In vivo analysis of intestinal permeability following hemorrhagic shock

AIM: To determine the time course of intestinal permeability changes to proteolytically-derived bowel peptides in experimental hemorrhagic shock. METHODS: We injected fluorescently-conjugated casein protein into the small bowel of anesthetized Wistar rats prior to induction of experimental hemorrhagic shock. These molecules, which fluoresce when proteolytically cleaved, were used as markers for the ability of proteolytically cleaved intestinal products to access the central circulation. Blood was serially sampled to quantify the relative change in concentration of proteolytically-cleaved particles in the systemic circulation. To provide spatial resolution of their location, particles in the mesenteric microvasculature were imaged using in vivo intravital fluorescent microscopy. The experiments were then repeated using an alternate measurement technique, fluorescein isothiocyanate(FITC)-labeled dextrans 20, to semi-quantitatively verify the ability of bowel-derived low-molecular weight molecules(< 20 k D) to access the central circulation.RESULTS: Results demonstrate a significant increase in systemic permeability to gut-derived peptides within 20 min after induction of hemorrhage(1.11 ± 0.19 vs 0.86 ± 0.07, P < 0.05) compared to control animals. Reperfusion resulted in a second, sustained increase in systemic permeability to gut-derived peptides in hemorrhaged animals compared to controls(1.2 ± 0.18 vs 0.97 ± 0.1, P < 0.05). Intravital microscopy of the mesentery also showed marked accumulation of fluorescent particles in the microcirculation of hemorrhaged animals compared to controls. These results were replicated using FITC dextrans 20 [10.85 ± 6.52 vs 3.38 ± 1.11 fluorescent intensity units(× 105, P < 0.05, hemorrhagic shock vs controls)], confirming that small bowel ischemia in response to experimental hemorrhagic shock results in marked and early increases in gut membrane permeability. CONCLUSION: Increased small bowel permeability in hemorrhagic shock may allow for systemic absorption of otherwise retained proteolytically-generated peptides, with consequent hemodynamic instability and remote organ failure.

Molecular and Cellular Immuno-Engineering

Genetically-encoded biosensors based on fluorescence proteins(FPs)and fluorescence resonance energy transfer(FRET)have enabled the specific targeting and visualization of signaling events in live cells with high spatiotemporal resolutions.Single-molecule FRET biosensors have been successfully developed to monitor the activity of a variety of signaling molecules,including tyrosine/serine/threonine kinases.We have a developed a general high-throughput screening(HTS)method based on directed evolution to develop sensitive and specific FRET biosensors.We have first applied a yeast library and screened for a mutated binding domain for phosphorylated peptide sequence.When this mutated binding domain and the peptide sequence are connected by a linker and then concatenated in between a pair of FRET FPs,a drastic increase in sensitivity can be achieved.It has also been increasingly clear that controlling protein functions using lights and chemical compounds to trigger allosteric conformational changes can be applied to manipulate protein functions and control cellular behaviors.In this work,we first engineered a novel class of machinery molecules which can provide a surveillance of the intracellular space,visualizing the spatiotemporal patterns of molecular events and automatically triggering corresponding molecular actions to guide cellular functions.We have adopted a modular assembly approach to develop these machinery molecules.We engineered such a molecule for the sensing of intracellular tyrosine phosphorylation based on fluorescence resonance energy transfer(FRET)and the consequent activation of a tyrosine phosphatase(PTP)Shp2,which plays a critical and positive role in various pathophysiological processes[1-3].We have further integrated this machinery molecule to the'don’t eat me'CD47 receptor SIRPa on macrophages such that the engagement of SIRPa and its activation of naturally negative signals will be rewired to turn on the positive Shp2 action to facilitate phagocytosis of red blood cells and target tumor cells,initiated by the specific antigen-targeting antibodies and their interaction with Fcg receptors.Because of the modular design of our engineered molecule,our approach can be extended to perform a broad range of cell-based imaging and immunotherapies,and hence highlight the translational power in bridging the fundamental molecular engineering to clinical medicine.We have also integrated with lights and ultrasound to manipulate the molecular activation of genes and enzymes,which allowed us to control the cellular functions of immunocells with high precision in space and time.

Warmest Congratulations to the Journal of Medical Biomechanics on Its 30^(th) Anniversary

It is my great pleasure and privilege to write this message of congratulation to commemorate the auspicious occasion of the 30^(th) anniversary of the Journal of Medical Biomechanics(JMB),which was first published in 1986 as the Journal of Biomechanics.The JMB,which is sponsored by Shanghai Jiao Tong University and supervised by the Ministry of Education of People’s Republic of China,has made outstanding accomplishments over the thirty-year period since its inception.It is

力传导中亚细胞水平的分子FRET成像（英文）

体内细胞受到含有化学和力学因素的生理和病理生理的刺激,故研究这些因素在细胞和器官水平如何调节功能就尤为重要。有关细胞和器官对化学因素的反应已开展诸多研究,而力学因素的影响却鲜有报道。近年来,荧光蛋白和显微镜技术的发展已成为阐明力传导过程的有用工具,先进的信号活细胞成像技术促进了力学生物学中分子机制的时空因素研究。本文综述荧光蛋白的基本知识以及其在生物学研究中的应用,特别讨论了以荧光共振能量迁移(fluorescence proteins and microscopy,FRET)技术为基础的生物传感器的发展和特征。基因编码的FRET生物传感器能够实现分子时空活动的成像和定量,使得活细胞中生物化学信号在力学刺激下的反应和传导可视化。同时,本文重点阐述分子水平力学刺激下的活细胞信号传导。

Multifunctional stimuli responsive polymer-gated iron and gold-embedded silica nano golf balls:Nanoshuttles for targeted on-demand theranostics

Multi-functional nanoshuttles for remotely targeted and on-demand delivery of therapeutic molecules and imaging to defined tissues and organs hold great potentials in personalized medicine, including precise early diagnosis, efficient prevention and therapy without toxicity. Yet, in spite of 25 years of research, there are still no such shuttles available. To this end, we have designed magnetic and gold nanoparticles （NP）-embedded silica nanoshuttles （MGNSs） with nanopores on their surface. Fluorescently labeled Doxombicin （DOX）, a cancer drug, was loaded in the MGNSs as a payload. DOX loaded MGNSs were encapsulated in heat and pH sensitive polymer P（NIPAM-co- MAA） to enable controlled release of the payload. Magnetically-guided transport of MGNSs was examined in： （a） a glass capillary tube to simulate their delivery via blood vessels; and （b） porous hydrogels to simulate their transport in composite human tissues, including bone, cartilage, tendon, muscles and blood-brain barrier {BBB）. The viscoelastic properties of hydrogels were examined by atomic force microscopy （AFM）. Cellular uptake of DOX- loaded MGNSs and the subsequent pH and temperature-mediated release were demonstrated in differentiated human neurons derived from induced pluripotent stem cells （iPSCs） as well as epithelial HeLa cells. The presence of embedded iron and gold NPs in silica shells and polymer-coating are supported by SEM and TEM. Fluorescence spectroscopy and microscopy documented DOX loading in the MGNSs. Time-dependent transport of MGNSs guided by an external magnetic field was observed in both glass capillary tubes and in the porous hydrogel. AFM results affirmed that the stiffness of the hydrogels model the rigidity range from soft tissues to bone. pH and temperature-dependent drug release analysis showed stimuli responsive and gradual drug release. Cells＇ viability MTT assays showed that MGNSs are non-toxic. The cell death from on-demand DOX release was observed in both neurons and epithelial cells even though the drug release efficiency was higher in neurons. Therefore, development of smart nanoshuttles have significant translational potential for controlled delivery of theranostics＇ payloads and precisely guided transport in specified tissues and organs （for example, bone, cartilage, tendon, bone marrow, heart, lung, liver, kidney, and brain） for highly efficient personalized medicine applications.

冯元桢先生百岁大寿贺喜

我最初认识冯先生是经由读他的奠基文章和经典书籍以及听他的卓越演讲,也曾经在开会时简短见过。第一次和冯先生真正见面是1965年在美国新泽西州大西洋城参加微循环学会会议的时候。我和内人匡政及两个女儿在一家饭馆吃饭,冯先生在另一桌和微循环学会理事们开会用餐。他过来和我们打招呼,结果就坐下来和我们一起吃,谈得很高兴(图1),他没有回去参加理事会,使我非常感动,永远铭记在心。这件事明确地显示冯先生对年轻后辈的关怀提携。

Malignant field expression signatures in biopsy samples at diagnosis predict the likelihood of lethal disease in patients with localized prostate cancer

Aim:Overtreatment of early-stage low-risk prostate cancer patients represents a significant problem in disease management and has significant socio-economic implications.Changes in prostate cancer screening and treatment practices in the United States have been associated with the recent decline in overall incidence and concomitant significant increase of the annual incidence of metastatic prostate cancer has been documented.Therefore,development of genetic and molecular markers of clinically significant disease in patients diagnosed with low grade localized prostate cancer would have a major impact in disease management.Methods:Identification of gene expression signatures(GES)associated with lethal prostate cancer has been performed using microarray analyses of biopsy specimens obtained at the time of diagnosis from 281 patients with Gleason 6(G6)and G7 tumors in a Swedish watchful waiting cohort with up to 30 years follow-up.The performance of GES has been validated in independent cohort of 568 prostate cancer patients of the Cancer Genome Anatomy Project Prostate Cancer database.Results:GES c omprising 98 genes identified 89%and 100%of all death events 4 years after diagnosis in G7 and G6 patients,respectively.At 6 years follow-up,83%and 100%of all deaths events were captured in G7 and G6 patients,respectively.Remarkably,the 98-gene signature appears to perform successfully in patients stratification with as little as 2%of cancer cells in a specimen,strongly indicating that it captures a malignant field effect in human prostates harboring cancer cells of different degrees of aggressiveness.In G6 and G7 tumors from prostate cancer patients of age 65 or younger,GES identified 86%of all death events during the entire follow-up period.In G6 and G7 tumors from prostate cancer patients of age 70 or younger,GES identified 90%of all death events 6 years after diagnosis.Conclusion:Classification performance of the reported in this study 98-genes GES of lethal prostate cancer appeared suitable to meet design and feasibility requirements of a prospective 4 to 6 years clinical trial,which is essential for regulatory approval of diagnostic and prognostic tests in clinical setting.Prospectively validated GES of lethal PC in biopsy specimens of G6 and G7 tumors will help physicians to identify,at the time of diagnosis,patients who should be considered for exclusion from active surveillance programs and who would most likely benefit from immediate curative interventions.

Inhibitory effect of lanosterol on cataractous lens of cynomolgus monkeys using a subconjunctival drug release system Keke

Background:To evaluate the effect of lanosterol on cataractous lens of cynomolgus monkeys using a subconjunctival drug release system.Methods:Nine elder cynomolgus monkeys were used,consisting of three monkeys without cataract as controls,three monkeys with naturally occurring cortical cataract,and three monkeys with nuclear cataract as intervention groups.Nanoparticulated thermogel with lanosterol and fluorescein was administered by subconjunctival injection in the monkeys with cataract.Fluorescence changes of injected thermogel and cataract progression were observed.Lanosterol concentration in aqueous humor,solubility changes in lens proteins,and oxidative stress levels were analyzed in the lenses of the control and intervention groups.Results:Injected thermogel showed decreased fluorescence during follow up.Lanosterol concentration in aqueous humor increased in the first 2 weeks and then gradually decreased,which was in accordance with the changes in cortical lens clarity.However,lenses with nuclear opacification showed little change.In the cortical region of lenses with cortical cataract,solubility ofα-crystallin was significantly increased after administration of lanosterol,as well as the reduction of oxidative stress.Conclusions:We demonstrated the effect of lanosterol on cataract progression based on in vivo models of primates.Lanosterol showed a short-term and reliable reversal effect on reducing cataract severity in cortical cataract in the early stages,possibly due to the increase in the solubility of lens proteins and changes in the oxidative stress status.Lanosterol administration using subconjunctival drug release system could be a promising nonsurgical approach for future clinical studies of cataract prevention and treatment.