Bi-FoRe: an efficient bidirectional knockin strategy to generate pairwise conditional alleles with fluorescent indicators

Gene expression labeling and conditional manipulation of gene function are important for elaborate dissection of gene function. However, contemporary generation of pairwise dual-function knockin alleles to achieve both conditional and geno-tagging effects with a single donor has not been reported. Here we first developed a strategy based on a flipping donor named FoRe to generate conditional knockout alleles coupled with fluorescent allele-labeling through NHEJ-mediated unidirectional targeted insertion in zebrafish facilitated by the CRISPR/Cas system. We demonstrated the feasibility of this strategy at sox10 and isl1 loci, and successfully achieved Cre-induced conditional knockout of target gene function and simultaneous switch of the fluorescent reporter, allowing generation of genetic mosaics for lineage tracing. We then improved the donor design enabling efficient one-step bidirectional knockin to generate paired positive and negative conditional alleles, both tagged with two different fluorescent reporters. By introducing Cre recombinase, these alleles could be used to achieve both conditional knockout and conditional gene restoration in parallel; furthermore, differential fluorescent labeling of the positive and negative alleles enables simple, early and efficient real-time discrimination of individual live embryos bearing different genotypes prior to the emergence of morphologically visible phenotypes. We named our improved donor as Bi-FoRe and demonstrated its feasibility at the sox10 locus. Furthermore, we eliminated the undesirable bacterial backbone in the donor using minicircle DNA technology. Our system could easily be expanded for other applications or to other organisms, and coupling fluorescent labeling of gene expression and conditional manipulation of gene function will provide unique opportunities to fully reveal the power of emerging single-cell sequencing technologies.

Advances in the research and applications of orange fluorescent protein / 生物工程学报

Fluorescent proteins can be used as probes to investigate intercellular molecular interactions and trace the pathway of specific metabolites, thus providing a detailed and accurate description of various metabolic processes and cellular pathways in living cells. Nowadays, the existing fluorescent proteins cover almost all spectral bands from ultraviolet to far-red. These fluorescent proteins have been applied in many fields of bioscience with the help of high-resolution microscopy, making great contributions to the development of biology. It is generally agreed that orange fluorescent proteins refer to the fluorescent proteins at the spectral range of 540-570 nm. In recent years, researches on orange fluorescent proteins have made great progress, and they have been widely applied in the field of biology and medicine as reporter protein and fluorescence resonance energy transfer as fluorescent receptor. This paper reviews the studies in the field of orange fluorescent proteins over the last 15 years, with the special focus on the development and application of orange fluorescent proteins to provide the basis for the future studies.

Construction and application of mCherry red fluorescent protein fusion expression system in lactic acid bacteria / 生物工程学报

In order to enrich the library of domestic research about new red fluorescent marker in lactic acid bacteria (LAB), we described a new fusion expression system in Lactobacillus plantarum WCFS1 based on the pSIP vector. This system contained red fluorescent protein mCherry as a marker and bile salt hydrolase gene (bsh) as a reporter gene. Moreover, in this study, four different promoters (PsppA, PldhL, P32 and PslpA) were used to regulate the expression of the fusion protein mCherry-BSH, completing the inducible and constitutive expression in lactic acid bacteria. The recombinant protein mCherry-BSH presented activity of red fluorescence and bile salt hydrolase (BSH). The successful construction of the fusion expression system in LAB using a red fluorescent protein mCherry provides favorable conditions for the distribution, intestinal colonization and survival rate of lactic acid bacteria, so as to reveal the function mechanism of its probiotic characteristics; and the system also could lay the foundation for researches on protein expression, cellular localization and properties identification of active protein in lactic acid bacteria.

Taurine Transporter dEAAT2 is Required for Auditory Transduction in Drosophila / 神经科学通报·英文版

Drosophila dEAAT2, a member of the excitatory amino-acid transporter (EAAT) family, has been described as mediating the high-affinity transport of taurine, which is a free amino-acid abundant in both insects and mammals. However, the role of taurine and its transporter in hearing is not clear. Here, we report that dEAAT2 is required for the larval startle response to sound stimuli. dEAAT2 was found to be enriched in the distal region of chordotonal neurons where sound transduction occurs. The Ca imaging and electrophysiological results showed that disrupted dEAAT2 expression significantly reduced the response of chordotonal neurons to sound. More importantly, expressing dEAAT2 in the chordotonal neurons rescued these mutant phenotypes. Taken together, these findings indicate a critical role for Drosophila dEAAT2 in sound transduction by chordotonal neurons.

A Critical Time-Window for the Selective Induction of Hippocampal Memory Consolidation by a Brief Episode of Slow-Wave Sleep / 神经科学通报·英文版

Although extensively studied, the exact role of sleep in learning and memory is still not very clear. Sleep deprivation has been most frequently used to explore the effects of sleep on learning and memory, but the results from such studies are inevitably complicated by concurrent stress and distress. Furthermore, it is not clear whether there is a strict time-window between sleep and memory consolidation. In the present study we were able to induce time-locked slow-wave sleep (SWS) in mice by optogenetically stimulating GABAergic neurons in the parafacial zone (PZ), providing a direct approach to analyze the influences of SWS on learning and memory with precise time-windows. We found that SWS induced by light for 30 min immediately or 15 min after the training phase of the object-in-place task significantly prolonged the memory from 30 min to 6 h. However, induction of SWS 30 min after the training phase did not improve memory, suggesting a critical time-window between the induction of a brief episode of SWS and learning for memory consolidation. Application of a gentle touch to the mice during light stimulation to prevent SWS induction also failed to improve memory, indicating the specific role of SWS, but not the activation of PZ GABAergic neurons itself, in memory consolidation. Similar influences of light-induced SWS on memory consolidation also occurred for Y-maze spatial memory and contextual fear memory, but not for cued fear memory. SWS induction immediately before the test phase had no effect on memory performance, indicating that SWS does not affect memory retrieval. Thus, by induction of a brief-episode SWS we have revealed a critical time window for the consolidation of hippocampus-dependent memory.

Spinal CCL2 Promotes Central Sensitization, Long-Term Potentiation, and Inflammatory Pain via CCR2: Further Insights into Molecular, Synaptic, and Cellular Mechanisms / 神经科学通报·英文版

Mounting evidence supports an important role of chemokines, produced by spinal cord astrocytes, in promoting central sensitization and chronic pain. In particular, CCL2 (C-C motif chemokine ligand 2) has been shown to enhance N-methyl-D-aspartate (NMDA)-induced currents in spinal outer lamina II (IIo) neurons. However, the exact molecular, synaptic, and cellular mechanisms by which CCL2 modulates central sensitization are still unclear. We found that spinal injection of the CCR2 antagonist RS504393 attenuated CCL2- and inflammation-induced hyperalgesia. Single-cell RT-PCR revealed CCR2 expression in excitatory vesicular glutamate transporter subtype 2-positive (VGLUT2) neurons. CCL2 increased NMDA-induced currents in CCR2/VGLUT2 neurons in lamina IIo; it also enhanced the synaptic NMDA currents evoked by dorsal root stimulation; and furthermore, it increased the total and synaptic NMDA currents in somatostatin-expressing excitatory neurons. Finally, intrathecal RS504393 reversed the long-term potentiation evoked in the spinal cord by C-fiber stimulation. Our findings suggest that CCL2 directly modulates synaptic plasticity in CCR2-expressing excitatory neurons in spinal lamina IIo, and this underlies the generation of central sensitization in pathological pain.

Spatiotemporal Imaging of Cellular Energy Metabolism with Genetically-Encoded Fluorescent Sensors in Brain / 神经科学通报·英文版

The brain has very high energy requirements and consumes 20% of the oxygen and 25% of the glucose in the human body. Therefore, the molecular mechanism underlying how the brain metabolizes substances to support neural activity is a fundamental issue for neuroscience studies. A well-known model in the brain, the astrocyte-neuron lactate shuttle, postulates that glucose uptake and glycolytic activity are enhanced in astrocytes upon neuronal activation and that astrocytes transport lactate into neurons to fulfill their energy requirements. Current evidence for this hypothesis has yet to reach a clear consensus, and new concepts beyond the shuttle hypothesis are emerging. The discrepancy is largely attributed to the lack of a critical method for real-time monitoring of metabolic dynamics at cellular resolution. Recent advances in fluorescent protein-based sensors allow the generation of a sensitive, specific, real-time readout of subcellular metabolites and fill the current technological gap. Here, we summarize the development of genetically encoded metabolite sensors and their applications in assessing cell metabolism in living cells and in vivo, and we believe that these tools will help to address the issue of elucidating neural energy metabolism.

Total clipless cholecystectomy by means of harmonic sealing / Colecistectomia laparoscópica totalmente ultrassônica sem uso de clipe metálico

BACKGROUND: In traditional laparoscopic cholecistectomy, the cystic duct and artery are commonly closed by metallic clips just before their division. Although the placement of these clips for occluding cystic artery and duct can be considered safe, biliary leaks and bleeding may occur especially by its dislodgement. AIM: To report a prospective case-series in total clipless cholecystectomy by means of harmonic shears for closure and division of the artery and cystic duct as well removal of the gallbladder from the liver. METHODS: Was evaluate a series of 125 patients who underwent laparoscopic cholecystectomy where the sealing and division of cystic artery and duct was carried out only by harmonic shears. The intact extracted gallbladder was submitted to a reverse pressure test for assessment of the technique safety by means of CO2 insuflation. RESULTS: The most common indication for surgery was gallstones. The mean operative time was 26 min and all gallbladders were dissected intact from the liver bed. There was no mortality and the overall morbidity rate was 0.8% with no hemorrhage or leaks. The reverse pressure test showed that all specimens support at least 36-mmHg of pressure without leaking. CONCLUSION: The harmonic shears is effective and safe in laparoscopic cholecystectomy as a sole instrument for sealing and division of the artery and cystic duct. The main advantages could be related to the safety and decreased operative time. .

RACIONAL: A colecistectomia laparoscópica na técnica tradicional oclui o ducto cístico e a artéria cística por clipes cirúrgicos, que podem se deslocar ou desprender no pós-operatório, possibilitando a ocorrência de fístula biliar ou hemorragia. OBJETIVO: Relato prospectivo de série de casos de colecistectomias laparoscópicas sem uso de clipe cirúrgico, sendo que a ligadura e secção da artéria cística e do ducto cístico foram realizadas por meio de bisturi ultrassônico. MÉTODO: Foram incluídos 125 pacientes submetidos à colecistectomia laparoscópica sem utilização de clipe cirúrgico metálico, onde a ligadura da artéria e do ducto cístico e também a remoção da vesícula biliar de seu leito hepático foram realizadas por meio de tesoura ultrassônica. Realizou-se teste de pressão reversa na vesícula biliar removida intacta do leito hepático para verificar a segurança da técnica. RESULTADOS: A principal indicação cirúrgica foi a colelitíase. O tempo cirúrgico médio foi de 26 min e todas as vesículas biliares foram retiradas intactas do leito hepático. Não houve mortalidade e a taxa global de morbidade foi de 0,8%, sem hemorragias ou fístulas. O teste de pressão reversa mostrou que o ducto cístico ocluído pelo bisturi harmônico suportou ao pelo menos 36 mmHg de pressão sem que ocorresse nenhum vazamento. CONCLUSÃO: O bisturi harmônico é eficaz e seguro em colecistectomias laparoscópicas eletivas como um instrumento único para ocluir e seccionar tanto a artéria cística quanto o ducto cístico. Vantagens podem ser apontadas ao método com relação a sua segurança e diminuição do tempo cirúrgico. .

Tumor angiogenesis promoted by fusion of glioma stem/progenitor cells with bone marrow mesenchymal stem cells / 中华肿瘤杂志

<p><b>OBJECTIVE</b>The aim of this study was to clarify whether the fusion of bone marrow mesenchymal stem cells (MSCs) with tumor cells can promote tumor angiogensis.</p><p><b>METHODS</b>Human glioma stem/progenitor cells (GSPCs) (SU3 cells) were transfected with red fluorescent protein (RFP) gene. Bone marrow mesenchymal stem cells (MSCs) were harvested from nude mice with whole-body green fluorescent protein (GFP) gene expression. Then the two kinds of cells were co-cultured in vitro. At the same time SU3-RFP was transplanted into the brain of GFP-expressing nude mice to establish xenograft tumors. The co-cultured cells, GFP/RFP double positive (yellow) cells and blood vessels obtained from the xenograft tumors were observed under fluorescent microscope and laser scanning confocal microscope.</p><p><b>RESULTS</b>After five passages in vitro, MSCs maintained the proliferative activity and highly expressed CD105. CD105 was also expressed in the femurs of GFP-expressing nude mice, tumor cells, blood vessels of SU3 xenograft tumors, and clinical malignant gliomas. When MSCs were co-cultured with SU3-RFP, the ratio of yellow cells co-expressing RFP and GFP was significantly increased after extended time and continuous passages. According to the flow cytometry, yellow cells co-expressing RFP and GFP were 83.7% of the cultured cells. In tissue slices of the xenograft tumors, bundles of yellow vessel-like structure and cross-sectioned yellow vascular wall structures including vascular wall stroma cells were observed with RFP and GFP expression, and were identified as de novo formed vessels derived from fusion of MSCs with SU3-RFP cells.</p><p><b>CONCLUSION</b>Cell fusion occurs between tumor cells and host MSCs and it promotes tumor angiogenesis.</p>

Sheep gastrointestinal helminthiasis in the Sertão region of Paraíba State, Northeastern Brazil: prevalence and risk factors / Helmintoses gastrintestinais de ovinos no Sertão do Estado da Paraíba, Nordeste do Brasil: prevalência e fatores de riscos

In this study, we aimed to establish the prevalence and risk factors relating to gastrointestinal helminthiasis, and to characterize the sanitary management practiced among sheep herds in the Sertão region of the state of Paraíba, northeastern Brazil, based on factors that condition the ways of controlling these parasites in these herds. The research was carried out between April and July 2012. We visited 54 farms, where fecal and blood samples were individually collected from 465 animals. On each farm, a questionnaire was applied to gather information on variables relating to potential risk factors. The prevalence of sheep gastrointestinal helminthiasis in the region was 75.9%. At least one animal tested positive for this helminthiasis on 53 (98.1%) of the 54 farms evaluated. The eggs per gram of feces (EPG) analysis showed the following infection burdens: 51.8% with mild infection, 27.1% moderate infection, 9.9% heavy infection and 11.2% fatal infection. Among the sheep farms visited, anthelmintics were used on 81.5% (p <0.05). The most relevant risk factor in this study was the farm area, because it defines the area available for grazing animals. Properties with many animals and little pasture area, which are the most abundant type in the Sertão region of Paraíba, tend to have high prevalence of gastrointestinal helminthiasis, because the animals are more prone to reinfection. The Sertão region of Paraíba presents high prevalence of gastrointestinal helminthiasis among sheep, and the farm area is the most relevant risk factor for the development of these parasites.

Objetivou-se determinar a prevalência e os fatores de risco para as helmintoses gastrintestinais, caracterizando o manejo sanitário sob fatores condicionantes das formas de controle dessas parasitoses em rebanhos de ovinos da região do Sertão da Paraíba. A pesquisa foi desenvolvida no período de abril a julho de 2012. Foram visitadas propriedades, utilizando-se 465 animais, sendo coletadas individualmente amostras de fezes e sangue durante as visitas. Em cada propriedade, foi aplicado questionário para a coleta de informações acerca de variáveis que atuariam como possíveis fatores de risco. Observou-se que a prevalência das helmintoses gastrintestinais de ovinos na região do Sertão da Paraíba foi de 75,9%. Pelo menos um animal foi positivo para essas helmintoses, em 53 (98,1%) das 54 propriedades avaliadas. A análise de OPG (Ovos Por Gramas de Fezes) demonstrou que 51,8% dos animais apresentaram infecção leve, 27,1% infecção moderada, 9,9% infecção pesada e 11,2% infecção fatal. A utilização de anti-helmínticos ocorreu em 81,5% das propriedades (p <0,05). O fator de risco mais relevante neste estudo foi a área da propriedade, porque delimita a área de pastejo do animal. Propriedades com muitos animais e pouca área de pastejo, que são as mais abundantes no Sertão da Paraíba, tendem a apresentar alta prevalência de helmintoses gastrintestinais, pois os animais estão mais propensos à reinfecção. A região do Sertão da Paraíba apresenta uma elevada prevalência de helmintoses gastrintestinais em ovinos, e a área das propriedades é o fator de risco mais relevante para o desenvolvimento dessas parasitoses.

Survival of bone marrow mesenchymal stem cells and periodontal ligament stem cells in cell sheets / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To evaluate the survival of bone marrow mesenchymal stem cells (BMMSC) and periodontal ligament stem cells (PDLSC) in BMMSC/PDLSC cell sheets which transplanted ectopically into subcutaneous dorsum of nude mice.</p><p><b>METHODS</b>The canine BMMSC and PDLSC from primary culture were tranfected with lentiviral vectors carrying green fluorescent protein (GFP) gene (Lentivirus-GFP) or red fluorescent protein (RFP) gene (Lentivirus-RFP) respectively. The immunophenotypes of GFP-labeled BMMSC and RFP-labeled PDLSC were identified by flow cytometry. Adipogenic and osteogenic differentiation of them were detected by alizarin red or oil red O respectively. Then, both GFP-labeled BMMSC cell sheets and RFP-labeled PDLSC cell sheets were fabricated respectively using normal culture dish (6 cm) after stimulation of extracellular matrix formation. Each was enveloped by collagen membrane (Bio-Gide) and then transplanted into the subcutaneous dorsum of nude mice. In vivo non-invasive biofluorescence imaging(BFI) was performed at 1, 2, 4 and 8 w post-tranplantation to trace and quantify the survival and growth of RFP-labeled PDLSC and GFP-labeled BMMSC via the BFI system of the NightOWL. The fluorescence intensity change of GFP/RFP signal was monitored and compared. The mice were sacrificed 8 weeks after cell sheets transplantation and the survival of stem cells was verified by fluorescence immunohistochemistry.</p><p><b>RESULTS</b>The flow cytometry showed that GFP-labeled BMMSC positively expressed CD29, CD44, CD34, STRO-1 were 93.07%, 92.84%, 3.23%, 67.67%, and RFP-labeled PDLSCs were 89.91%, 88.47%, 6.04%, 74.11%, respectively. Both of them had the potency of differentiating into osteoblasts and adipocytes. The stemness of both of them was almost same. After being transplanted into nude mice, the signal strength of GFP(BMMSC) was weaker and weaker in 1, 2, 4 and 8 w [(83.1±3.1)×10(6), (65.1±2.3)×10(6), (51.5 ± 2.3)×10(6), (33.8 ± 2.0)×10(6) ph/s, respectively.]. The signal strength of RFP(PDLSC) was weakenedin 1, 2 and 4 w [(53.8±3.0)×10(6), (42.2±2.6)×10(6), (34.5±2.1)×10(6) ph/s], then recovered in 8 w ([ 45.1±2.9)×10(6) ph/s]. The signal strength of RFP(PDLSC) was signifcantly stronger in 8 w than in 4 w(P < 0.01). The survival of RFP-labeled PDLSC was significant higher than that of GFP-labeled BMMSC. After 8 weeks, lots of RFP-labeled PDLSC were observed by microscope, but less GFP-labeled BMMSC were observed.</p><p><b>CONCLUSIONS</b>Histometric analysis revealed that the survival of stem cells in the RFP-labeled PDLSC cell sheets was significantly higher than that of in the GFP-labeled BMMSCs cell sheets.</p>

Construction and investigation of a recombinant eukaryotic expression vector for expressing the ORF3 protein of hepatitis E virus in BHK-21 fibroblasts / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To construct a eukaryotic expression vector to express the hepatitis E virus protein open reading frame 3 (ORF3) and investigate the intracellular location of the expressed protein using the baby hamster kidney (BHK-21) fibroblast cell line.</p><p><b>METHODS</b>The ORF3 gene was amplified by RT-PCR, cloned into the HindIII and EcoRI sites in the multicloning site of the pDsRed-Monomer-N1mammalian expression vector that encodes a red fluorescent protein (DsRed), and confirmed by restriction enzyme digestion and sequencing. The recombinant plasmid was then transfected into BHK-21 cells via the Lipofectamine 2000 reagent; the subsequent ORF3 gene overexpression was confirmed by RT-PCR and the protein expression and location was detected by Western blotting and immunofluorescence assay.Results TThe pDsRed-Monomer-N1-ORF3 recombinant plasmid was successfully constructed. After transfection into BHK-21 ceils, the ORF3 gene was transcribed and expressed, and the ORF3 protein was mainly located in the cytoplasm, where it could react with a specific antibody.</p><p><b>CONCLUSION</b>The ORF3-DsRed fusion protein was mainly located in the cytoplasm of BHK-21 fibroblasts, and may represent a useful tool for research on the role of this protein in HEV infection.</p>

Establishment and characterization of dual-color fluorescence nude mouse models of glioma / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To establish red-green dual-color fluorescence glioma model in nude mice and to explore its practical values.</p><p><b>METHODS</b>CM-DiI-stained rat glioma C6 cells (C6-CM- DiI cells) expressing red fluorescence were inoculated into the brain of athymic nude mice expressing green fluorescence protein (NC-C57BL/6J-EGFP). Then the whole-body dual-color fluorescence imaging was detected dynamically. Finally whole brains of the tumor-bearing mice were removed and 5 µm thick serial frozen slices were made. Light microscopy, fluorescence microscopy and confocal laser scanning microscopy were performed to observe the transplanted tumor tissue structure and fluorescent cells.</p><p><b>RESULTS</b>Tumor mass with red fluorescence increased gradually under continuous in-vivo fluorescence imaging monitoring. Under the fluorescence microscope, cells with red, green and yellow fluorescence were observed in the frozen sections of transplanted tumor tissue and the mutual structural relationship among them could be defined. The tumor cells migration, implantation and cell fusion between transplanted tumor cells and host cells could be observed. It could be distinguished according to the fluorescence, that blood vessels of tumor-origin displayed red fluorescence, blood vessels of host-origin displayed green fluorescence and mosaic blood vessels appeared yellow fluorescence. It was depicted that host innate astrocytes and oligodendrocytes in the microenvironment at the tumor periphery could be activated and dedifferentiated into nestin-positive cells.</p><p><b>CONCLUSIONS</b>In contrast to traditional animal model, the dual-color fluorescence imaging of nude mouse models of glioma possesses enormous advantages in investigating tumor mass in-vivo fluorescence imaging, tumor cells migration and metastasis, tumor angiogenesis and reactive activation of host innate cells in the microenvironment at tumor periphery, thus, has highly practical application value.</p>

Genetic approach to track neural cell fate decisions using human embryonic stem cells

With their capability to undergo unlimited self-renewal and to differentiate into all cell types in the body, human embryonic stem cells (hESCs) hold great promise in human cell therapy. However, there are limited tools for easily identifying and isolating live hESC-derived cells. To track hESC-derived neural progenitor cells (NPCs), we applied homologous recombination to knock-in the mCherry gene into the Nestin locus of hESCs. This facilitated the genetic labeling of Nestin positive neural progenitor cells with mCherry. Our reporter system enables the visualization of neural induction from hESCs both in vitro (embryoid bodies) and in vivo (teratomas). This system also permits the identification of different neural subpopulations based on the intensity of our fluorescent reporter. In this context, a high level of mCherry expression showed enrichment for neural progenitors, while lower mCherry corresponded with more committed neural states. Combination of mCherry high expression with cell surface antigen staining enabled further enrichment of hESC-derived NPCs. These mCherry(+) NPCs could be expanded in culture and their differentiation resulted in a down-regulation of mCherry consistent with the loss of Nestin expression. Therefore, we have developed a fluorescent reporter system that can be used to trace neural differentiation events of hESCs.

DsRed2 transient expression in Culex quinquefasciatus mosquitoes

Culex quinquefasciatus mosquitoes have been successfully genetically modified only once, despite the efforts of several laboratories to transform and establish a stable strain. We have developed a transient gene expression method, in Culex, that delivers plasmid DNA directly to the mosquito haemolymph and additional tissues. We were able to express DsRed2 fluorescent protein in adult Cx. quinquefasciatus mosquitoes by injecting plasmids directly into their thorax. The expression of DsRed2 in adult Cx. quinquefasciatus mosquitoes is an important stepping stone to genetic transformation and the potential use of new control strategies and genetic interactions.

Avaliação da qualidade microbiológica de bebida láctea e creme de leite UAT por ATP-Bioluminescência / Evaluation of microbiological quality of UHT milk drink and UHT milk cream by ATP-Bioluminescence

Embora métodos tradicionais sejam utilizados na avaliação microbiológica de produtos UAT, metodologias rápidas, baseadas em ATP-Bioluminescência, têm sido desenvolvidas. Os resultados da aplicação dessa técnica em 54 amostras de bebida láctea UAT achocolatada e 12 de creme de leite UAT foram comparados com os resultados de métodos microbiológicos, utilizando-se diferentes meios de cultura e tempos de incubação das referidas amostras. A técnica de ATP-Bioluminescência foi aplicada por meio do sistema MLS, e os resultados foram expressos em unidades relativas de luz (RLU). Em todos os tempos de incubação - 48, 72 e 168 horas - , as amostras apresentaram contagens baixas de microrganismos mesófilos e psicrotróficos aeróbios quando analisadas em meio PCA, BHI, PetrifilmTM AC e por ATP-Bioluminescência (<150 RLU), demonstrando alta especificidade da técnica. Apenas uma amostra de creme de leite UAT apresentou contagem de mesófilos aeróbios acima do padrão estabelecido pela legislação brasileira (<100 UFC/mL) quando analisada em meio PCA (260 UFC/mL) e PetrifilmTM AC (108 UFC/mL), no tempo de 168 horas. Essa alta contagem de microrganismos mesófilos aeróbios também foi detectada pela técnica de ATP-Bioluminescência (416 RLU). Os resultados da técnica de ATP-Bioluminescência foram iguais aos resultados em meio PCA, BHI e PetrifilmTM AC.

Although traditional methods are used for the microbiological evaluation of UHT products, rapid methodologies based on ATP-Bioluminescence have been developed. The results of applying this technique in 54 samples of chocolate UHT milk drink and 12 of UHT milk cream were compared with the results of microbiological methods, using different culture media and incubation times for the referred samples. The ATP-Bioluminescence technique was applied through the MLS system and the results were expressed as relative light units (RLU). In all incubation times - 48, 72, and 168 hours - , the samples showed lower counts of mesophilic and psychrotrophic aerobic microorganisms when analyzed using PCA, BHI, PetrifilmTM AC and ATP-Bioluminescence (<150RLU), demonstrating the technique's high specificity. Only one sample of UHT milk cream showed a mesophilic aerobic count above the standard established by Brazilian legislation (<100CFU/mL) when analyzed in PCA (260 CFU/mL) and PetrifilmTM AC (108CFU/mL) at 168 hours. This high count of aerobic mesophilic microorganisms was also detected by the ATP-Bioluminescence (416 RLU) technique. The results of the ATP-Bioluminescence technique were equal to the results in PCA, BHI and PetrifilmTM AC.

Construction and preliminary applications of a Saccharomyces cerevisiae detection plasmid using for screening promoter elements / 药学学报

Synthetic biology of natural products is the design and construction of new biological systems by transferring a metabolic pathway of interest products into a chassis. Large-scale production of natural products is achieved by coordinate expression of multiple genes involved in genetic pathway of desired products. Promoters are cis-elements and play important roles in the balance of the metabolic pathways controlled by multiple genes by regulating gene expression. A detection plasmid of Saccharomyces cerevisiae was constructed based on DsRed-Monomer gene encoding for a red fluorescent protein. This plasmid was used for screening the efficient promoters applying for multiple gene-controlled pathways. First of all, eight pairs of primers specific to DsRed-Monomer gene were synthesized. The rapid cloning of DsRed-Monomer gene was performed based on step-by-step extension of a short region of the gene through a series of PCR reactions. All cloned sequences were confirmed by DNA sequencing. A vector named pEASYDs-M containing full-length DsRed-Monomer gene was constructed and was used as the template for the construction of S. cerevisiae expression vector named for pYeDP60-Ds-M. pYeDP60-Ds-M was then transformed into S. cerevisiae for heterologous expression of DsRed-Monomer gene. SDS-PAGE, Western blot and fluorescence microscopy results showed that the recombinant DsRed-Monomer protein was expressed successfully in S. cerevisiae. The well-characterized DsRed-Monomer gene was then cloned into a yeast expression vector pGBT9 to obtain a promoter detection plasmid pGBT9Red. For determination efficacy of pGBT9Red, six promoters (including four inducible promoters and two constitutive promoters) were cloned by PCR from the S. cerevisiae genome, and cloned into pGBT9Red by placing upstream of DsRed-Monomer gene, separately. The fluorescence microscopy results indicated that the six promoters (GAL1, GAL2, GAL7, GAL10, TEF2 and PGK1) can regulate the expression of DsRed-Monomer gene. The successful construction of pGBT9Red lays the foundation for further analysis of promoter activity and screening of promoter element libraries.

Applications of FRET technology in the study of mechanotransduction / 生物医学工程学杂志

Mechanical force has essential effects on cellular behaviors such as proliferation, migration and differentiation, and the mechanism of mechanotransduction is still one of the hot spots in mechanobiology study. Traditional methods could not provide accurate evaluation of the protein activation signal upon mechanical stress application. The development of fluorescence protein technology greatly promoted the understanding of mechanotransduction. In particular, genetically-encoded biosensors based on fluorescence resonance energy transfer (FRET) technique has achieved a real-time dynamic observation of living cell signal protein activity, which provides a powerful tool for the in-depth study of biomechanics. In this paper, we provide a summary on recent progress of FRET application in biomechanics. Firstly we introduce the FRET technology, and then we summarize three methods to integrate the mechanical stimulation with the FRET imaging system on cell experiments. After that, the important progress of biomechanical research on signal pathway made by FRET technology, such as cytoskeleton, Rho family, calcium and cellular physical stress visualization, are also discussed. Finally, we point out the bottleneck of the future development in FRET technology, and also make the prospect of the application of FRET in mechanotransduction. In summary, FRET technology provides a powerful tool for the studies of mechanotransduction, which will advance our systematic understanding on the molecular mechanisms about how cells respond to mechanical stimulation.

Host glial cell canceration induced by glioma stem cells in GFP/RFP dual fluorescence orthotopic glioma models in nude mice / 中华肿瘤杂志

<p><b>OBJECTIVE</b>During the process of tissue remodeling in human tumor transplantation models, the roles of the inoculated tumor cells and host tissue in tumor progression is still largely unknown. The aim of this study was to investigate the relationships and interactions between these two sides using GFP-RFP double fluorescence tracing technique.</p><p><b>METHODS</b>Red fluorescence protein (RFP) gene was stably transfected into glioma stem cell line SU3, then SU3-RFP cells were transplanted into the brain of athymic nude mice with green fluorescence protein (GFP) expression. After the intracerebral tumors were formed, the relationship and interaction between GFP cells and RFP cells were analyzed. Highly proliferative GFP cells were screened out, and monocloned with micro-pipetting. DNA content assay, chromosome banding and carcinogenicity test of the GFP cells were performed to observe the GFP cells' cancerous phenotype in nude mice.</p><p><b>RESULTS</b>In the transplantable tumor tissue, besides a great quantity of RFP cells, there were still a proportion of GFP cells and GFP/RFP fusion cells. The proportion of RFP cells, GFP cells and GFP/RFP cells were (88.99 ± 1.46)%, (5.59 ± 1.00)%, and (4.11 ± 1.020)%, respectively. Two monoclonal host GFP cells (H1 and H9) were cloned, which demonstrated the properties of immortality, loss of contact inhibition, and ultra-tetraploid when cultured in vitro. Both H1 and H9 cells expressed CNP, a specific marker of oligodendrocytes. The GFP cells also demonstrated 100% tumorigenic rate and high invasive properties in vivo.</p><p><b>CONCLUSIONS</b>In this glioma transplantation model, the transplanted tumor tissues contained not only transplanted glioma stem cells but also cancerous host GFP cells. Our findings offer important clues to further research on the relationships among different members in the tumor microenvironment.</p>