predominant in the ovaries and oocytes, indicating its essential roles in oocyte meiosis. It was also revealed that p60 katanin­like 1 was concentrated at the spindle poles and co­localized and interacted with γ­tubulin, indicating that it may be involved in pole organization. Next, specific siRNA was used to deplete p60 katanin­like 1; the spindle organization was severely disrupted and characterized by an abnormal width:length ratio, multipolarity and extra aster microtubules out of the main spindles. Finally, it was determined that p60 katanin­like 1 knockdown retarded oocyte meiosis, reduced fertilization, and caused abnormal mitochondrial distribution. Collectively, these results indicated that p60 katanin­like 1 is essential for oocyte meiosis by ensuring the integrity of the spindle poles.

Fast-scanning photoacoustic microscopy with a side-looking fiber optic ultrasound sensor.

Optical-resolution photoacoustic microscopy (OR-PAM) images biological tissue with sub-cellular resolution and optical absorption contrast. OR-PAM is limited by the tradeoff among imaging speed, field of view, and sensitivity. In this work, we present an OR-PAM technique based on an unfocused side-looking fiber optic ultrasound (FOUS) sensor, which achieves high imaging speed, large field of view, and good sensitivity for in vivo imaging. The FOUS sensor is developed based on a dual-polarized fiber laser and read out with real-time frequency demodulation. Via minimizing the readout noise, the sensor offers a noise-equivalent pressure of 43.6 Pa, enabling high detection sensitivity over a large field of view. High imaging speed is achieved via scanning the laser beam with a 2D galvo mirror in the ultrasound detection area. Microvascular imaging with a frame rate of 2 Hz over a 2 × 2 mm2 area has been demonstrated in the mouse ear. The new OR-PAM technique may be used in the visualization of biological and physiologic dynamics.

Molecular insights into the mechanism and the efficiency-structure relationship of phosphorus removal by coagulation.

Types and structures of phosphorus compounds influence the removal of phosphorus by coagulation. Until now, the molecular-level interaction between coagulants and phosphorus (especially organophosphates) and the relationship between removal efficiency and phosphorus structure have not been clear. This work investigated the removal of phosphorus with different structures using conventional coagulants (poly aluminum chloride (PACl) and polymerized ferric sulfate (PFS)) and a novel covalently-bound inorganic-organic hybrid coagulant (CBHyC). CBHyC removed more than 98% of phosphate and most of organophosphates, had more stable performance than PACl and PFS, and was less affected by pH, initial phosphorus concentration, and co-occurring materials. Molecular dynamics simulation demonstrated that CBHyC removed phosphorus mainly through electrostatic attraction and hydrophobic interaction. Furthermore, this work established QSAR (quantitative structure activity relationship) models for removal efficiency and organophosphate structure for the first time. The model showed that atomic charges of phosphorus atoms (QP) and hydrogen atoms (QH+) in the system and the energy gap (ΔEMO) affected electronegativity and hydrophobicity, thus influencing organophosphate removal efficiency. The model had high fitting precision and good predictive ability and has the potential to greatly reduce the cost of optimizing processes and conditions for phosphorus removal.

[Major Ionic Features and Possible Controls in the Groundwater in the Hamatong River Basin].

In order to study the major ion chemistry and controls of groundwater, 59 groundwater samples were collected and their major ions measured in the Hamatong River Basin. The hydrogeochemical characteristics of groundwater in this basin were analyzed by means of mathematical statistics, Piper triangular diagrams, Gibbs figures, and ionic relations, and the water chemical evolution and ion sources of the Hamatong River Basin were determined. The results showed that Ca2+ was the main cation in the groundwater, accounting for 22.1% to 72.4% of the total cations, with an average value of 48.7%. HCO3- was the main anion, accounting for 35.3% to 97.5% of the total anions, and with an average value of 80%. Total dissolved solids concentration ranged from 93.3 mg·L-1 to 521.1 mg·L-1 with a median value of 219.1 mg·L-1. The hydrochemical types of groundwater are HCO3-Ca, HCO3-Ca·Mg, and HCO3-Ca·Na. Chemical weathering rates of carbonates and silicates were estimated, and the chemical composition of groundwater samples located in the middle of Gibbs model indicated that the major chemical process of groundwater was controlled by rock weathering. Silicate weathering is believed to significantly contribute to dissolved solute compositions, and carbonate weathering played an important role as the source of dissolved ions.

GTPase-activating protein Elmod2 is essential for meiotic progression in mouse oocytes.

Meiotic failure in oocytes is the major determinant of human zygote-originated reproductive diseases, the successful accomplishment of meiosis largely relay on the normal functions of many female fertility factors. Elmod2 is a member of the Elmod family with the strongest GAP (GTPase-activating protein) activity; although it was identified as a possible maternal protein, its actual physiologic role in mammalian oocytes has not been elucidated. Herein we reported that among Elmod family proteins, Elmod2 is the most abundant in mouse oocytes, and that inhibition of Elmod2 by specific siRNA caused severe meiotic delay and abnormal chromosomal segregation during anaphase. Elmod2 knockdown also significantly decreased the rate of oocyte maturation (to MII, with first polar body extrusion), and significantly greater numbers of Elmod2-knockdown MII oocytes were aneuploid. Correspondingly, Elmod2 knockdown dramatically decreased fertilization rate. To investigate the mechanism(s) involved, we found that Elmod2 knockdown caused significantly more abnormal mitochondrial aggregation and diminished cellular ATP levels; and we also found that Elmod2 co-localized and interacted with Arl2, a GTPase that is known to maintain mitochondrial dynamics and ATP levels in oocytes. In summary, we found that Elmod2 is the GAP essential to meiosis progression of mouse oocytes, most likely by regulating mitochondrial dynamics.

Preclinical Study of Cell Therapy for Osteonecrosis of the Femoral Head with Allogenic Peripheral Blood-Derived Mesenchymal Stem Cells.

PURPOSE: To explore the value of transplanting peripheral blood-derived mesenchymal stem cells from allogenic rabbits (rPBMSCs) to treat osteonecrosis of the femoral head (ONFH). MATERIALS AND METHODS: rPBMSCs were separated/cultured from peripheral blood after granulocyte colony-stimulating factor mobilization. Afterwards, mobilized rPBMSCs from a second passage labeled with PKH26 were transplanted into rabbit ONFH models, which were established by liquid nitrogen freezing, to observe the effect of rPBMSCs on ONFH repair. Then, the mRNA expressions of BMP-2 and PPAR-γ in the femoral head were assessed by RT-PCR. RESULTS: After mobilization, the cultured rPBMSCs expressed mesenchymal markers of CD90, CD44, CD29, and CD105, but failed to express CD45, CD14, and CD34. The colony forming efficiency of mobilized rPBMSCs ranged from 2.8 to 10.8 per million peripheral mononuclear cells. After local transplantation, survival of the engrafted cells reached at least 8 weeks. Therein, BMP-2 was up-regulated, while PPAR-γ mRNA was down-regulated. Additionally, bone density and bone trabeculae tended to increase gradually. CONCLUSION: We confirmed that local transplantation of rPBMSCs benefits ONFH treatment and that the beneficial effects are related to the up-regulation of BMP-2 expression and the down-regulation of PPAR-γ expression.

[The interaction of vascular endothelial growth factor and interleukin-6 in multiple myeloma].

OBJECTIVE: To study the interaction between human multiple myeloma (MM) cell line and MM-bone marrow stromal cells (BMSCs) modulated by mutual stimulation of vascular endothelial growth factor (VEGF) and interleukin-6 (IL-6), and to analyze the role of VEGF and IL-6 in the pathogenesis of MM. METHODS: MM patient (MM-BMSCs) and normal donor-BMSCs (N-BMSCs) were established from the MNCs of MM and normal bone marrow. ELISA was performed to detect the expression of VEGF and IL-6 in culture supernatants of human MM cell lines U266, MM-BMSCs, N-BMSCs, and co-cultures of U266 and BMSCs in vitro. VEGF and IL-6 were detected in culture supernatants with or without IL-6, anti-IL-6, VEGF, anti-VEGF antibody. RESULTS: Human MM cell line U266 secreted VEGF, but did not secrete IL-6. BMSCs from MM patients and normal donors secreted both VEGF and IL-6. BMSCs stimulated with recombinant human VEGF induced a time and dose-dependent increase in IL-6 secretion. The effects of VEGF were canceled by monoclonal anti-VEGF antibody. Exogenous IL-6 stimulated VEGF secretion of BMSCs. Importantly, when U266 cell were adhered to BMSCs, there was significant increase of VEGF (2.5 - 5.0 fold, P < 0.05) and IL-6 (5.5 - 9.0 fold, P < 0.05) secretion. The secretion of VEGF or IL-6 in BMSCs alone or co-cultures of BMSCs with U266 were inhibited by anti-human IL-6 or anti-human VEGF antibody. U266 cell stimulated with recombinant human IL-6 induced a dose-dependent increase in VEGF secretion, which was inhibited in the presence of a monoclonal antihuman IL-6 antibody. CONCLUSIONS: The interaction between myeloma cells and marrow stromal cells modulates the secretion of VEGF and IL-6, facilitates myeloma cells growth and angiogenesis in MM. It plays an important role in the pathogenesis of MM. This study provides a theoretic basis for target therapy in the treatment of bone marrow microenvironment.

[Enrichment and Biological Characteristics of Peripheral Blood-derived Mesenchymal Stem Cells in Rats].

OBJECTIVE: To explore the effective method for enrichment of rat peripheral blood-derived mesenchymal stem cells(PBMSC) and study the cell biological characteristics. METHODS: Peripheral mononuclear cells were isolated by density gradient centrifugation from blood of 4 week old rats after G-CSF mobilization. Thereafter, the fibroblast-like cells were acquired by plastic-adherent culture, and the proliferation curve was assayed. For analyzing surface markers of the second generation cultured isolated PBMSC, both flow cytometry(CD90, CD44, CD29, CD45, CD11b and CD79a) and immunocytochemical staining(CD73, CD105, CD34 and HLA-DR) methods were used. Furthermore, the differentiation capacities of PBMSC into osteocytes, chondrocytes and adipocytes were identified. RESULTS: (1) The adherent cells displayed typical colony-forming unit fibroblast(CFU-F) growth pattern after 6-7 day of primary culture and reached 80% confluence after 21 days of culture. The passaged PBMSC possessed high proliferative capacity and spindle growth pattern and was able to grown into exponential phase next day with a doubling time of 39.2 h. (2) PBMSC expressed mesenchymal markers such as CD90, CD44, CD29, CD73 and CD105, but failed to expressed markers of CD45, CD11b, CD79a, CD34 and HLA-DR. (3) After 21 days of culture in osteogenic, chondrogenic and adipogenic differentiation media, calcifying nodules, intracellular glycosaminoglycans and lipid droplets could be found by alizarin red, alcian blue and oil red-O staining, respectively. CONCLUSION: PBMSC can be enriched from rat peripheral blood with high purity and abundance by our methods. The growth and phenotypic characteristics of the isolated PBMSC are consistent with that of well-known MSC, and these cells possess the capability to multi-lineage mesoderm differentiation.