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Mass cytometry (CyTOF) is a new technology that allows the investigation of protein expression at single cell level with high resolution. While several protocols are available to investigate leukocyte expression, platelet staining and analysis with CyTOF have been described only from whole blood. Moreover, available protocols do not allow sample storage but require fresh samples to be obtained, processed, and measured immediately. We provide a structured and reproducible method to stain platelets from platelet-rich plasma to study thrombocyte protein expression and reactivity using mass cytometry. With our method, it is possible to acquire a large number of events allowing deep bioinformatic investigation of platelet expression heterogeneity. Integrated in our protocol is also a previously established freezing protocol that allows the storage of stained samples and to delay their measurement. Finally, we provide a structured workflow using different platelet stimulators and a freely available bioinformatic pipeline to analyze platelet expression. Our protocol unlocks the potential of CyTOF analysis for studying platelet biology in health and disease.

Lentiviral vectors (LVs) are robust delivery vehicles for gene therapy as they can efficiently integrate transgenes into host cell genomes. However, LVs with lengthy or complex expression cassettes typically are produced at low titers and have reduced gene transfer capacity, creating barriers for clinical and commercial applications. Modifications of the packaging cell line and methods may be able to produce complex vectors at higher titer and infectivity and may improve production of many different LVs. In this study, we identified two host restriction factors in HEK293T packaging cells that impeded LV production, 2′-5′-oligoadenylate synthetase 1 (OAS1) and low-density lipoprotein receptor (LDLR). Knocking out these two genes separately led to ∼2-fold increases in viral titer. We created a monoclonal cell line, CRISPRed HEK293T to Disrupt Antiviral Response (CHEDAR), by successively knocking out OAS1, LDLR, and PKR, a previously identified factor impeding LV titers. Packaging in CHEDAR yielded ∼7-fold increases in physical particles, full-length vector RNA, and vector titers. In addition, overexpressing transcription elongation factors, SPT4 and SPT5, during packaging improved the production of full-length vector RNA, thereby increasing titers by ∼2-fold. Packaging in CHEDAR with over-expression of SPT4 and SPT5 led to ∼11-fold increases of titers. These approaches improved the production of a variety of LVs, especially vectors with low titers or with internal promoters in the reverse orientation, and may be beneficial for multiple gene therapy applications.

Because the tooth surface micromorphology affects the performance of the gear and the finite element analysis and the grinding process of tooth surface through finite element analysis and simulation is complicated and time-consuming, the paper proposed a three-dimensional (3D) ultrasonic assistant grinding (RUAG) geometric simulation method to discover the micromorphology formation mechanism. Based on the motion path of a single grain, the 3D models of gear blank and the grinder was built. It helps to simulate the RUAG process. The morphologies of the simulated tooth surface were compared under conventional grinding (CG) and RUAG, which was consistent with the practice. It is proved that the geometric simulation method intuitively shows the micro dents on the tooth surface under different grinding parameters. The simulation method is helpful to analyze the surface morphology under different RUAG machining parameters and saves the cost and time of the actual production and processing.

Targeted epitope-based mass spectrometry imaging (MSI) utilizes laser cleavable mass-tags bound to targeting moieties for detecting proteins in tissue sections. Our work constitutes the first proof-of-concept of a novel laser desorption ionization (LDI)-MSI strategy using photocleavable Ru(ii) polypyridine complexes as mass-tags for imaging of integrins avß3 in human cancer tissues.

Human platelets differ considerably with regard to their size, RNA content and thrombogenicity. Reticulated platelets (RPs) are young, hyper-reactive platelets that are newly released from the bone marrow. They are larger and contain more RNA compared to older platelets. In comparison to more mature platelets, they exhibit a significantly higher thrombogenicity and are known to be elevated in patients with an increased platelet turnover such as, diabetics and after acute myocardial infarction. Several studies have shown that RPs correlate with an insufficient antiplatelet response to aspirin and specific P2Y 12 receptor inhibitors. In addition, RPs are promising novel biomarkers for the prediction of adverse cardiovascular events in cardiovascular disease. However, the reason for RPs intrinsic hyper-reactivity and their association with ischemic events is not completely understood and the biology of RPs is still under investigation. We here present a structured review of preclinical and clinical findings concerning the role of RPs in cardiovascular disease.

The precisely ground tooth surface of the spiral bevel gear still shows the thermal crack, and its micromorphology is the cause of high-frequency noise. The ultrasonic assistant grinding (RUAG) technology has the unique advantage of solving these problems. Therefore, this paper designed the split-type rotary device in which the disc grinder and amplitude horn are core components and bolted together. A deformation coefficient was proposed to calculate the strain stress of the disc grinder. Combined with the stepped horn resonant theory, the theoretical model of a multi-step rotating body can be used to calculate the frequency and amplitude of the grinder and horn. The harmonic response simulation was adopted to verify the theoretical results. The experiment proved that the device’s frequency, node, and amplitude are reasonable. The comparison results showed that the simulated frequency and amplitude were closer to the practical one. Moreover, the difference between them increased with the dimension of the device.

Protein-based adhesives with their robust adhesion performance and excellent biocompatibility have been extensively explored over years. In particular, the unique adhesion behaviours of mussel and sandcastle worm inspired the development of synthetic adhesives. However, the chemical synthesized adhesives often demonstrate weak underwater adhesion performance and poor biocompatibility/biodegradability, limiting their further biomedical applications. In sharp contrast, genetically engineering endows the protein-based adhesives the ability to maintain underwater adhesion property as well as biocompatibility/biodegradability. Herein, we outline recent advances in the design and development of protein-based adhesives by genetic engineering. We summarize the fabrication and adhesion performance of elastin-like polypeptide-based adhesives, followed by mussel foot protein (mfp) based adhesives and other sources protein-based adhesives, such as, spider silk spidroin and suckerin. In addition, the biomedical applications of these bioengineered protein-based adhesives are presented. Finally, we give a brief summary and perspective on the future development of bioengineered protein-based adhesives.

The recent development of mass spectrometry imaging (MSI) technology allowed to obtain extremely detailed images of the spatial distribution of proteins in tissue at high spatial resolution reaching cell dimensions, high target specificity and a large dynamic concentration range. This review focusses on the development of two main MSI principles, namely targeted and untargeted detection of protein distribution in tissue samples, with special emphasis on the improvements in analyzed mass range and spatial resolution over the last 10 years. Untargeted MSI of in situ digested proteins with matrix-assisted laser desorption ionization is the most widely used approach, but targeted protein MSI technologies using laser ablation inductively coupled plasma (LA-ICP) and photocleavable mass tag chemical labeling strategies are gaining momentum. Moreover, this review also provides an overview of the effect of sample preparation on image quality and the bioinformatic challenge to identify proteins and quantify their distribution in complex MSI data.

Summary Lentiviral vectors (LVs) commonly used for the treatment of hemoglobinopathies often have low titers and sub-optimal gene transfer efficiency for human hematopoietic stem and progenitor cells (HSPCs), hindering clinical translation and commercialization for ex vivo gene therapy. We observed that a high percentage of β-globin LV viral genomic RNAs were incomplete toward the 3′ end in packaging cells and in released vector particles. The incomplete vector genomes impeded reverse transcription in target cells, limiting stable gene transfer to HSPCs. By combining three modifications to vector design and production (shortening the vector length to 5.3 kb; expressing HIV-1 Tat protein during packaging; and packaging in PKR−/− cells) there was a 30-fold increase in vector titer and a 3-fold increase in vector infectivity in HSPCs. These approaches may improve the manufacturing of β-globin and other complex LVs for enhanced gene delivery and may facilitate clinical applications., Graphical Abstract, Highlights • Vector genomes are truncated in a length-dependent manner during packaging • Truncated RNAs cannot be reverse transcribed, impeding titer and infectivity • Protein kinase R inhibits virion formation for bidirectional lentiviral vectors • Three strategies to improve lentiviral vector titer by 30× and infectivity by 3×, In this article, Kohn and colleagues investigated the mechanisms which lead to poor vector production and infectivity of long lentiviral vectors. This is a major problem for many clinical gene therapy trials. This work substantially adds to the mechanistic understanding of the problems associated with complex lentiviral vector production, and the combination of three modifications results in a significant gain in titers and infectivity.

In this paper, the decision-making problem of speed-up scheme for existing high-speed railway is studied. Considering the different running speed of different parts of high-speed railway, the speed-up scheme of existing line is formulated. Taking the factors affecting the decision-making of railway speed-up scheme into consideration, the efficiency, cost and influence are determined as the evaluation indexes, and the grey target decision-making method in grey system theory is selected to evaluate each scheme. In this problem, qualitative analysis can be used for the optimal decision-making of the initial scheme. The corresponding situations under each index are comprehensively considered and sorted. The value is assigned to each qualitative evaluation to form the effect vector of each situation. In the spherical grey target formed by grey target decision method, the optimal scheme can be obtained by comparing the off-target distance of each effect vector corresponding to the scheme. It is proved that the grey target decision-making method can also obtain the optimal scheme through qualitative analysis when it is difficult to make quantitative analysis.

This thesis described the synthesis, carachterization, and application of bioconjugated metal-based compounds. The bioconjugated Pd-based metallocages are non-toxic to healthy tissue cells. Their well defined structure make them ideal tools for targeted delivery of anticancer drug cisplatin. The bioconjugated Ru-based compounds exhibit high photocleavable property which can be used as mass-tag for the targted mass spectrometry imaging of tumor tissue.

Cisplatin occupies a crucial role in the treatment of various malignant tumours. However, its efficacy and applicability are heavily restricted by severe systemic toxicities and drug resistance. Our study exploits the active targeting of supramolecular metallacages to enhance the activity of cisplatin in cancer cells while reducing its toxicity. Thus, Pd2L4 cages (L = ligand) have been conjugated to four integrin ligands with different binding affinity and selectivity. Cage formation and encapsulation of cisplatin was proven by NMR spectroscopy. Upon encapsulation, cisplatin showed increased cytotoxicity in vitro, in melanoma A375 cells overexpressing αvβ3 integrins. Moreover, ex vivo studies in tissue slices indicated reduced toxicity towards healthy liver and kidney tissues for cage-encapsulated cisplatin. Analysis of metal content by ICP-MS demonstrated that encapsulated drug is less accumulated in these organs compared to the 'free' one.

Supramolecular Pd2L4 cages (L = ligand) hold promise as drug delivery systems. With the idea of achieving targeted delivery of the metallacages to tumor cells, the bioconjugation of exo-functionalized self-assembled Pd2L4 cages to peptides following two different approaches is reported for the first time. The obtained bioconjugates were analyzed and identified by high-resolution mass spectrometry.

As a derivative demand of economic development, freight transport plays an important role in economic growth. In recent years, the experience of some countries shows that freight demand remains stable or even declines with GDP growth. This study uses the panel data of 30 provinces (cities and autonomous regions) in China from 1997 to 2017 to analyze the evolution of the relationship between freight demand and economic development, using the decoupling index and transport intensity measurement models. We find that there is an inverted U-shaped relationship between the turnover of freight traffic per person and economic development, and the development processes of Beijing, Shanghai, and Tianjin fully conform to this feature. China's economic development as a whole is decoupled from freight development, while its transport intensity is weakening; however, there are obvious regional variations. This indicates that the contribution of high freight-demand sectors to economic development has declined, which is undoubtedly the result of multiple factors such as the industrial structure and geographical characteristics of each region. This study provides an opinion in support of region-dependent infrastructure improvements, for managers to realize the “win-win” development of transportation and the economy.

The lack of selectivity for cancer cells and the resulting\ud negative impact on healthy tissue is a severe drawback of actual\ud cancer chemotherapy. Tethering of cytotoxic drugs to targeting\ud vectors such as peptides, which recognize receptors overexpressed\ud on the surface of tumor cells, is one possible strategy to overcome\ud such a problem. The pentapeptide cyc(RGDfK) targets the integrin\ud receptor αvβ3, important for tumor growth and metastasis formation.\ud In this work, two terpyridine based Ru(II) complexes were prepared\ud and for the first time conjugated to cyc(RGDfK) via amide bond\ud formation resulting in a monomeric and a dimeric bioconjugate. Both\ud Ru(II) complexes bind strongly and selectively to integrin αvβ3, with\ud the dimeric molecule displaying a 20-fold higher affinity to the\ud receptor than the monomeric one. However, the cytotoxicity of the\ud complexes and related bioconjugates against human A549 and\ud SKOV-3 cell lines is still not sufficient for application as anticancer\ud agents. Nevertheless, considering the high selectivity for integrin\ud receptor αvβ3, the synthesis of Ru-based bioconjugates with\ud cyc(RGDfK) paves a promising way towards the design of effective\ud targeted anticancer agents.

Cataluminescence (CTL) is a kind of chemiluminescence during catalytic reaction on surface of catalysts under a heated condition. Due to the low catalytic reactivity of CO, normally low intensity of CTL is obtained during heterogeneously catalytic oxidation of CO under heated conditions (normally higher than 150°C), even catalyzed by precious-metal-based catalysts. Therefore, seeking enhanced CTL of CO at room temperature and using low-cost catalysts becomes significant. Here, CTL generated from CO oxidation was firstly reported at room temperature, which was carried out in a non-thermal plasma-assisted (NTPA) catalysis system. With air acting as discharge gas, carrier gas as well as oxidant, a Mn/SiO2 nanomaterials-based NTPA catalysis system was fabricated for CO catalytic oxidation at room temperature, whose temperature was much lower than previous CTL methods. Relatively high and selective CTL responses were acquired during CO oxidation on surface of Mn/SiO2 nanomaterials, whereas no significant CTL signal was recorded without plasma assistance or on other metals-doped SiO2 catalysts. Without any excitation light source or heating element, a low cost and simple CO sensor was fabricated by using common and easily synthesized catalysts. The present work has greatly simplified the constructions, and enlarged CTL applications.

In this article, a Venturi electrosonic spray ionization (V-ESSI) cataluminescence (CTL) sensor array was reported for discriminating saccharides in solution. Integrating electrosonic spray ionization (ESSI), a liquid system of Venturi self-pumping injection for the CTL reaction, was fabricated for enhancing CTL reactivity of aqueous samples. Comparing with simple Venturi injection by air and Venturi easy ambient sonic-spray ionization without electric assistance (V-EASI), the remarkable enhancement of CTL signals resulted from V-ESSI. This system showed higher cross-reactive CTL responses catalyzed by alkaline earth metal-nanomaterials than other catalysts, giving different signals for a given saccharide on different catalysts and different responses for different saccharides on the same catalyst. Then, a 4 × 2 CTL sensor array was used for obtaining "fingerprints" of distinct CTL response patterns. Analyzed by linear discriminant analysis (LDA), this V-ESSI CTL sensor array not only achieved the well discrimination of different saccharides (99.9% of total variation) but also discriminated four groups of urine sugar-level for urine samples from diabetic patients (98.1% of discrimination accuracy). It had good reproducibility and gave a linear range of 22.5-67558 μg/mL (R0.99) for xylose with a detection limit of 7.4 μg/mL on MgO. As a new artificial tongue, this system provided a simple, rapid, low cost, low energy consumption, and environmentally friendly pathway for aqueous sample discrimination. It has dramatically expanded applications of the CTL-based senor array and will be applicable to clinical diagnoses, environment monitoring, industrial controls, food industry, and various marine monitoring.

Exo-functionalized Pd2L4 cage compounds with attached Ru(II) pyridine complexes were prepared via coordination-driven self-assembly. Unlike most of the previously reported palladium( II) cages, one of these metallocages exhibits an exceptionally high quantum yield of 66%. The presented approach is promising to obtain luminescent coordination complexes for various applications.

Combining plasma activation and cross-reactivity of sensor array, we have developed a plasma-assisted cataluminescence (PA-CTL) sensor array for fast sensing and discrimination of gaseous hydrocarbons, which can be potentially used for fast diagnosis of lung cancer. Based on dielectric barrier discharge, a low-temperature plasma is generated to activate gaseous hydrocarbons with low cataluminescence (CTL) activities. Extremely increased CTL responses have been obtained, which resulted in a plasma assistance factor of infinity (∞) for some hydrocarbons. On a 4 × 3 PA-CTL sensor array made from alkaline-earth nanomaterials, gaseous hydrocarbons showed robust and unique CTL responses to generate characteristic patterns for fast discrimination. Because of the difference in the component of hydrocarbons in breath, exhaled breath samples from donors with and without lung cancer were tested, and good discrimination has been achieved by this technique. In addition, the feasibility of multidimentional detection based on temperature was confirmed. It had good reproducibility and gave a linear range of 65-6500 ng/mL or 77-7700 ppmv (R > 0.98) for CH(4) with a detection limit of 33 ng/mL (38 ppmv) on MgO. The PA-CTL sensor array is simple, low-cost, thermally stable, nontoxic, and has an abundance of alkaline-earth nanomaterials to act as sensing elements. This has expanded the applications of CTL-based senor arrays and will show great potential in clinical fast diagnosis.

Faithful chromosome segregation in meiosis requires crossover (CO) recombination, which is regulated to ensure at least one CO per homolog pair. We investigate the failure to ensure COs in juvenile male mice. By monitoring recombination genome-wide using cytological assays and at hotspots using molecular assays, we show that juvenile mouse spermatocytes have fewer COs relative to adults. Analysis of recombination in the absence of MLH3 provides evidence for greater utilization in juveniles of pathways involving structure-selective nucleases and alternative complexes, which can act upon precursors to generate noncrossovers (NCOs) at the expense of COs. We propose that some designated CO sites fail to mature efficiently in juveniles owing to inappropriate activity of these alternative repair pathways, leading to chromosome mis-segregation. We also find lower MutLγ focus density in juvenile human spermatocytes, suggesting that weaker CO maturation efficiency may explain why younger men have a higher risk of fathering children with Down syndrome.

A novel biodegradable brain drug delivery system, the lactoferrin (Lf) conjugated polyethylene glycol-polylactide-polyglycolide (PEG-PLGA) nanoparticle (Lf-NP) was constructed in this paper with its in vitro and in vivo delivery properties evaluated by a fluorescent probe coumarin-6. Lf was thiolated and conjugated to the distal maleimide function surrounding on the pegylated nanoparticle to form Lf-NP. TEM observation and ELISA analysis confirmed the existence of active Lf on the surface of Lf-NP. The results of qualitative and quantitative uptake studies of coumarin-6 incorporated Lf-NP showed a more pronounced accumulation of Lf-NP in bEnd.3 cells than that of unconjugated nanoparticle (NP). Further uptake inhibition study indicated that the increased uptake of Lf-NP was via an additional clathrin mediated endocytosis processes. Following intravenous administration, a near 3 fold of coumarin-6 was found in the mice brain carried by Lf-NP compared to that carried by NP. Intravenous injection of urocortin loaded Lf-NP effectively attenuated the striatum lesion caused by 6-hydroxydopamine in rats as indicated by the behavioral test, the immunohistochemistry test and striatal transmitter content detection results. The cell viability test and CD68 immunohistochemistry demonstrated the acceptable toxicity of the system. All these results demonstrated that Lf-NP was a promising brain drug delivery system with reasonable toxicity.

Based on cataluminescence from plasma-assisted catalytic oxidation, a low temperature CO sensor was fabricated. With Ag doped alkaline-earth catalyst as sensing element, air as discharge gas, carrier gas and oxidant supplier, significant cataluminescence was achieved at low temperature, demonstrating a potential low-consumption and portable sensor of CO.