Unraveling the diversity of hyphal explorative traits among Rhizophagus irregularis genotypes.

Differences in functioning among various genotypes of arbuscular mycorrhizal (AM) fungi can determine their fitness under specific environmental conditions, although knowledge of the underlying mechanisms still is very fragmented. Here we compared seven homokaryotic isolates (genotypes) of Rhizophagus irregularis, aiming to characterize the range of intraspecific variability with respect to hyphal exploration of organic nitrogen (N) resources, and N supply to plants. To this end we established two experiments (one in vitro and one in open pots) and used 15N-chitin as the isotopically labeled organic N source. In Experiment 1 (in vitro), mycelium of all AM fungal genotypes transferred a higher amount of 15N to the plants than the passive transfer of 15N measured in the non-mycorrhizal (NM) controls. Noticeably, certain genotypes (e.g., LPA9) showed higher extraradical mycelium biomass production but not necessarily greater 15N acquisition than the others. Experiment 2 (in pots) highlighted that some of the AM fungal genotypes (e.g., MA2, STSI) exhibited higher rates of targeted hyphal exploration of chitin-enriched zones, indicative of distinct N exploration patterns from the other genotypes. Importantly, there was a high congruence of hyphal exploration patterns between the two experiments (isolate STSI always showing highest efficiency of hyphal exploration and isolate L23/1 being consistently the lowest), despite very different (micro) environmental conditions in the two experiments. This study suggests possible strategies that AM fungal genotypes employ for efficient N acquisition, and how to measure them. Implications of such traits for local mycorrhizal community assembly still need to be understood.

Arbuscular mycorrhizal fungi suppress ammonia-oxidizing bacteria but not archaea across agricultural soils.

Arbuscular mycorrhizal (AM) fungi are supposedly competing with ammonia-oxidizing microorganisms (AO) for soil nitrogen in form of ammonium. Despite a few studies directly addressing AM fungal and AO interactions, mostly in artificial cultivation substrates, it is not yet clear whether AM fungi can effectively suppress AO in field soils containing complex indigenous microbiomes. To fill this knowledge gap, we conducted compartmentalized pot experiments using four pairs of cropland and grassland soils with varying physicochemical properties. To exclude the interference of roots, a fine nylon mesh was used to separate the rhizosphere and mesh bags, with the latter being filled with unsterile field soils. Inoculation of plants with AM fungus Rhizophagus irregularis LPA9 suppressed AO bacteria (AOB) but not archaea (AOA) in the soils, indicating how soil nitrification could be suppressed by AM fungal presence/activity. In addition, in rhizosphere filled with artificial substrate, AM inoculation did suppress both AOB and AOA, implying more complex interactions between roots, AO, and AM fungi. Besides, we also observed that indigenous AM fungi contained in the field soils eventually did colonize the roots of plants behind the root barrier, and that the extent of such colonization was higher if the soil has previously been taken from cropland than from grassland. Despite this, the effect of experimental AM fungal inoculation on suppression of indigenous AOB in the unsterile field soils did not vanish. It seems that studying processes at a finer temporal scale, using larger buffer zones between rhizosphere and mesh bags, and/or detailed characterization of indigenous AM fungal and AO communities would be needed to uncover further details of the biotic interactions between the AM fungi and indigenous soil AO.

Recognition of refractory Mycoplasma pneumoniae pneumonia among Myocoplasma pneumoniae pneumonia in hospitalized children: development and validation of a predictive nomogram model.

BACKGROUD: The current diagnostic criteria for refractory Mycoplasma pneumoniae pneumonia (RMPP) among Mycoplasma pneumoniae Pneumonia (MPP) are insufficient for early identification, and potentially delayed appropriate treatment. This study aimed to develop an effective individualized diagnostic prediction nomogram for pediatric RMPP. METHODS: A total of 517 hospitalized children with MPP, including 131 with RMPP and 386 without RMPP (non-RMPP), treated at Lianyungang Maternal and Child Health Care Hospital from January 2018 to December 2021 were retrospectively enrolled as a development (modeling) cohort to construct an RMPP prediction nomogram. Additionally, 322 pediatric patients with MPP (64 with RMPP and 258 with non-RMPP, who were treated at the Affiliated Hospital of Xuzhou Medical University from June 2020 to May 2022 were retrospectively enrolled as a validation cohort to assess the prediction accuracy of model. Univariable and multivariable logistic regression analyses were used to identify RMPP risk factors among patients with MPP. Nomogram were generated based on these risk factors using the rms package of R, and the predictive performance was evaluated based on receiver operating characteristic (ROC) curves and using decision curve analysis (DCA). RESULTS: Multivariate analysis revealed five significant independent predictors of RMPP among patients with MPP: age (hazard ratio [HR] 1.16, 95% confidence interval [CI] 1.08-1.33, P = 0.038), fever duration (HR 1.34, 95%CI 1.20-1.50, P < 0.001), lymphocyte count (HR 0.45, 95%CI 0.23-0.89, P = 0.021), serum D-dimer (D-d) level (HR 1.70, 95%CI 1.16-2.49, P = 0.006), and pulmonary imaging score (HR 5.16, 95%CI 2.38-11.21, P < 0.001). The area under the ROC curve was 90.7% for the development cohort and 96.36% for the validation cohort. The internal and external verification calibration curves were almost linear with slopes of 1, and the DCA curve revealed a net benefit with the final predictive nomogram. CONCLUSION: This study proposes a predictive nomogram only based on five variables. The nomogram can be used for early identification of RMPP among pediatric patients with MPP, thereby facilitating more timely and effective intervention.

[Prediction of epitope region and preparation of mouse polyclonal antibody of human Shisa-like protein 1(SHISAL1)].

Objective To investigate antigen optimization of Shisa like protein 1 (SHISAL1) for preparing mouse anti-human SHISAL1 polyclonal antibody and to identify the specificity of the prepared antibody. Methods Bioinformatics was employed to predict the antigenic epitope region of SHISAL1 protein, and then a polypeptide composed of amino acid residues from the site of 28 to 97 of SHISAL1, termed SHISAL1-N, was selected as the antigen. The coding region of SHISAL1-N was cloned by molecular cloning technique, and then it was inserted into pET-28a to generate pET28a-SHISAL1-N recombinant plasmid. The two recombinant plasmids pET28a-SHISAL1-N and pET28a-SHISAL1 were transformed into BL21 (DE3) bacteria and induced to express by IPTG. The two proteins were purified and immunized to female Kunming mice, respectively. The specificities and sensitivities of the acquired antibodies were detected by Western blot analysis, immunoprecipitation and immunofluorescent cytochemical staining. Results pET28a-SHISAL1-N recombinant plasmid was successfully constructed, and the two fused proteins, SHISAL1 and SHISAL1-N, were induced to express. Moreover, two types of SHISAL1 mouse polyclonal antibodies, derived from SHISAL1-N and SHISAL1 antigens, were obtained. Western blot results showed that the antibody prepared from SHISAL1 antigen was less specific and sensitive compared with the antibody prepared from SHISAL1-N antigen which could specifically identify different endogenous SHISAL1 protein. Immunoprecipitation results showed that SHISAL1-N antibody could specifically pull down SHIISAL1 protein in hepatocellular carcinoma cells and immunofluorescence results demonstrated that SHISAL1-N antibody could specifically bind to SHISAL1 protein in the cytoplasm. Conclusion We have optimized the SHISAL1 antigen and prepared the mouse anti-human SHISAL1 polyclonal antibodies successfully, which can be used for Western blot analysis, immunoprecipitation and immunofluorescence cytochemical staining.

Influence of Waste Toner on Asphalt Binder: Chemical and Rheological Characterization.

The growing amount of waste toner (WT) has posed a significant environmental challenge. Meanwhile, researchers are interested in the feasibility of utilizing waste toner as an asphalt binder modifier because its primary chemical components (Styrene-acrylic copolymer and carbon black) are known to improve asphalt properties. The objective of this study was to evaluate the chemical and rheological properties of the waste-toner-modified asphalt binder and hence determine the suitability of integrating waste toner for asphalt modification. The waste-toner-modified asphalt (TMA) binders were produced by blending base asphalt with two types of waste toners of different gradation sizes. Microscopic tests such as x-ray fluorescence (XRF), attenuated total reflectance transform infrared spectroscopy (ATR-FTIR), and scanning electron microscopy with energy dispersive X-ray (SEM-EDS) and fluorescence microscope, as well as rheology tests such as multiple stress creep recovery (MSCR) tests, oscillation tests, and bending beam rheometer tests were performed. The FTIR results showed that there was a chemical reaction between waste toners and base asphalt binder. A fluorescence effect was observed on the binders produced with different toners used in this research. The binder modified with an optimal content of 8%WTs revealed better high and low-temperature properties. Additionally, 8%WTs used in this research could change the PG70-22 binder to PG76-22 binder. The rutting properties of asphalt material were improved for its improved elasticity. In addition, the 200-mesh TMA binders were desirable with respect to waste toner particle size. Overall, there is a benefit to using waste toner in the asphalt industry.

Case report: Fatal infantile hypertonic myofibrillar myopathy with compound heterozygous mutations in the CRYAB gene.

Background: Fatal infantile hypertonic myofibrillar myopathy (FIHMM) is an autosomal recessive hereditary disease characterized by amyotrophy, progressive flexion contracture and ankylosis of the trunk and limb muscles, apnea and respiratory failure, and increased creatine phosphate levels. It is caused by mutations in the CRYAB gene, and only around 18 cases including genetic mutations have been reported worldwide. All patients with FIHMM develop respiratory distress, progressive stiffness of the limbs, and have a poor prognosis. However, no effective treatment for CRYAB-associated respiratory failure has been reported. Here, we report a case of FIHMM with a novel heterozygous missense mutation. Case Presentation: A 2-year-old female developed scoliosis of the lumbar spine and restrictive ventilatory dysfunction in infancy. She was admitted to the hospital with labored breathing on the third day after the second injection of inactivated poliomyelitis vaccine. Acute respiratory failure, pneumothorax, and cardiac arrest arose in the patient during hospitalization, and progressive stiffness of the trunk and limb muscles appeared, accompanied by obvious abdominal distension and an increase in phosphocreatine kinase levels. Screenings for genetic metabolic diseases in the blood and urine were normal. Electromyography revealed mild myogenic damage. A muscle biopsy indicated the accumulation of desmin, α-crystallin, and myotilin in the musculus biceps brachii, and dense granules were observed in muscle fibers using electron microscopy. Mutation analysis of CRYAB revealed a novel heterozygous missense mutation in the proband, c.302A > C (p.His101Pro) and c.3G > A (p.Met1Ile), which inherited from her asymptomatic, heterozygous carrier parents, respectively. The proband was finally diagnosed as FIHMM. One month after the FIHMM diagnosis, the child died of respiratory failure. Conclusion: We report a case of FIHMM with a novel heterozygous missense mutation of CRYAB. This finding might improve our understanding of FIHMM and highlight a novel mutation in the Chinese population.

A McAb-Based Direct Competitive ELISA to Detect O:9 Salmonella Infection in Chicken.

Salmonella enteritidis and Salmonella pullorum belonging to Group O9 Salmonella are major causative agents of infectious diseases in chicken. O9 antigen as a part of lipopolysaccharide (LPS) is a predominant detected target for Salmonella infection. To identify the infection, an anti-O9 monoclonal antibody (McAb)-based direct competitive enzyme-linked assay (O9 Dc-ELISA) was developed after constraints were optimized; the establishment and application of O9 Dc-ELISA, compared to two commercial kits and plate agglutination test (PAT), showed that O9 Dc-ELISA could screen out more positive samples than the PAT method could and produce the same agreement rates with commercial kits in terms of sensitivity in addition to strong specificity to clinical serum samples.

Spatio-temporal variabilities of soil phosphorus pool and phosphorus uptake with maize stover biochar amendment for 5 years of maize.

Phosphorus reuse by application of biochar is a recent concept that needs to be supported by long-term field data. To monitor biochar's long-term effects on P turnover, one-off biochar was applied in 2013 with mineral NPK fertilizers being applied every year since then. Biochar application rates included 0 t ha-1 (CK), 15.75 t ha-1 (BC1), 31.5 t ha-1 (BC2), and 47.25 t ha-1 (BC3). Over the 5 years' field experiment, P distribution in soil profile, inorganic and organic P fractions in bulk, and rhizosphere soil and maize P uptake were determined. The results showed that biochar reduced the inorganic P fractions (Ca2-P, Ca8-P, Al-P, Fe-P and O-P by 4.8-33.7%, 8.8-59.0%, 13.7-28.6%, 8.4-17.6%, and 3.3-25.5%, respectively), and increased organic P fractions (MLOP and HROP by 67.2-11.6% and 18.8-87.7%, respectively) in bulk soil, while in rhizosphere soil, Fe-P and MLOP were decreased by 13.4-34.5% and 67.2-111.6%, respectively, in 2017. After the application of biochar for 5 years, moderately labile organic phosphorus (MLOP), moderately resistant organic phosphorus (MROP), and highly resistant organic phosphorus (HROP) with different biochar treatments were enhanced by 12.8-42.7%, 20.1-48.0%, and 5.5-66.6%, respectively, but Ca8-P, Al-P, O-P, and Ca10-P were all decreased by 18.6-24.9%, 16.4-21.4%, and 3.3-23.48%, respectively. Total P storage in 0-100 cm was declined by biochar. Increases in maize P uptake in the stover (38.6-71.3%) and grain (20.9-25.5%) were occurred after 31.5 t ha-1 and 47.25 t ha-1 biochar addition. To sum up, biochar is found to regulate the distribution, storage, and transformation of soil P, which lead to increase in maize P uptake.

Crop types have stronger effects on soil microbial communities and functionalities than biochar or fertilizer during two cycles of legume-cereal rotations of dry land.

The addition of biochar to agricultural fields has been widely studied, but most of these studies have emphasized its effects by growing a single type of crop over short- to long-term time spans. Additionally, a limited number of studies have focused on the soil microbial community composition with respect to biochar addition in legume-cereal crop rotation. In this study, we examined soil microbial community structures by adding biochar (0, 5, and 10 t ha-1) and fertilizer (nitrogen-N, phosphorous-P and potassium-K) during 2 cycles of mash bean and wheat rotations. The results showed that the bacterial (16S rRNA) gene abundance was often increased by biochar addition in the presence of mash bean (Vigna mungo L.) but not wheat. When the soil received fertilizer, the bacterial gene abundance was less responsive to biochar addition. Fungal (ITS rRNA) copy numbers were enhanced by biochar and fertilizer in presence of wheat but were decreased in the presence of mash bean. Fertilizer addition also resulted in less change in ITS genes after biochar addition. Microbial functional groups including Gram+, Gram- and Pseudomonas bacteria were stimulated by biochar or fertilizer only in mash bean soils, while mycorrhizae were significantly increased by biochar in wheat soils. Although biochar addition affected soil properties, microbial community assays were not greatly altered by these physicochemical properties. In conclusion, the crop type played a decisive role, rather than biochar or fertilizer addition, in shaping microbial community structures (16S and ITS phyla) during crop rotation.

[Preparation and application of mouse polyclonal antibodies against human Shisa like 1 (SSL1)].

Objective To prepare polyclonal antibodies against Shisa like 1 protein (SSL1) and study the localization of SSL1 in hepatocellular carcinoma SMMC-7721 cells. Methods Human SSL1 gene was cloned from HepG2 cells by reverse transcription PCR, and then inserted into prokaryotic expression vector pET-28a to generate the SSL1 expression vector. The recombinant plasmid pET28a-SSL1 was then transformed into E. coli BL21 (DE3) and induced to express by IPTG. Polyclonal antibody against SSL1 was generated by immunizing Kunming mouse with the purified protein by the routine method. The specificity of polyclonal antibody was verified by Western blot analysis. The expression of SSL1 in SMMC-7721 cells was detected by immunofluorescent cytochemistry. Golgi complexes were signed by Golgi-Tracker Red to analyze the subcellular localization of SSL1 protein in SMMC-7721 cells. Results The SSL1 gene was cloned and the recombinant vector pET28a-SSL1 was successfully constructed. Pure SSL1 protein expression in E. coli BL21 was confirmed and polyclonal antibodies against protein SSL1 was obtained in immunized Kunming mice. Immunofluorescent cytochemistry showed that SSL1 was expressed in the cytoplasm, and was co-localized with Golgi-Tracker Red in SMMC-7721 cells. Conclusion We have obtained SSL1 polyclonal antibodies with high specificity, which was proved situated in Golgi bodies of SMMC-7721 cells.

[Correlation between galectin-3 level in bronchoalveolar lavage fluid and cellular immunity in children with refractory Mycoplasma pneumoniae pneumonia].

OBJECTIVE: To study the correlation of galectin-3 level in bronchoalveolar lavage fluid (BALF) with Mycoplasma pneumoniae (MP) load and cellular immunity of neutrophils and macrophages in the airway in children with refractory MP pneumonia (RMPP). METHODS: A total of 64 children with RMPP who were hospitalized from January 2013 to January 2017 were enrolled. In addition to the conservative medical treatment, all the 64 children with RMPP were given bronchoalveolar lavage in the acute stage (5-7 days after admission) and 48 out of the 64 children were given bronchoalveolar lavage in the recovery stage (10-14 days after admission). Four milliliters of BALF of the affected lung lobe or segment were collected. ELISA was used to measure the level of galectin-3 in BALF supernatant. RT-PCR was used to measure MP load. Hematoxylin and eosin staining was used to measure the percentage of neutrophils and macrophages. Six children with bronchial foreign bodies were enrolled as the control group. RESULTS: The RMPP group had a significantly higher level of galectin-3 in BALF in both the acute and recovery stages than the control group (P<0.01), and the level of galectin-3 in the acute stage was significantly higher than in the recovery stage (P<0.01). The RMPP group had a significantly higher percentage of neutrophils in BALF in both the acute and recovery stages than the control group (P<0.01), and the percentage of neutrophils in the acute stage was significantly higher than in the recovery stage (P<0.01). The RMPP group had a significantly lower percentage of macrophages in BALF in both the acute and recovery stages than the control group (P<0.01), but there was no significant difference in the percentage of macrophages between the acute and recovery stages (P>0.05). The RMPP group had a significantly higher MP load in BALF in both the acute and recovery stages than the control group (P<0.01), and the MP load in the acute stage was significantly higher than in the recovery stage (P<0.01). In the children with RMPP, galectin-3 level in BALF in the acute stage was positively correlated with MP load and the percentage of neutrophils (rs=0.789 and 0.726 respectively; P<0.01). CONCLUSIONS: Galectin-3 is involved in the process of airway inflammation in children with RMPP, and the level of galectin-3 in BALF is positively correlated with MP load. RMPP is a cellular immune inflammatory lesion with the increase of neutrophils and the reduction in macrophages. Galectin-3 is closely associated with neutrophil chemotaxis and luminal infiltration in children with RMPP. MP load gradually decreases with the recovery from RMPP, but it is not completely eliminated by the immune system in the recovery stage. MP infection can increase the consumption of macrophages in children with RMPP.

Using a modified soil quality index to evaluate densely tilled soils with different yields in Northeast China.

Northeastern China has long-term densely tilled soils that supply approximately 20% of the annual total national grains. There are very few reports on the agricultural soil quality subjecting to the predatory tillage. Here, the soil quality index (SQI) of a brunisolic soil was calculated using the minimum data set (MDS) and integrated quality index (IQI). The topsoil layer was divided into plow layer (11.9 ± 1.9 cm) and plow pan (11.4 ± 2.6 cm) in fields of high yields (HYB), medium yields (MYB), and low yields (LYB). Our results showed that the MDS of the topsoil layer only contained chemical indicators. The bulk density (BD), as one of the most important soil quality indicators, was found of no significant differences in the topsoil layers. In different layers (i.e., the topsoil layer, plow layer, and plow pan), the value of SQI presented a consistent tendency of HYB > MYB > LYB (p < 0.05). The correlation between SQI and yield was higher in the plow layer (0.60) and plow pan (0.63) than the topsoil layer (0.47). This further verified the reasonability of using soil stratification for SQI calculation. Our findings indicate the potential of using soil quality assessments to examine soil productivity (e.g., fertilizer deficiency) in crop lands with soil stratification.

A comprehensive review on self-healing of asphalt materials: Mechanism, model, characterization and enhancement.

Self-healing has great potential to extend the service life of asphalt pavement, and this capability has been regarded as an important strategy when designing a sustainable infrastructure. This review presents a comprehensive summary of the state-of-the-art investigations concerning the self-healing mechanism, model, characterization and enhancement, ranging from asphalt to asphalt pavement. Firstly, the self-healing phenomenon as a general concept in asphalt materials is analyzed including its definition and the differences among self-healing and some viscoelastic responses. Additionally, the development of self-healing in asphalt pavement design is introduced. Next, four kinds of possible self-healing mechanism and corresponding models are presented. It is pointed out that the continuum thermodynamic model, considering the whole process from damage initiation to healing recovery, can be a promising study field. Further, a set of self-healing multiscale characterization methods from microscale to macroscale as well as computational simulation scale, are summed up. Thereinto, the computational simulation shows great potential in simulating the self-healing behavior of asphalt materials from mechanical and molecular level. Moreover, the factors influencing self-healing capability are discussed, but the action mechanisms of some factors remain unclear and need to be investigated. Finally, two extrinsic self-healing technologies, induction heating and capsule healing, are recommended as preventive maintenance applications in asphalt pavement. In future, more effective energy-based healing systems or novel material-based healing systems are expected to be developed towards designing sustainable long-life asphalt pavement.

Phosphorus recovery and reuse by pyrolysis: Applications for agriculture and environment.

Phosphorus ore extraction for soil fertilization supports the demand of modern agriculture, but extractable resource limitations, due to scarcity, impose a P reuse and recycling research agenda. Here we propose to integrate biochar production (pyrogenic carbon) with municipal and agricultural waste management systems, to recover and reuse phosphorous that would otherwise be lost from the ecological food web. A meta-analysis and available data on total P in biochar indicated that P-enriched feedstocks include animal manure, human excreta, and plant-biomass collected from P-polluted sites. Phosphorus in biochar could participate in P equilibriums in soils and is expected to supply P. The release, sorption and desorption of P by biochar will codetermine the potential of P replenishment by biochar and P loss from biochar-amended soils. Abiotic and biotic factors are expected to affect sorption/desorption of P between biochar and soil aggregates, and P acquisition by plants. Chemical extraction, using acid or alkaline solutions, is considered as a means for P retrieval from high P biochar, especially for biochar with high heavy metal contents. To bridge the gap between academia and practice, this paper proposes future development for phosphorus acclamation by pyrolysis: 1) identification of high-P bio-waste for pyrolysis; 2) retrieval of P by using biochar as soil amendment or by chemical leaching; 3) biochar modification by inorganic nutrients, P solubilizing microorganisms and other organic matter; and 4) compatible pyrolysis equipment fit to the current waste management context, such as households, and waste water treatment plants.

Biochar as a novel niche for culturing microbial communities in composting.

Biochar has been applied as a bulk agent or an additive to compost. The mixture of biochar and compost has been considered to exert synergistic effect as a soil amendment. In a composting system, the macro-porous sites of biochar may act as a novel niche that selects and cultures the microorganisms from the bulk compost. A variety of volatile organic carbons (VOCs) such as aromatic hydrocarbons and aliphatics were detected in biochar pellets (BC) pyrolyzed at 100°C. In the mesosphilic phase, the water-soluble carbon (WSC) and water-soluble phenols (WSP) in biochar increased from 2.1 to 26mgkg(-1) and 5.9 to 101µgkg(-1), respectively. These labile carbons however, were subjected to a rapid metabolism over the composting course. We further compared the responses of microbial community in BC to those in the bulk organic matter. Both Shannon-Wiener and Richness indexes of bacterial communities were higher in BC than in the adjacent compost (ADJ) and the bulk organic matter (control). As for fungal communities, the two indexes were higher in BC than ADJ and control only in the mature phase. During the composting course, the bacterial activity was higher than the fungal counterpart in terms of the changes of corresponding biomarkers, glucosamine and muramic acids. The results suggested that the diversified labile carbons sources including VOCs and WSC in BC could influence the structure of microbial community and resulted in an enhanced carbon catabolic capacity.

Overexpression of hnRNPC2 induces multinucleation by repression of Aurora B in hepatocellular carcinoma cells.

Heterogeneous ribonuclear protein C2 (hnRNPC2), an RNA binding protein, is a component of hnRNPC which is upregulated in many tumors. Multinucleation exists in many tumors and is positively correlated with tumor grade. To uncover the correlation between hnRNPC2 and multi-nucleation in hepatocellular carcinoma SMMC-7721 cells, we constructed a pEGFP-hnRNPC2 vector and transfected it into cancer cells. Our results revealed that overexpression of hnRNPC2 induced multinucleation in SMMC-7721 cells. Tracking tests indicated that the induced multinucleated cells were unable to recover to mononuclear cells and finally died as a result of defects in cell division. Furthermore, Aurora B, which was localized at the midbody and plays a role in cytokinesis, was repressed in hnRNPC2-overexpressing cells, whose knockdown by RNA interference also induced multinucleation in SMMC-7721 cells. Quantitative polymerase chain reaction (qPCR) and mRNA-protein co-immunoprecipitation results revealed that Aurora B mRNA did not decrease in hnRNPC2-overexpressing cells, instead it bound more hnRNPC2 and less eIF4E, an mRNA cap binding protein and translational initiation factor. Moreover, hnRNPC2 bound more eIF4E in hnRNPC2-overexpressing cells. These results indicate that hnRNPC2 repressed Aurora B binding with eIF4F, which must bind with Aurora B mRNA in order to initiate its translation. This induced multinucleation in hepatocellular carcinoma cells. In addition, hnRNPC2 accelerated hepatocellular carcinoma cell proliferation. Collectively, these data suggest that hnRNPC2 may be a potential target for hepatocellular carcinoma cell diagnosis and treatment.

Cancer cell growth suppression by a 62nt AU-rich RNA from C/EBPß 3'UTR through competitive binding with HuR.

AU-rich elements are functional motifs in the 3'untranslated region of mRNA and are binding sites for the RNA binding protein HuR, an mRNA stabilizer and translation enhancer implicated in carcinogenesis. It is not clear whether, and, if so, how the AU-rich elements function in cells when they are separated from their mRNA and form an independent RNA species. Here, we show that a short RNA with AU-rich elements derived from C/EBPß 3'UTR suppressed growth in a human liver cancer cell line. It specifically bound HuR, and it competed with C/EBPß mRNA in order to bind to HuR. Our results provide evidence that the cancer cell growth suppression by this 62nt RNA containing AU-rich elements may be due to competitive binding to HuR. This work may open new options for the development of novel anti-cancer drugs.

Tumor suppression by RNA from C/EBPß 3'UTR through the inhibition of protein kinase Cε activity.

BACKGROUND: Since the end of last century, RNAs from the 3'untranslated region (3'UTR) of several eukaryotic mRNAs have been found to exert tumor suppression activity when introduced into malignant cells independent of their whole mRNAs. In this study, we sought to determine the molecular mechanism of the tumor suppression activity of a short RNA from 3'UTR of C/EBPß mRΝΑ (C/EBPß 3'UTR RNA) in human hepatocarcinoma cells SMMC-7721. METHODOLOGY/PRINCIPAL FINDINGS: By using Western blotting, immunocytochemistry, molecular beacon, confocal microscopy, protein kinase inhibitors and in vitro kinase assays, we found that, in the C/EBPß 3'UTR-transfectant cells of SMMC-7721, the overexpressed C/EBPß 3'UTR RNA induced reorganization of keratin 18 by binding to this keratin; that the C/EBPß 3'UTR RNA also reduced phosphorylation and expression of keratin 18; and that the enzyme responsible for phosphorylating keratin 18 is protein kinase Cε. We then found that the C/EBPß 3'UTR RNA directly inhibited the phosphorylating activity of protein kinase Cε; and that C/EBPß 3'UTR RNA specifically bound with the protein kinase Cε-keratin 18 conjugate. CONCLUSION/SIGNIFICANCE: Together, these facts suggest that the tumor suppression in SMMC-7721 by C/EBPß 3'UTR RNA is due to the inhibition of protein kinase Cε activity through direct physical interaction between C/EBPß 3'UTR RNA and protein kinase Cε. These facts indicate that the 3'UTR of some eukaryotic mRNAs may function as regulators for genes other than their own.

[Construction of human-bovine interspecies embryos and investigation of interspecies embryonic mitochondrial source].

UNLABELLED: Obtaining human blastocysts is a prerequisite for cell replacement therapy using embryonic stem cells. We established an interspecies somatic cell nuclear transfer (iSCNT) technique for producing blastocysts without sacrificing human oocytes. Human foetal fibroblasts were used as donor cells injected into the enucleated bovine oocytes in nuclear transfer, whereas bovine foetal fibroblasts were used to produce intraspecies embryos. We also examined the fate of human and bovine mitochondrial DNA (mtDNA) during preimplantation development after nuclear transfer by PCR. PCR analysis for the detection of human and bovine mtDNA was done at the 2,8-morula, and blastocyst stages of the embryos. RESULT: 2.8% interspecies embryos developed to blastocysts after cultured in an SOF medium, while blastocyst rate of intraspecies embryos were 10.1%. Both human and bovine mtDNAs existed until the morula stage, whereas only the bovine mtDNA was found at the blastocyst stage. These results indicated that interspecies cloning without using human oocytes could generate human blastocysts. Because of the incoordination between bovine mtDNA and human nuclear gene, developmental rate of interspecies embryos was significantly lower than intraspecie. Whether the embryonic stem cell could be used for cell replacement therapy need further research.

[Construction of universal vector for gene targeting and analysis of its function].

To make a universal gene targeting vector fitting for most gene and delete positive selection gene after targeting successfully, a vector named pA2T was constructed by inserting one neomycin gene (neo) for positive selection and two same herpes simplex virus thymidine kinase gene HSV-tk1 and HSV-tk2 for negative selection into the vector of pGEM-3Z, and two locus of crossing-over (x) in P1 (LoxP) and two different multiple cloning sites (MCS) were inserted into two flanks of neo separately. There were eight rare cloning sites between neo and HSV-tk1 and five rare cloning sites between neo and HSV-tk2, and neo, HSV-tk1 and HSV-tk2 could be translated respectively in the pA2T. Transfection of the pA2T into goat fetus fibroblast cells with Lipofectamine 2000 conferred resistance to geneticin (G418) and resistance to ganciclovir (GAC) in the cells, which suggested the positive and negative selectable markers could express in the cells and thus the vector pA2T could be used as a universal gene targeting vector. Transformation of the pA2T into the BM25.8 expressing Cre recombinase conferred neo was deleted in the pA2T, which suggested the LoxP was active. Thus, this vector can be inserted by most gene sequences as homologous sequences and positive selection gene can be deleted after targeting successfully, which is very convenience for the production of transgenic animals using gene targeting method.