Improvement of Opal Multiplex Immunofluorescence Workflow for Human Tissue Sections

The Opal multiplex technique is an established methodology for the detection of multiple biomarkers in one section. The protocol encompasses iterative single stainings and heating-mediated removal of the primary and secondary antibodies after each staining round, leaving untouched the Opal fluorophores which are deposited onto the antigen of interest. According to our experience, repetitive heating of skin sections often results in tissue damage, indicating an urgent need for milder alternatives to strip immunoglobulins. In this study, we demonstrate that considerable heating-related damage was found not only in skin but also in tissues of different origin, mostly characterized by low cell density. Importantly, the morphology remained fully intact when sections were repetitively exposed to β-mercaptoethanol-containing stripping buffer instead of multiple heating cycles. However, target epitopes appeared sensitive at a differential degree to multiple treatments with stripping buffer, as shown by loss in staining intensity, but in all cases, the staining intensity could be restored by increment of the primary antibody concentrations. Application of β-mercaptoethanol-containing stripping buffer instead of heating for antibody removal markedly improved the quality of the Opal multiplex technique, as a substantial higher number of differently colored cells could be visualized within a well-conserved morphological context:     

GC32细胞对三种虫媒病毒的敏感性研究

用胃癌细胞株ＧＣ３２,对乙型脑炎、基孔肯雅、兰加特病毒进行敏感性研究。并用ＢＨＫ２１细胞作对照,发现ＧＣ３２细胞对两种病毒的敏感性与对照细胞ＢＨＫ２１极为接近。ＢＨＫ２１和ＧＣ３２细胞对基孔肯雅、乙型脑炎和兰加特的免疫荧光实验,于感染后４８ｈ或７２ｈ都出现＋＋＋～＋＋＋＋的阳性结果。两种细胞对基孔肯雅和乙型脑炎病毒都形成空斑,ＢＨＫ２１细胞对基孔肯雅和乙型脑炎病毒的空斑形成单位分别为５．６５和５．３６;ＧＣ３２对基孔肯雅和乙型脑炎病毒的空斑形成单位分别为６．４８和５．６１。实验表明,ＧＣ３２细胞可以作为有关病毒实验的理想细胞株。     

Detailed analysis of inflammatory and neuromodulatory cytokine secretion from human NT2 astrocytes using multiplex bead array

Astrocytes are a very important cell type in the brain fulfilling roles in both neuroimmunology and neurotransmission. We have conducted the most comprehensive analysis of secreted cytokines conducted to date (astrocytes of any source) to determine whether astrocytes derived from the human Ntera2 (NT2) cell-line are a good model of human primary astrocytes. We have compared the secretion of cytokines from NT2 astrocytes with those produced in astrocyte enriched human brain cultures and additional cytokines implicated in brain injury or known to be expressed in the human brain. The concentration of cytokines was measured in astrocyte conditioned media using multiplex bead array (MBA), where 18 cytokines were measured simultaneously. Resting NT2 astrocytes produced low levels (～1-30 pg/ml) of MIP1α, IL-6 and GM-CSF and higher levels of MCP-1, IP-10 and IL-8 (1-11 ng/ml) under non-inflammatory conditions. All of these in addition to IL-1β, TNFα, and IL-13, were increased by pro-inflammatory activation (TNFα or IL-1β stimulation). In contrast, IL-2, IL-4, IL-5, IL-7, IL-10, IL-12, LTα, and IFNγ were not detected in astrocyte conditioned media under any of the culture conditions tested. NT2 astrocytes were unresponsive to IL-2 and the adenyl cyclase agonist, forskolin. Interestingly, IFNγ stimulation selectively increased IP-10 secretion only. As astrocytes stimulated with IL-1β or TNFα produced several chemokines in the ng/ml range, we next assessed the chemoattractant properties of these cells. Conditioned media from TNFα-stimulated astrocytes significantly chemoattracted leukocytes from human blood. This study provides the most comprehensive analysis of cytokine production by human astrocytes thus far, and shows that NT2 astrocytes are highly responsive to pro-inflammatory mediators including TNFα and IL-1β, producing cytokines and chemokines capable of attracting leukocytes from human blood. We conclude that in the absence of adult human primary astrocytes that NT2-astrocytes may provide a valuable alternative to study the immunological behaviour of human astrocytes.     

Hemagglutinin of Influenza Virus Partitions into the Nonraft Domain of Model Membranes

The HA of influenza virus is a paradigm for a transmembrane protein thought to be associated with membrane-rafts, liquid-ordered like nanodomains of the plasma membrane enriched in cholesterol, glycosphingolipids, and saturated phospholipids. Due to their submicron size in cells, rafts can not be visualized directly and raft-association of HA was hitherto analyzed by indirect methods. In this study, we have used GUVs and GPMVs, showing liquid disordered and liquid ordered domains, to directly visualize partition of HA by fluorescence microscopy. We show that HA is exclusively (GUVs) or predominantly (GPMVs) present in the liquid disordered domain, regardless of whether authentic HA or domains containing its raft targeting signals were reconstituted into model membranes. The preferential partition of HA into ld domains and the difference between lo partition in GUV and GPMV are discussed with respect to differences in packaging of lipids in membranes of model systems and living cells suggesting that physical properties of lipid domains in biological membranes are tightly regulated by protein-lipid interactions.     

Combined hydrophobic-metal binding fusion tags for applications in aqueous two-phase partitioning

In this work, we studied the influence of fusion affinity tags containing both hydrophobic and histidines residues on the partitioning of the green fluorescent protein, GFPuv, in aqueous two-phase system. The tags were fused to the N-terminal of GFPuv and tested by immobilized metal affinity partitioning, in a PEG/salt system. The presence of both types of residues in the tag increased the partitioning greatly. Particularly, four engineered tags (H6, FH6, WH6, and YH6) containing a hexa-histidine sequence as well as different hydrophobic residues, all increased partitioning more than twice, reaching K values around 20, as compared to another construct (His6-GFP) containing an isolated hexa-histidine sequence. YH6, also proved be beneficial for protein expression.     

Efficient generation of transgenic chickens using the spermatogonial stem cells in vivo and ex vivo transfection

The highly efficient novel methods to produce transgenic chickens were established by directly in-jecting the recombinant plasmid containing green fluorescent protein (GFP) gene into the cock's testis termed as testis-medianted gene transfer (TMGT), and transplanting transfected spermatogonial stem cells (TTSSCs). For the TMGT approach,four dosages of pEGFP-N1 DNA/cationic polymer complex were injected intratesticularly. The results showed: (1) 48 h after the injection,the percentages of testis cells expressing GFP were 4.0%, 8.7%, 10.2% and 13.6% in the 50, 100, 150 and 200 μg/mL group, re-spectively. The difference from the four dosage groups was significant (P<0.05). On day 25 after the injection, a dosage-dependent and time-dependent increase in the number of transgenic sperm was observed. The percentages of gene expression reached the summit and became stable from day 70 to 160, being 12.7%, 12.8%, 15.9% and 19.1%, respectively. The difference from the four dosage groups was also significant (P<0.05). (2) 70 d after the injection, strong green fluorescent could be observed in the seminiferous tubules by whole-mount in-situ hybridization. (3) 70 d after the injection, the semen was collected and used to artificially inseminate wild-type females. The blastoderms of F1 and F2 transgenic chicken expressed GFP were 56.2% (254/452) and 53.2% (275/517), respectively. The detec-tion of polymerase chain reaction (PCR) of F1 and F2 transgenic chicken blood genomic DNA showed that 56.5% (3/23) of F1 and 52.9% (9/17) of F2 were positive. Southern blot showed GFP DNA was in-serted in their genomic DNAs. (4) Frozen whole mount tissue sections of F1 and F2 transgenic chicken liver, heart, kidney and muscle showed that the rates of green fluorescent positive were between 50.0% and 66.7%. (5) With the TTSSCs method, SSCs ex vivo transfected with GFP were transplanted into recipient roosters whose endogenic SSCs had been resoluted. The donor SSCs settled and GFP ex-pression became readily detectable in the frozen whole mount tissue sections of recepient testes. Moreover, sperms carrying GFP could be produced normally. The results of artificially inseminating wild-type females with these sperms showed 12.5% (8/64) of offspring embryo expressed GFP and 11.1% (2/18) hatched chicks were tested transgenic. Our data therefore suggest TMGT and TTSSCs are the feasible methods for the generation of transgenic chickens.     

The Tiam1 PDZ Domain Couples to Syndecan1 and Promotes Cell-Matrix Adhesion

The T-cell lymphoma invasion and metastasis gene 1 (Tiam1) is a guanine exchange factor (GEF) for the Rho-family GTPase Rac1 that is crucial for the integrity of adherens junctions, tight junctions, and cell-matrix interactions. This GEF contains several protein-protein interaction domains, including a PDZ domain. Earlier studies identified a consensus PDZ-binding motif and a synthetic peptide capable of binding to the Tiam1 PDZ domain, but little is known about its ligand specificity and physiological role in cells. Here, we investigated the structure, specificity, and function of the Tiam1 PDZ domain. We determined the crystal structures of the Tiam1 PDZ domain free and in complex with a “model” peptide, which revealed the structural basis for ligand specificity. Protein database searches using the consensus PDZ-binding motif identified two eukaryotic cell adhesion proteins, Syndecan1 and Caspr4, as potential Tiam1 PDZ domain binding proteins. Equilibrium binding experiments confirmed that C-terminal peptides derived from Syndecan1 and Caspr4 bound the Tiam1 PDZ domain. NMR chemical shift perturbation experiments indicated that the Tiam1 PDZ/Syndecan1 and PDZ/Caspr4 complexes were structurally distinct and identified key residues likely to be responsible for ligand selectivity. Moreover, cell biological analysis established that Syndecan1 is a physiological binding partner of Tiam1 and that the PDZ domain has a function in cell-matrix adhesion and cell migration. Collectively, our data provide insight into the structure, specificity, and function of the Tiam1 PDZ domain. Importantly, our data report on a physiological role for the Tiam1 PDZ domain and establish a novel link between two previously unrelated signal transduction pathways, both of which are implicated in cancer.     

A nucleolar protein RRS1 contributes to chromosome congression

We report here the functional analysis of human Regulator of Ribosome Synthesis 1 (RRS1) protein during mitosis. We demonstrate that RRS1 localizes in the nucleolus during interphase and is distributed at the chromosome periphery during mitosis. RNA interference experiments revealed that RRS1-depleted cells show abnormalities in chromosome alignment and spindle organization, which result in mitotic delay. RRS1 knockdown also perturbs the centromeric localization of Shugoshin 1 and results in premature separation of sister chromatids. Our results suggest that a nucleolar protein RRS1 contributes to chromosome congression.     

Characteristics of sugarcane white leaf phytoplasma transmission by the leafhopper Matsumuratettix hiroglyphicus

The leafhopper Matsumuratettix hiroglyphicus (Matsumura) (Hemiptera: Cicadellidae) is the most important vector of sugarcane white leaf (SCWL) phytoplasma that significantly affects the sugarcane crop in Asia. Here, we aimed to study the characteristics of SCWL phytoplasma transmission by M. hiroglyphicus. To this end, the stylet penetration activities performed during the acquisition access period (AAP) and inoculation access period (IAP) were investigated by the direct current electrical penetration graph technique and confirmed by quantitative polymerase chain reaction (qPCR). Additionally, the latent period (LP) of SCWL phytoplasma in the vector was determined by qPCR and localised by fluorescent in situ hybridisation. The results indicated that the acquisition of SCWL phytoplasma occurred during phloem ingestion (waveform D), whereas its inoculation was associated with salivation into the phloem sieve element (waveform C). The minimum AAP was 15 min and the minimum duration of phloem ingestion was 2.35 min. The minimum LP of SCWL phytoplasma in the vector was at least 14 days; then, SCWL phytoplasma moved to the salivary glands of the insect, enabling the transmission of the pathogen to the host plants. The minimum IAP for a successful transmission of SCWL phytoplasma to the host plants was 11–12 min, with a minimum duration of salivation into phloem of 1.35 min. The female vectors had higher SCWL phytoplasma copy numbers than the male vectors, and displayed faster AAP, IAP, and LP. Overall, our findings provide important information related to the feeding behaviour of M. hiroglyphicus and its effect on the transmission of SCWL phytoplasma.