Interaction Mode between Inclusion Complex of Vitamin K3 with γ- Cyclodextrin and Herring-Sperm DNA

Methods including spectroscopy, electronic chemistry and thermodynamics were used to study the inclusion effect between γ-cyclodextrin (CD) and vitamin K₃(K₃), as well as the interaction mode between herring-sperm DNA (hsDNA) and γ-CD-K₃ inclusion complex. The results from ultraviolet spectroscopic method indicated that VK₃ and γ-CD formed 1:1 inclusion complex, with the inclusion constant K_f = 1.02 × 10⁴ L/mol, which is based on Benesi–Hildebrand's viewpoint. The outcomes from the probe method and Scatchard methods suggested that the interaction mode between γ-CD-K₃ and DNA was a mixture mode, which included intercalation and electrostatic binding effects. The binding constants were K ^θ_25°C = 2.16 × 10⁴ L/mol, and K^θ_37°C = 1.06 × 10⁴ L/mol. The thermodynamic functions of the interaction between γ-CD-K₃ and DNA were Δ_rH_m^θ = ?2.74 × 10⁴ J/mol, Δ_rS_m^θ = 174.74 J·mol^?1K^?1, therefore, both Δ_rH_m^θ (enthalpy) and Δ_rS_m^θ (entropy) worked as driven forces in this action.     

Fluorescence spectroscopy and docking study in two flavonoids,isolated tectoridin and its aglycone tectorigenin,interacting with human serum albumin: a comparison study

Two flavonoids, tectoridin (TD) isolated from rhizomes of Iris tectorum and hydrolyzed aglycone tectorigenin (TG) were prepared and characterized to compare their different interaction ability with human serum albumin (HSA). Based on the results, the affinity of TG–HSA was stronger than that of TD–HAS, and TG combined more closely with HSA than did TD. HSA fluorescence was quenched by TD/TG. The interactions between TD/TG and HSA involved static quenching. The thermodynamic parameters indicated that both binding processes were spontaneous; hydrogen binding and van der Waals force were the main forces between TD and HSA, whereas a hydrophobic interaction was the main binding force between TG and HSA. Synchronous and 3D fluorescence spectra showed that the binding of TD/TG to HSA induced conformational changes. Moreover, a docking study confirmed the experimental results. Copyright © 2015 John Wiley & Sons, Ltd.     

Construction of photodynamic‐effect immunofluorescence probes by a complex of quantum dots,immunoglobulin G and chlorin e6 and their application in HepG2 cell killing

In this study, tri‐functional immunofluorescent probes (Ce6–IgG–QDs) based on covalent combinations of quantum dots (QDs), immunoglobulin G (IgG) and chlorin e6 (Ce6) were developed and their photodynamic ability to induce the death of cancer cells was demonstrated. Strategically, one type of second‐generation photosensitizer, Ce6, was first coupled with anti‐IgG antibody using the EDC/NHS cross‐linking method to construct the photosensitive immunoconjugate Ce6–IgG. Then, a complex of Ce6–IgG–QDs immunofluorescent probes was obtained in succession by covalently coupling Ce6–IgG to water soluble CdTe QDs. The as‐manufactured Ce6–IgG–QDs maintained the bio‐activities of both the antigen–antibody‐based tumour targeting effects of IgG and the photodynamic‐related anticancer activities of Ce6. By way of polyclonal antibody interaction with rabbit anti‐human epidermal growth factor receptor (anti‐EGFR antibody, N‐terminus), Ce6–IgG–QDs were labelled indirectly onto the surface of human hepatocarcinoma (HepG2) cells in cell recognition and killing experiments. The results indicated that the Ce6–IgG–QDs probes have excellent tumour cell selectivity and higher photosensitivity in photodynamic therapy (PDT) compared with Ce6 alone, due to their antibody‐based specific recognition and location of HepG2 cells and the photodynamic effects of Ce6 killed cells based on efficient fluorescence resonance energy transfer between QDs and Ce6. Copyright © 2015 John Wiley & Sons, Ltd.     

红缘天牛成虫对杏树挥发物的生理和行为反应

为了研究红缘天牛成虫对寄主植物杏树衰弱树及木段挥发物的生理及行为反应,本文用6种挥发物,分别以不同浓度的单一组份对红缘天牛进行了电生理和行为测试;又在这6种单组份化合物对天牛行为选择表现为引诱活性的浓度范围内选择EAG值最高的浓度,以4-6种化合物等体积制成7种组合配方进行EAG和行为测试。结果表明:单一组份R-柠檬烯、反-2-己烯醛、丁酸丁酯、S-柠檬烯、异戊醇和3-蒈烯等6种单一化合物在预设的8个浓度梯度范围内,都能引起红缘天牛产生一定的生理反应,但行为选择实验的结果没有统计学意义。7种组合配方中,R4对红缘天牛成虫引诱活性最强(P0.01),引诱率达到76.67%,R1次之(P0.05),R4与R1的区别在于增加了丁酸丁酯;即丁酸丁酯在R4配方中具有明显的增效作用。     

绿僵菌及其与dsRNA混合使用对褐飞虱的防治效果

褐飞虱Nilaparvata lugens Stl是我国重要的迁飞性水稻害虫,本文研究了金龟子绿僵菌Metarhizium anisopliae及其与dsRNA混合使用对褐飞虱的防治效果。绿僵菌悬浮液1.6×108孢子/m L至8×106孢子/m L对褐飞虱2龄、4龄和成虫进行喷药,发现1.6×107孢子/m L对各个虫态虫龄均有良好致死效果,并且成虫和4龄若虫均好于2龄若虫。在交配行为上来看,绿僵菌处理过的褐飞虱成虫活跃度非常低,从配对开始一直到交配结束的各个阶段都受到明显影响,处理组3 h的交配率只有3.70%,而对照组的交配率为24.44%。还把褐飞虱几丁质合成酶基因A的dsRNA与绿僵菌混合使用防治褐飞虱2龄和4龄若虫,结果表明0.5μg/μL ds CHSA与绿僵菌混合使用的防治效果最好,2龄若虫的死亡率为89.63%,4龄若虫的死亡率达到93.94%。而0.2μg/μL ds CHSA与绿僵菌的混合,对2龄和4龄若虫的致死率为65.56%-76.52%。研究结果为褐飞虱的生物防治提供了新的思路。     

Immobilized Metal Affinity Chromatography Coupled to Multiple Reaction Monitoring Enables Reproducible Quantification of Phospho-signaling

A major goal in cell signaling research is the quantification of phosphorylation pharmacodynamics following perturbations. Traditional methods of studying cellular phospho-signaling measure one analyte at a time with poor standardization, rendering them inadequate for interrogating network biology and contributing to the irreproducibility of preclinical research. In this study, we test the feasibility of circumventing these issues by coupling immobilized metal affinity chromatography (IMAC)-based enrichment of phosphopeptides with targeted, multiple reaction monitoring (MRM) mass spectrometry to achieve precise, specific, standardized, multiplex quantification of phospho-signaling responses. A multiplex immobilized metal affinity chromatography- multiple reaction monitoring assay targeting phospho-analytes responsive to DNA damage was configured, analytically characterized, and deployed to generate phospho-pharmacodynamic curves from primary and immortalized human cells experiencing genotoxic stress. The multiplexed assays demonstrated linear ranges of ≥3 orders of magnitude, median lower limit of quantification of 0.64 fmol on column, median intra-assay variability of 9.3%, median inter-assay variability of 12.7%, and median total CV of 16.0%. The multiplex immobilized metal affinity chromatography- multiple reaction monitoring assay enabled robust quantification of 107 DNA damage-responsive phosphosites from human cells following DNA damage. The assays have been made publicly available as a resource to the community. The approach is generally applicable, enabling wide interrogation of signaling networks.Cell signaling research is faced with the challenging task of interrogating increasingly large numbers of analytes in “systems biology” approaches, while maintaining the high standards of integrity and reproducibility traditionally associated with the scientific approach. For example, studies interrogating complex systems, such as protein signaling networks, require quantification technologies capable of sensitive, specific, multiplexable, and reproducible application. However, recent reports have highlighted alarmingly high rates of irreproducibility in fundamental biological and pre-clinical studies (1, 2), as well as poor performance of affinity reagents used in traditional proteomic assay and detection platforms (3, 4). There is an imminent need for high quality assays, including highly characterized standards and detailed documentation of processes and procedures (5). To improve the translation of cell signaling discoveries into clinical application, we need reproducible and transferable technologies that enable higher throughput quantification of protein phosphorylation.Signaling dynamics through post-translational modifications (e.g. phosphorylation) are predominantly measured by Western blotting. Although this technique has led to many discoveries and is the de facto “gold standard,” it suffers from many drawbacks. Western blotting is a low throughput approach applied to individual analytes (i.e. no multiplexing) and is susceptible to erroneous interpretation when applied quantitatively (6). Alternative immunoassay platforms have emerged (e.g. immunohistochemistry, ELISA, mass cytometry, and bead-based or planar arrays), but suffer from similar limitations, namely specificity issues (because of cross-reactivity of antibodies), poor standardization, and difficulties in multiplexing.One alternative for quantifying phosphorylation is targeted, multiple reaction monitoring (MRM)¹ MS, a widely deployed technique in clinical laboratories for quantification of small molecules (7, 8). MRM is now also well established for precise and specific quantification of endogenous, proteotypic peptides relative to spiked-in stable isotope-labeled internal standards (9–11), and MRM can be applied to phosphopeptides (12–18). MRM assays can be run at high multiplex levels (19–21) and can be standardized to be highly reproducible across laboratories (22–24), even on an international stage (25). Because phosphorylation typically occurs at sub-stoichiometric levels and because phosphopeptides must compete for ionization with more abundant peptides, mass spectrometry-based analysis of phosphorylation requires an analyte enrichment step. Immuno-affinity enrichment approaches using anti-phospho-tyrosine antibodies (26) or panels of antibodies targeting signaling nodes (27) have been implemented with shotgun mass spectrometry. Although anti-peptide antibodies can also be used to enrich individual phosphopeptides upstream of MRM (28), the generation of these reagents is time-consuming and costly, limiting widespread uptake.Phosphopeptide enrichment based on metal affinity chromatography has recently matured into a reproducible approach (29). Immobilized metal affinity chromatography (IMAC) is widely used in discovery phosphoproteomic studies to enrich phosphopeptides upstream of shotgun-based mass spectrometry (30, 31). We hypothesized that a subset of the cellular phosphoproteome with favorable binding characteristics to the IMAC resin might be reproducibly recovered for quantification when coupled with quantitative MRM mass spectrometry, enabling robust IMAC-MRM assays without the need for an antibody.In this report, we: (1) demonstrate the feasibility of generating analytically robust, multiplex IMAC-MRM assays for quantifying cellular phospho-signaling, (2) present a semi-automated, 96-well format magnetic bead-based protocol for IMAC enrichment, (3) provide a catalogue of phosphopeptides that are highly amenable to IMAC-MRM quantification, and (4) make publicly available standard operating protocols (SOP) and fit-for-purpose analytical validation data for IMAC-MRM assays targeting 107 phospho-analytes, providing a community resource for study of the DNA damage response. The data suggest that the IMAC-MRM approach is generally applicable to signaling pathways, enabling wider interrogation of signaling networks.     

N-terminal Huntingtin Knock-In Mice: Implications of Removing the N-terminal Region of Huntingtin for Therapy

The Huntington’s disease (HD) protein, huntingtin (HTT), is a large protein consisting of 3144 amino acids and has conserved N-terminal sequences that are followed by a polyglutamine (polyQ) repeat. Loss of Htt is known to cause embryonic lethality in mice, whereas polyQ expansion leads to adult neuronal degeneration. Whether N-terminal HTT is essential for neuronal development or contributes only to late-onset neurodegeneration remains unknown. We established HTT knock-in mice (N160Q-KI) expressing the first 208 amino acids of HTT with 160Q, and they show age-dependent HTT aggregates in the brain and neurological phenotypes. Importantly, the N-terminal mutant HTT also preferentially accumulates in the striatum, the brain region most affected in HD, indicating the importance of N-terminal HTT in selective neuropathology. That said, homozygous N160Q-KI mice are also embryonic lethal, suggesting that N-terminal HTT alone is unable to support embryonic development. Using Htt knockout neurons, we found that loss of Htt selectively affects the survival of developing neuronal cells, but not astrocytes, in culture. This neuronal degeneration could be rescued by a truncated HTT lacking the first 237 amino acids, but not by N-terminal HTT (1–208 amino acids). Also, the rescue effect depends on the region in HTT known to be involved in intracellular trafficking. Thus, the N-terminal HTT region may not be essential for the survival of developing neurons, but when carrying a large polyQ repeat, can cause selective neuropathology. These findings imply a possible therapeutic benefit of removing the N-terminal region of HTT containing the polyQ repeat to treat the neurodegeneration in HD.     

High glucose concentration induces endothelial cell proliferation by regulating cyclin‐D2‐related miR‐98

Cyclin D2 is involved in the pathology of vascular complications of type 2 diabetes mellitus (T2DM). This study investigated the role of cyclin‐D2‐regulated miRNAs in endothelial cell proliferation of T2DM. Results showed that higher glucose concentration (4.5 g/l) significantly promoted the proliferation of rat aortic endothelial cells (RAOECs), and significantly increased the expression of cyclin D2 and phosphorylation of retinoblastoma 1 (p‐RB1) in RAOECs compared with those under low glucose concentration. The cyclin D2‐3′ untranslated region is targeted by miR‐98, as demonstrated by miRNA analysis software. Western blot also confirmed that cyclin D2 and p‐RB1 expression was regulated by miR‐98. The results indicated that miR‐98 treatment can induce RAOEC apoptosis. The suppression of RAOEC growth by miR‐98 might be related to regulation of Bcl‐2, Bax and Caspase 9 expression. Furthermore, the expression levels of miR‐98 decreased in 4.5 g/l glucose‐treated cells compared with those treated by low glucose concentration. Similarly, the expression of miR‐98 significantly decreased in aortas of established streptozotocin (STZ)‐induced diabetic rat model compared with that in control rats; but cyclin D2 and p‐RB1 levels remarkably increased in aortas of STZ‐induced diabetic rats compared with those in healthy control rats. In conclusion, this study demonstrated that high glucose concentration induces cyclin D2 up‐regulation and miR‐98 down‐regulation in the RAOECs. By regulating cyclin D2, miR‐98 can inhibit human endothelial cell growth, thereby providing novel therapeutic targets for vascular complication of T2DM.     

Role of long non‐coding RNA MIAT in proliferation,apoptosis and migration of lens epithelial cells: a clinical and in vitro study

Age‐related cataract is among the most common chronic disorders of ageing and is the world's leading blinding disorder. Long non‐coding RNAs play important roles in several biological processes and complicated diseases. However, the role of lncRNAs in the setting of cataract is still unknown. Here, we extracted total RNAs from the transparent and age‐matched cataractous human lenses, and determined lncRNA expression profiles using microarray analysis. We found that 38 lncRNAs were differentially expressed between transparent and cataractous lenses. 17 of 20 differentially expressed lncRNAs were further verified by quantitative RT‐PCRs. One top abundant lncRNA, MIAT, was specifically up‐regulated both in the plasma fraction of whole blood and aqueous humor of cataract patients. MIAT knockdown could affect the proliferation, apoptosis and migration of Human lens epithelial cells (HLECs) upon oxidative stress. Posterior capsule opacification (PCO) is a common complication of cataract surgery, which is associated with abnormal production of inflammatory factors. MIAT knockdown could repress tumour necrosis factor‐α‐induced abnormal proliferation and migration of HLECs, suggesting a potential role of MIAT in PCO‐related pathological process. Moreover, we found that MIAT acted as a ceRNA, and formed a feedback loop with Akt and miR‐150‐5p to regulate HLEC function. Collectively, this study provides a novel insight into the pathogenesis of age‐related cataract.     

Functional TRPV2 and TRPV4 channels in human cardiac c‐kit+ progenitor cells

The cellular physiology and biology of human cardiac c‐kit⁺ progenitor cells has not been extensively characterized and remains an area of active research. This study investigates the functional expression of transient receptor potential vanilloid (TRPV) and possible roles for this ion channel in regulating proliferation and migration of human cardiac c‐kit⁺ progenitor cells. We found that genes coding for TRPV2 and TRPV4 channels and their proteins are significantly expressed in human c‐kit⁺ cardiac stem cells. Probenecid, an activator of TRPV2, induced an increase in intracellular Ca²⁺ (Ca²⁺_i), an effect that may be attenuated or abolished by the TRPV2 blocker ruthenium red. The TRPV4 channel activator 4α‐phorbol 12‐13‐dicaprinate induced Ca²⁺_i oscillations, which can be inhibited by the TRPV4 blocker RN‐1734. The alteration of Ca²⁺_i by probenecid or 4α‐phorbol 12‐13‐dicprinate was dramatically inhibited in cells infected with TRPV2 short hairpin RNA (shRNA) or TRPV4 shRNA. Silencing TRPV2, but not TRPV4, significantly reduced cell proliferation by arresting cells at the G0/G1 boundary of the cell cycle. Cell migration was reduced by silencing TRPV2 or TRPV4. Western blot revealed that silencing TRPV2 decreased expression of cyclin D1, cyclin E, pERK1/2 and pAkt, whereas silencing TRPV4 only reduced pAkt expression. Our results demonstrate for the first time that functional TRPV2 and TRPV4 channels are abundantly expressed in human cardiac c‐kit⁺ progenitor cells. TRPV2 channels, but not TRPV4 channels, participate in regulating cell cycle progression; moreover, both TRPV2 and TRPV4 are involved in migration of human cardiac c‐kit⁺ progenitor cells.