Biophysical Response to Pulsed Laser Microbeam‐Induced Cell Lysis and Molecular Delivery

Cell lysis and molecular delivery in confluent monolayers of PtK₂ cells are achieved by the delivery of 6 ns, λ = 532 nm laser pulses via a 40×, 0.8 NA microscope objective. With increasing distance from the point of laser focus we find regions of (a) immediate cell lysis; (b) necrotic cells that detach during the fluorescence assays; (c) permeabilized cells sufficient to facilitate the uptake of small (3 kDa) FITC‐conjugated Dextran molecules in viable cells; and (d) unaffected, viable cells. The spatial extent of cell lysis, cell detachment, and molecular delivery increased with laser pulse energy. Hydrodynamic analysis from time‐resolved imaging studies reveal that the maximum wall shear stress associated with the pulsed laser microbeam‐induced cavitation bubble expansion governs the location and spatial extent of each of these regions independent of laser pulse energy. Specifically, cells exposed to maximum wall shear stresses τ_{w, max} > 190 ± 20 kPa are immediately lysed while cells exposed to τ_{w, max} > 18 ± 2 kPa are necrotic and subsequently detach. Cells exposed to τ_{w, max} in the range 8–18 kPa are viable and successfully optoporated with 3 kDa Dextran molecules. Cells exposed to τ_{w, max} < 8 ± 1 kPa remain viable without molecular delivery. These findings provide the first direct correlation between pulsed laser microbeam‐induced shear stresses and subsequent cellular outcome. (© 2008 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

In Search of Mitochondrial Mechanisms: Interfield Excursions between Cell Biology and Biochemistry

Developing models of biological mechanisms, such as those involved in respiration in cells, often requires collaborative effort drawing upon techniques developed and information generated in different disciplines. Biochemists in the early decades of the 20th century uncovered all but the most elusive chemical operations involved in cellular respiration, but were unable to align the reaction pathways with particular structures in the cell. During the period 1940–1965 cell biology was emerging as a new discipline and made distinctive contributions to understanding the role of the mitochondrion and its component parts in cellular respiration. In particular, by developing techniques for localizing enzymes or enzyme systems in specific cellular components, cell biologists provided crucial information about the organized structures in which the biochemical reactions occurred. Although the idea that biochemical operations are intimately related to and depend on cell structures was at odds with the then-dominant emphasis on systems of soluble enzymes in biochemistry, a reconceptualization of energetic processes in the 1960s and 1970s made it clear why cell structure was critical to the biochemical account. This paper examines how numerous excursions between biochemistry and cell biology contributed a new understanding of the mechanism of cellular respiration.     

Models and Methods to Evaluate Transport of Drug Delivery Systems Across Cellular Barriers

Sub-micrometer carriers (nanocarriers; NCs) enhance efficacy of drugs by improving solubility, stability, circulation time, targeting, and release. Additionally, traversing cellular barriers in the body is crucial for both oral delivery of therapeutic NCs into the circulation and transport from the blood into tissues, where intervention is needed. NC transport across cellular barriers is achieved by: (i) the paracellular route, via transient disruption of the junctions that interlock adjacent cells, or (ii) the transcellular route, where materials are internalized by endocytosis, transported across the cell body, and secreted at the opposite cell surface (transyctosis). Delivery across cellular barriers can be facilitated by coupling therapeutics or their carriers with targeting agents that bind specifically to cell-surface markers involved in transport. Here, we provide methods to measure the extent and mechanism of NC transport across a model cell barrier, which consists of a monolayer of gastrointestinal (GI) epithelial cells grown on a porous membrane located in a transwell insert. Formation of a permeability barrier is confirmed by measuring transepithelial electrical resistance (TEER), transepithelial transport of a control substance, and immunostaining of tight junctions. As an example, ~200 nm polymer NCs are used, which carry a therapeutic cargo and are coated with an antibody that targets a cell-surface determinant. The antibody or therapeutic cargo is labeled with ¹²⁵I for radioisotope tracing and labeled NCs are added to the upper chamber over the cell monolayer for varying periods of time. NCs associated to the cells and/or transported to the underlying chamber can be detected. Measurement of free ¹²⁵I allows subtraction of the degraded fraction. The paracellular route is assessed by determining potential changes caused by NC transport to the barrier parameters described above. Transcellular transport is determined by addressing the effect of modulating endocytosis and transcytosis pathways.     

一氧化氮合酶的若干研究进展

一氧化氮合酶(NOS)是一氧化氮(NO)生物学与医学研究的重要内容.近年来,对NOS酶本质及其生化与分子生物学特性甚至某些分子遗传学方面的认识都在迅速发展和深化.研究表明,干预NOS-NO途径的某些环节,如酶激活、NO合成、释放与转运甚至有关酶的编码基因及其表达,将为某些临床问题的解决提供新的思路和手段．     

杆状病毒介导的哺乳动物细胞基因转移及其影响因素

杆状病毒作为优良的哺乳动物细胞基因转移载体已在基因治疗、疫苗开发、药物筛选以及基因调控研究等方面发挥巨大作用。但杆状病毒对哺乳动物细胞的嗜性不够广泛、转导效率和转基因表达水平偏低,以及表达持续时间短暂等问题制约其进一步发展,因此如何突破这个制约的瓶颈成为学者们新的关注焦点。针对以上问题,围绕杆状病毒介导哺乳动物细胞基因转移的各种影响因素进行综述。     

生物化学与分子生物学课程思政教育的探索与实践——以成都医学院为例

在全面推进高校课程思政建设的背景下,为落实立德树人的根本任务,各高校纷纷开展专业课程思政建设。以成都医学院的《生物化学与分子生物学》课程为例,文章深度挖掘该门课程所蕴含的思政元素,最终确定了7个典型的思政元素,并进行了相应的课程设计,采用案例式教学方法将其在实践中教学。课后,对成都医学院2019级临床医学、麻醉学、儿科学、医学影像学、药学和中药学等6个专业的888名学生发放调查问卷,检测其实施效果。结果表明,所开展的案例式思政教学能够较好地增强学生对该门课程的认可度,帮助学生树立良好的价值观念,初步达到了专业课思政教学的目的。最后,结合实施效果,本文从教学内容和方法的改进、课程思政教学团队的打造和思政教学评价机制的完善等方面展开了积极的探索,以期为专业课程全面实施课程思政提供借鉴。     

Molecular Engineered Hole‐Extraction Materials to Enable Dopant‐Free,Efficient p‐i‐n Perovskite Solar Cells

Two hole‐extraction materials (HEMs), TPP‐OMeTAD and TPP‐SMeTAD, have been developed to facilitate the fabrication of efficient p‐i‐n perovskite solar cells (PVSCs). By replacing the oxygen atom on HEM with sulfur (from TPP‐OMeTAD to TPP‐SMeTAD), it effectively lowers the highest occupied molecular orbital of the molecule and provides stronger Pb? S interaction with perovskites, leading to efficient charge extraction and surface traps passivation. The TPP‐SMeTAD‐based PVSCs exhibit both improved photovoltaic performance and reduced hysteresis in p‐i‐n PVSCs over those based on TPP‐OMeTAD. This work not only provides new insights on creating perovskite‐HEM heterojunction but also helps in designing new HEM to enable efficient organic–inorganic hybrid PVSCs.     

动态调控:一种高效的细胞工厂工程化代谢改造策略

动态调控作为代谢工程优化中最有效的策略之一,通常包含输入信号发生器、生物传感器和执行机构3个部分。输入信号可以是细胞代谢物和环境条件变化,如化学分子、核糖核酸、温度、光信号等。而生物传感器是能够响应输入信号变化,并转化成特定信号输出的基因元件,其输出信号可以直接调控基因表达,也可以作为其他感应元件的输入。重点介绍了动态调控的基本原理及分类及其在微生物细胞工厂工程化改造中的应用实例,主要包括动态调控的优势和研究进展、动态调控系统的构建与表征,同时,也着重讨论了动态调控在细胞工厂改造中潜在的机遇与可能面临的挑战。     

Mesoporous Silica Nanoparticles for Cancer Therapy: Energy-Dependent Cellular Uptake and Delivery of Paclitaxel to Cancer Cells

Biocompatible mesoporous silica nanoparticles, containing the fluorescence dye fluorescein isothiocyanate (FITC), provide a promising system to deliver hydrophobic anticancer drugs to cancer cells. In this study, we investigated the mechanism of uptake of fluorescent mesoporous silica nanoparticles (FMSN) by cancer cells. Incubation with FMSN at different temperatures showed that the uptake was higher at 37°C than at 4°C. Metabolic inhibitors impeded uptake of FMSN into cells. The inhibition of FMSN uptake by nocodazole treatment suggests that microtubule functions are required. We also report utilization of mesoporous silica nanoparticles to deliver a hydrophobic anticancer drug paclitaxel to PANC-1 cancer cells and to induce inhibition of proliferation. Mesoporous silica nanoparticles may provide a valuable vehicle to deliver hydrophobic anticancer drugs to human cancer cells.     

Deep Profiling of Cellular Heterogeneity by Emerging Single‐Cell Proteomic Technologies

The ability to comprehensively profile cellular heterogeneity in functional proteome is crucial in advancing the understanding of cell behavior, organism development, and disease mechanisms. Conventional bulk measurement by averaging the biological responses across a population often loses the information of cellular variations. Single‐cell proteomic technologies are becoming increasingly important to understand and discern cellular heterogeneity. The well‐established methods for single‐cell protein analysis based on flow cytometry and fluorescence microscopy are limited by the low multiplexing ability owing to the spectra overlap of fluorophores for labeling antibodies. Recent advances in mass spectrometry (MS), microchip, and reiterative staining‐based techniques for single‐cell proteomics have enabled the evaluation of cellular heterogeneity with high throughput, increased multiplexity, and improved sensitivity. In this review, the principles, developments, advantages, and limitations of these advanced technologies in analysis of single‐cell proteins, along with their biological applications to study cellular heterogeneity, are described. At last, the remaining challenges, possible strategies, and future opportunities that will facilitate the improvement and broad applications of single‐cell proteomic technologies in cell biology and medical research are discussed.     

Live‐cell imaging reveals sustained centromere binding of CENP‐T via CENP‐A and CENP‐B

At the centromere, a network of proteins, the kinetochore, assembles in order to grant correct chromatin segregation. In this study the dynamics and molecular interactions of the inner kinetochore protein CENP‐T were analyzed employing a variety of fluorescence microscopy techniques in living human cells. Acceptor‐bleaching FRET indicates that CENP‐T directly associates with CENP‐A and CENP‐B. CENP‐T exchange into centromeres is restricted to the S‐phase of the cell cycle as revealed by FRAP, suggesting a coreplicational loading mechanism, as we have recently also demonstrated for CENP‐I. These properties make CENP‐T one of the basic inner kinetochore proteins with most further proteins binding downstream, suggesting a fundamental role of CENP‐T in kinetochore function. (© 2008 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Strategic Halogen Substitution to Enable High‐Performance Small‐Molecule‐Based Tandem Solar Cell with over 15% Efficiency

Small molecules have been recently highlighted as active materials owing to their facile synthesisis method, well‐defined molecular structure, and highly reproducible performance. In particular, optimizing bulk heterojunction (BHJ) morphologies is important to achieving high performance in solution‐processable small molecule solar cells (SM‐SCs). Herein, a series of benzodithiophene‐based active materials with different halogen atoms substituted at the end‐group, are reported, as well as how these halogen atoms affect the morphology of BHJ architectures through microstructure analyses. Materials with chlorine atoms show a well‐mixed morphology and interpenetrating networks when blended with [6,6]‐phenyl‐C₇₁‐butyric acid methyl ester, facilitating effective charge transportation. This controlled morphology helps attain excellent performance with a power conversion efficiency (PCE) of 10.5% and a highest fill factor of 78.0% without additives. In addition, it can be applied to two‐terminal (2T)‐tandem solar cells, attaining an outstanding PCE of up to 15.1% with complementary absorption in the field of the 2T‐tandem solar cells introducing the SM‐SCs. These results suggest that tailoring interactions with halogen atoms is an effective way to control BHJ architectures, thereby achieving remarkable performance in SM‐SCs.     

Biodistribution of Small Interfering RNA at the Organ and Cellular Levels after Lipid Nanoparticle-mediated Delivery

Chemically stabilized small interfering RNA (siRNA) can be delivered systemically by intravenous injection of lipid nanoparticles (LNPs) in rodents and primates. The biodistribution and kinetics of LNP–siRNA delivery in mice at organ and cellular resolution have been studied using immunofluorescence (IF) staining and quantitative polymerase chain reaction (qPCR). At 0.5 and 2 hr post tail vein injection of Cy5-labeled siRNA encapsulated in LNP, the organ rank-order of siRNA levels is liver > spleen > kidney, with only negligible accumulation in duodenum, lung, heart, and brain. Similar conclusions were drawn by using qPCR to measure tissue siRNA levels as a secondary end point. siRNA levels in these tissues decreased by more than 10-fold after 24 hr. Within the liver, LNPs delivered siRNA to hepatocytes, Kupffer cells, and sinusoids in a time-dependent manner, as revealed by IF staining and signal quantitation methods established using OPERA/Columbus software. siRNA first accumulated in liver sinusoids and trafficked to hepatocytes by 2 hr post dose, corresponding to the onset of target mRNA silencing. Fluorescence in situ hybridization methods were used to detect both strands of siRNA in fixed tissues. Collectively, the authors have implemented a platform to evaluate biodistribution of siRNA across cell types and across tissues in vivo, with the objective of elucidating the pharmacokinetic and pharmacodynamic relationship to guide optimization of delivery vehicles.     

Physiological Response of Rabbit Bucks to Dietary Fumonisin: Performance,Haematology and Serum Biochemistry

Maize grains contaminated with fumonisin, a metabolite of Fusarium verticillioides was incorporated into matured male rabbits’ diet to evaluate its effects on performance, haematology and serum biochemistry in rabbits. Thirty individually caged crossbred adult rabbit bucks averaging 1.36 ± 0.01 kg (about 22–24-week-old) were randomly allotted to three treatment diets comprising a control diet (containing 0.35 ± 0.02 mg fumonisin/kg) and two test diets containing 12.30 ± 0.16 and 24.56 ± 0.14 mg fumonisin/kg, constituting treatments 1 (low infection), 2 (medium infection) and 3 (high infection), respectively, in a five-week feeding trial. Results showed that the dry matter intake (DMI) (g/rabbit) at the end of the feeding trial was significantly (P < 0.05) influenced. The DMI declined with increasing dietary fumonisin by a significant 80% and 95% (P < 0.05) for high and medium levels of dietary fumonisin, respectively, relative to the mean weekly DMI of 609.93 ± 45.08 g by rabbits fed diet with low level of fumonisin. The weekly weight gain tended to decrease with increased dietary fumonisin levels, while the haematological and serum biochemical components examined, were not statistically influenced among the diets when fed to male rabbits for a period of 5 weeks. Target audience: Livestock farmers, Feed millers, and Animal nutritionists.     

Cy‐preds: An algorithm and a web service for the analysis and prediction of cysteine reactivity

Cysteine (Cys) is a critically important amino acid, serving a variety of functions within proteins including structural roles, catalysis, and regulation of function through post‐translational modifications. Predicting which Cys residues are likely to be reactive is a very sought after feature. Few methods are currently available for the task, either based on evaluation of physicochemical features (e.g., pKa and exposure) or based on similarity with known instances. In this study, we developed an algorithm (named HAL‐Cy) which blends previous work with novel implementations to identify reactive Cys from nonreactive. HAL‐Cy present two major components: (i) an energy based part, rooted on the evaluation of H‐bond network contributions and (ii) a knowledge based part, composed of different profiling approaches (including a newly developed weighting matrix for sequence profiling). In our evaluations, HAL‐Cy provided significantly improved performances, as tested in comparisons with existing approaches. We implemented our algorithm in a web service (Cy‐preds), the ultimate product of our work; we provided it with a variety of additional features, tools, and options: Cy‐preds is capable of performing fully automated calculations for a thorough analysis of Cys reactivity in proteins, ranging from reactivity predictions (e.g., with HAL‐Cy) to functional characterization. We believe it represents an original, effective, and very useful addition to the current array of tools available to scientists involved in redox biology, Cys biochemistry, and structural bioinformatics. Proteins 2016; 84:278–291. © 2015 Wiley Periodicals, Inc.     

The natural heterogeneity of trypanosoma cruzi: Biological and medical implications

Trypanosoma cruzi is a heterogeneous group of parasites. The imposition of natural or artificial pressures can result in the selection of subsets of the population with concomitant changes in characteristics used to evaluate the group. In order to ascertain the extent of heterogeneity, stocks of single-cell clones were prepared from various sources. Selected cell biological, biochemical, immunochemical, parasitological, and histopathological parameters of these clones have been studied. A ten-fold difference in the rate of growth of the epimastigote stage of T cruzi clones has been observed. The extracellular growth rates of the clones correlate with the rate of growth of the obligate intracellular amastigote stage and consequently, the length of intracellular cycle of the parasite. A 40% difference in the amount of total DNA/parasite has been found between clones. Although the amount of DNA/kinetoplast and nucleus varies between clones, the major contribution to the differences in total DNA/parasite appears to be the nucleus. From 16 to 35 antigens have been demonstrated in the T cruzi clones assayed to date. Five to seven of these antigens are common to all of the stocks assayed. However, both isolate- and clone-specific antigens have also been demonstrated. The susceptibility of inbred strains of mice to T cruzi clones varies with the clone of the parasite. These data imply that the genetics of the parasite as well as the host modulate both the course and outcome of a T cruzi infection. The influence of monosaccharides on the receptor-mediated infection of vertebrate cells by trypomastigotes of T cruzi also varies between clones. The implications of these findings upon our concept and understanding of present and future problems in Chagas disease are discussed.     

Peptide ligand‐mediated endocytosis of nanoparticles to cancer cells: Cell receptor‐binding‐ versus cell membrane‐penetrating peptides

The endocytosis‐mediating performances of two types of peptide ligands, cell receptor binding peptide (CRBP) and cell membrane penetrating peptide (CMPP), were analyzed and compared using a common carrier of peptide ligands‐human ferritin heavy chain (hFTH) nanoparticle. Twenty‐four copies of a CMPP(human immunodeficiency virus‐derived TAT peptide) and/or a CRBP (peptide ligand with strong and specific affinity for either human integrin(α_vβ₃) or epidermal growth factor receptor I (EGFR) that is overexpressed on various cancer cells) were genetically presented on the surface of each hFTH nanopariticle. The quantitative level of endocytosis and intracellular localization of fluorescence dye‐labeled CRBP‐ and CMPP‐presenting nanoparticles were estimated in the in vitro cultures of integrin‐ and EGFR‐overexpressing cancer and human dermal fibroblast cells(control). From the cancer cell cultures treated with the CMPP‐ and CRBP‐presenting nanoparticles, it was notable that CRBPs resulted in quantitatively higher level of endocytosis than CMPP (TAT) and successfully transported the nanoparticles to the cytosol of cancer cells depending on concentration and treatment period of time, whereas TAT‐mediated endocytosis localized most of the nanoparticles within endosomal vesicles under the same conditions. These novel findings provide highly useful informations to many researchers both in academia and in industry who are interested in developing anticancer drug delivery systems/carriers.