Targeted Delivery of an Activatable Fluorescent Probe for the Detection of Furin Activity in Living Cells

Furin, a protein convertase, plays a crucial role in the progression of tumors. In this work, a new fluorescent probe consisting of a peptide, Arg‐Val‐Arg‐Arg (RVRR), and an aminoluciferin fluorophore was designed and prepared for the responsive and activatable detection of furin activity in vitro and in living cells. We demonstrated that this probe could be responsive toward furin with an “off–on” florescence signal and generated an approximately 3.58‐fold enhancement in the fluorescence intensity in vitro. Fluorescence imaging in MDA‐MB‐468 and LoVo cells showed that the probe could be cleaved by overexpressed furin with fluorescence turn‐on in MDA‐MB‐468 cells, and this probe was also found to be capable of discriminating between furin‐overexpressing and furin‐deficient cell lines. Our research indicates that this probe has great potential for the detection of furin activity in living cells.     

无线传感器网络在温室监控中的应用

采用无线传感器网络对温室进行监控,用同一套网络分别完成风、光、水、电、热和农药等的数据采集和环境控制,可有效提高农业集约化生产程度,简化系统复杂性,降低设备成本。     

Optimization of Caged Electrophiles for Improved Monitoring of Cysteine Reactivity in Living Cells

Cysteine residues play critical roles in protein function and are susceptible to numerous post‐translational modifications (PTMs) that serve to modulate the activity and localization of diverse proteins. Many of these PTMs are highly transient and labile, thus necessitating methods to study these modifications directly within the context of living cells. We previously reported a caged electrophilic probe, CBK1, that can be activated by UV for temporally controlled covalent modification of cysteine residues in living cells. To improve upon the number of cysteine residues identified in cellular cysteine‐profiling studies, the reactivity and uncaging efficiency of a panel of caged electrophiles were explored. We identified an optimized caged electrophilic probe, CIK4, that affords significantly improved coverage of cellular cysteine residues. The broader proteome coverage afforded by CIK4 renders it a useful tool for the biological investigation of cysteine‐reactivity changes and PTMs directly within living cells and highlights design elements that are critical to optimizing photoactivatable chemical probes for cellular labeling.     

Viral Transduction of Human Rod Opsin or Channelrhodopsin Variants to Mouse ON Bipolar Cells Does Not Impact Retinal Anatomy or Cause Measurable Death in the Targeted Cells

The viral gene delivery of optogenetic actuators to the surviving inner retina has been proposed as a strategy for restoring vision in advanced retinal degeneration. We investigated the safety of ectopic expression of human rod opsin (hRHO), and two channelrhodopsins (enhanced sensitivity CoChR-3M and red-shifted ReaChR) by viral gene delivery in ON bipolar cells of the mouse retina. Adult Grm6^Cre mice were bred to be retinally degenerate or non-retinally degenerate (homozygous and heterozygous for the rd1^Pde6b mutation, respectively) and intravitreally injected with recombinant adeno-associated virus AAV2/2(quad Y-F) serotype containing a double-floxed inverted transgene comprising one of the opsins of interest under a CMV promoter. None of the opsins investigated caused changes in retinal thickness; induced apoptosis in the retina or in transgene expressing cells; or reduced expression of PKCα (a specific bipolar cell marker). No increase in retinal inflammation at the level of gene expression (IBA1/AIF1) was found within the treated mice compared to controls. The expression of hRHO, CoChR or ReaChR under a strong constitutive promoter in retinal ON bipolar cells following intravitreal delivery via AAV2 does not cause either gross changes in retinal health, or have a measurable impact on the survival of targeted cells.     

Biomimetic Assembly of a Polydopamine Layer on Graphene as an Electron Gate for Fluorescent MicroRNA Detection in Living Cells

A novel strategy was developed for microRNA detection based on the fluorescence quenching of polydopamine (PDA)-coated reduced graphene oxide (RGO) nanosheets (RGO@PDA). Compared with graphene oxide (GO), the reduction of GO and modification of the surface of RGO by PDA not only improve the stability, dispersity, biocompatibility, and cellular uptake without degeneration of the unique electronic properties of graphene, but also add an electron gate for harvesting electrons, as well as enabling efficient and forward electron transfer to avoid unwanted electron transfer and realize highly sensitive miRNA detection; thus a lower detection limit can be achieved in this sensing system. Remarkably, nanoprobes consisting of RGO@PDA and fluorescein-labeled single-stranded DNA can naturally enter cancer cells without the aid of transfection agents, as well as resisting enzymatic lysis and showing almost no effect on the cell viability. More importantly, intense and time-dependent fluorescence responses were observed from the important tumor marker microRNA-21 (miR-21) in living cells; thus suggesting that the proposed sensing platform shows great promise for applications in disease diagnosis and fundamental research into biochemistry.     

Multiple Parallel Fusion Network for Predicting Protein Subcellular Localization from Stimulated Raman Scattering (SRS) Microscopy Images in Living Cells

Stimulated Raman Scattering Microscopy (SRS) is a powerful tool for label-free detailed recognition and investigation of the cellular and subcellular structures of living cells. Determining subcellular protein localization from the cell level of SRS images is one of the basic goals of cell biology, which can not only provide useful clues for their functions and biological processes but also help to determine the priority and select the appropriate target for drug development. However, the bottleneck in predicting subcellular protein locations of SRS cell imaging lies in modeling complicated relationships concealed beneath the original cell imaging data owing to the spectral overlap information from different protein molecules. In this work, a multiple parallel fusion network, MPFnetwork, is proposed to study the subcellular locations from SRS images. This model used a multiple parallel fusion model to construct feature representations and combined multiple nonlinear decomposing algorithms as the automated subcellular detection method. Our experimental results showed that the MPFnetwork could achieve over 0.93 dice correlation between estimated and true fractions on SRS lung cancer cell datasets. In addition, we applied the MPFnetwork method to cell images for label-free prediction of several different subcellular components simultaneously, rather than using several fluorescent labels. These results open up a new method for the time-resolved study of subcellular components in different cells, especially cancer cells.     

BP神经网络分光光度法在多组分体系分析中的应用研究

介绍了BP神经网络的基本原理，用BP神经网络分光光度法解决了多组分体系中各组分曲线相互重叠的问题，不需要用物理的、化学方法进行分离或掩蔽而测定出各组分的相对含量，具有简便、准确的优点。     

Cellular Response to Individual Components of the Platelet Concentrate

Platelet concentrates and especially their further product platelet lysate, are widely used as a replacement for cell culturing. Platelets contain a broad spectrum of growth factors and bioactive molecules that affect cellular fate. However, the cellular response to individual components of the human platelet concentrate is still unclear. The aim of this study was to observe cellular behavior according to the individual components of platelet concentrates. The bioactive molecule content was determined. The cells were supplemented with a medium containing 8% (v/v) of platelet proteins in plasma, pure platelet proteins in deionized water, and pure plasma. The results showed a higher concentration of fibrinogen, albumin, insulin growth factor I (IGF-1), keratinocyte growth factor (KGF), and hepatocyte growth factor (HGF), in the groups containing plasma. On the other hand, chemokine RANTES and platelet-derived growth factor bb (PDGF-bb), were higher in the groups containing platelet proteins. The groups containing both plasma and plasma proteins showed the most pronounced proliferation and viability of mesenchymal stem cells and fibroblasts. The platelet proteins alone were not sufficient to provide optimal cell growth and viability. A synergic effect of platelet proteins and plasma was observed. The data indicated the importance of plasma in platelet lysate for cell growth.     

Bovine Induced Pluripotent Stem Cells Are More Resistant to Apoptosis than Testicular Cells in Response to Mono-(2-ethylhexyl) Phthalate

Although the androgen receptor (AR) has been implicated in the promotion of apoptosis in testicular cells (TSCs), the molecular pathway underlying AR-mediated apoptosis and its sensitivity to environmental hormones in TSCs and induced pluripotent stem cells (iPSCs) remain unclear. We generated the iPSCs from bovine TSCs via the electroporation of OCT4. The established iPSCs were supplemented with leukemia inhibitory factor and bone morphogenetic protein 4 to maintain and stabilize the expression of stemness genes and their pluripotency. Apoptosis signaling was assessed after exposure to mono-(2-ethylhexyl) phthalate (MEHP), the active metabolite of di-(2-ethylhexyl) phthalate. Here, we report that iPSCs were more resistant to MEHP-induced apoptosis than were original TSCs. MEHP also repressed the expression of AR and inactivated WNT signaling, and then led to the commitment of cells to apoptosis via the cyclin dependent kinase inhibitor p21^CIP1. The loss of the frizzed receptor 7 and the gain of p21^CIP were responsible for the stimulatory effect of MEHP on AR-mediated apoptosis. Our results suggest that testicular iPSCs can be used to study the signaling pathways involved in the response to environmental disruptors, and to assess the toxicity of environmental endocrine disruptors in terms of the maintenance of stemness and pluripotency.     

Strategies for Site-Specific Labeling of Receptor Proteins on the Surfaces of Living Cells by Using Genetically Encoded Peptide Tags

Fluorescence microscopy imaging enables receptor proteins to be investigated within their biological context. A key challenge is to site-specifically incorporate reporter moieties into proteins without interfering with biological functions or cellular networks. Small peptide tags offer the opportunity to combine inducible labeling with small tag sizes that avoid receptor perturbation. Herein, we review the current state of live-cell labeling of peptide-tagged cell-surface proteins. Considering their importance as targets in medicinal chemistry, we focus on membrane receptors such as G protein-coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs). We discuss peptide tags that i) are subject to enzyme-mediated modification reactions, ii) guide the complementation of reporter proteins, iii) form coiled-coil complexes, and iv) interact with metal complexes. Given our own contributions in the field, we place emphasis on peptide-templated labeling chemistry.     

Zearalenone-Induced Mechanical Damage of Intestinal Barrier via the RhoA/ROCK Signaling Pathway in IPEC-J2 Cells

Zearalenone (ZEN) is a widespread contaminant of cereals and agricultural products which causes food safety issues. Ingesting food or feed contaminated with ZEN can disrupt the intestinal epithelial barrier function. The RhoA/ROCK signaling pathway plays a key role in regulating the epithelial barrier function, but studies on such roles have rarely focused on the intestine. The aim of this experiment was to investigate the exact mechanism of ZEN-induced intestinal barrier damage and whether the RhoA/ROCK signaling pathway is involved. The results showed that ZEN significantly induced alkaline phosphatase (AP) activity and FITC–dextran (4 kDa) passage across the epithelial barrier, which significantly reduced the transepithelial resistance (TEER). Meanwhile, ZEN could induce the significantly down-regulated mRNA expression of tight junction proteins (occludin, claudin-1, ZO-1, and claudin-3) and redistribution of ZO-1 immunofluorescence. Further studies demonstrated that ZEN exposure activated the RhoA/ROCK signaling pathway, significantly up-regulated the mRNA expression of ROCK1, the main effector of the signaling pathway, the protein expression of phosphorylated myosin light chain (MLC) and myosin light chain kinase (MLCK), and relatively increased the activity of ATP in cells, simultaneously remodeling the cytoskeleton (F-actin). Overall, our study indicated that ZEN induced intestinal barrier dysfunction by activating the RhoA/ROCK signaling pathway.     

Response of Mythimna unipuncta Males to Components of the Sesamia nonagrioides Pheromone

Several sympatric lepidopteran species feed on maize plants, and the different components of their species-specific female sex pheromones may play a role in attracting conspecifics and/or deter heterospecific males. In this study, we analyzed the content of Mythimna unipuncta pheromone glands and tested the response of males to components of their own pheromone blend and that of Sesamia nonagrioides in the wind tunnel. Whole pheromone glands, and lures where (Z)-9-hexadecenyl acetate, Z)-11-hexadecen-1-ol or (Z)-9-hexadecenyl acetate + (Z)-11-hexadecen-1-ol were added to the major component, (Z)-11-hexadecenyl acetate, elicited significantly higher responses by M. unipuncta males than lures with main component alone, although the levels varied with concentration. In the field a rather different outcome was observed, as the addition of other compounds found in the female pheromone gland did not improve trap catch over lures with only (Z)-11-hexadecenyl acetate. The addition of (Z)-11-hexadecenal, a compound of the S. nonagrioides pheromone, to (Z)-11-hexadecenyl acetate significantly reduced attraction of M. unipuncta males both in the wind tunnel and in the field, as well as the number of sympatric clover cutworm, Discestra trifolii, under field conditions. The addition of (Z)-9-hexadecenyl acetate, a minor component of the M. unipuncta pheromone blend, reduced the number of S. nonagrioides captured in field traps that were baited with the S. nonagrioides lure. The significance of such inhibition in the reproductive isolation of sympatric species that attack maize is discussed.     

小波分析及其在化工信号分析处理中的应用及展望

介绍了小波变换的基本理论和常用方法，小波变换在信号处理方面的应用进展。认为小波变换的时一频局部化的性质，使其成为信号处理的强有力的工具。总结了在化学工程领域小波分析方法在信号除噪、系统特征提取、奇异性检测、故障诊断、预测等方面的应用，提出小波分析方法在化工领域有广阔的应用前景。     

传播时间法超声流量计信号处理技术进展述评

主要从信号处理的角度,回顾了传播时间法超声流量计近五十年来的技术进展,分别评述了时差法、相差法和频差法中经典的检测技术,重点介绍了近年出现的一些新颖测量方法。这些新方法既有基于先进微电子工艺的测时,也有从雷达系统借鉴而来的脉冲压缩技术,还有利用时相域分析的信号处理方法等。最后预测了超声流量计的发展趋势。     

Sitagliptin Modulates the Response of Ovarian Cancer Cells to Chemotherapeutic Agents

The strong association between diabetes mellitus type 2 and cancer is observed. The incidence of both diseases is increasing globally due to the interaction between them. Recent studies suggest that there is also an association between cancer incidence and anti-diabetic medications. An inhibitor of dipeptidyl-peptidase 4 (DPP-4), sitagliptin, is used in diabetes treatment. We examined the influence of sitagliptin alone or in combination with a cytostatic drug (paclitaxel) on the development of epithelial ovarian cancer cells and the process of metastasis. We examined migration, invasiveness, apoptosis, and metalloproteinases (MMPs) and their inhibitors’ (TIMPs) production in two human ovarian cancer cell lines. Sitagliptin induced apoptosis by caspase 3/7 activation in paclitaxel-treated SKOV-3 and OVCAR-3 cells. Sitagliptin maintained paclitaxel influence on ERK and Akt signaling pathways. Sitagliptin additionally reduced migration and invasiveness of SKOV-3 cells. There were distinct differences of metalloproteinases production in sitagliptin-stimulated ovarian cancer cells in both cell lines, despite their identical histological classification. Only the SKOV-3 cell line expressed MMPs and TIMPs. SKOV-3 cells co-treated with sitagliptin and paclitaxel decreased concentrations of MMP-1, MMP-2, MMP-7, MMP-10, TIMP-1, TIMP-2. The obtained data showed that sitagliptin used with paclitaxel may be considered as a possibility of pharmacological modulation of intracellular transmission pathways to improve the response to chemotherapy.     

Neither Excessive Nitric Oxide Accumulation nor Acute Hyperglycemia Affects the N-Acetylaspartate Network in Wistar Rat Brain Cells

The N-acetylaspartate network begins in neurons with N-acetylaspartate production catalyzed by aspartate N-acetyltransferase from acetyl-CoA and aspartate. Clinical studies reported a significant depletion in N-acetylaspartate brain level in type 1 diabetic patients. The main goal of this study was to establish the impact of either hyperglycemia or oxidative stress on the N-acetylaspartate network. For the in vitro part of the study, embryonic rat primary neurons were treated by using a nitric oxide generator for 24 h followed by 6 days of post-treatment culture, while the neural stem cells were cultured in media with 25–75 mM glucose. For the in vivo part, male adult Wistar rats were injected with streptozotocin (65 mg/kg body weight, ip) to induce hyperglycemia (diabetes model) and euthanized 2 or 8 weeks later. Finally, the biochemical profile, NAT8L protein/Nat8l mRNA levels and enzymatic activity were analyzed. Ongoing oxidative stress processes significantly affected energy metabolism and cholinergic neurotransmission. However, the applied factors did not affect the N-acetylaspartate network. This study shows that reduced N-acetylaspartate level in type 1 diabetes is not related to oxidative stress and that does not trigger N-acetylaspartate network fragility. To reveal why N-acetylaspartate is reduced in this pathology, other processes should be considered.     

Plasma Membrane Receptors Involved in the Binding and Response of Osteoclasts to Noncellular Components of the Bone

Osteoclasts differentiate from hematopoietic cells and resorb the bone in response to various signals, some of which are received directly from noncellular elements of the bone. In vitro, adherence to the bone triggers the reduction of cell–cell fusion events between osteoclasts and the activation of osteoclasts to form unusual dynamic cytoskeletal and membrane structures that are required for degrading the bone. Integrins on the surface of osteoclasts are known to receive regulatory signals from the bone matrix. Regulation of the availability of these signals is accomplished by enzymatic alterations of the bone matrix by protease activity and phosphorylation/dephosphorylation events. Other membrane receptors are present in osteoclasts and may interact with as yet unidentified signals in the bone. Bone mineral has been shown to have regulatory effects on osteoclasts, and osteoclast activity is also directly modulated by mechanical stress. As understanding of how osteoclasts and other bone cells interact with the bone has emerged, increasingly sophisticated efforts have been made to create bone biomimetics that reproduce both the structural properties of the bone and the bone’s ability to regulate osteoclasts and other bone cells. A more complete understanding of the interactions between osteoclasts and the bone may lead to new strategies for the treatment of bone diseases and the production of bone biomimetics to repair defects.     

Substance P Activates the Wnt Signal Transduction Pathway and Enhances the Differentiation of Mouse Preosteoblastic MC3T3-E1 Cells

Recent experiments have explored the impact of Wnt/β-catenin signaling and Substance P (SP) on the regulation of osteogenesis. However, the molecular regulatory mechanisms of SP on the formation of osteoblasts is still unknown. In this study, we investigated the impact of SP on the differentiation of MC3T3-E1 cells. The osteogenic effect of SP was observed at different SP concentrations (ranging from 10⁻¹⁰ to 10⁻⁸ M). To unravel the underlying mechanism, the MC3T3-E1 cells were treated with SP after the pretreatment by neurokinin-1 (NK1) antagonists and Dickkopf-1 (DKK1) and gene expression levels of Wnt/β-catenin signaling pathway components, as well as osteoblast differentiation markers (collagen type I, alkaline phosphatase, osteocalcin, and Runx2), were measured using quantitative polymerase chain reaction (PCR). Furthermore, protein levels of Wnt/β-catenin signaling pathway were detected using Western blotting and the effects of SP, NK1 antagonist, and DKK1 on β-catenin activation were investigated by immunofluorescence staining. Our data indicated that SP (10⁻⁹ to 10⁻⁸ M) significantly up-regulated the expressions of osteoblastic genes. SP (10⁻⁸ M) also elevated the mRNA level of c-myc, cyclin D1, and lymphocyte enhancer factor-1 (Lef1), as well as c-myc and β-catenin protein levels, but decreased the expression of Tcf7 mRNA. Moreover, SP (10⁻⁸ M) promoted the transfer of β-catenin into nucleus. The effects of SP treatment were inhibited by the NK1 antagonist and DKK1. These findings suggest that SP may enhance differentiation of MC3T3-E1 cells via regulation of the Wnt/β-catenin signaling pathway.