Initial joint bleed volume in a delayed on-demand treatment setup correlates with subsequent synovial changes in hemophilic mice

Background: Hemophilic arthropathy is a debilitating morbidity of hemophilia caused by recurrent joint bleeds. We investigated if the joint bleed volume, before initiation of treatment, was linked to the subsequent degree of histopathological changes and the development of bone pathology in a mouse model of hemophilic arthropathy.Methods: FVIII knock-out(F8-KO) mice were dosed with a micro-CT blood pool agent prior to induction of hemarthrosis. Eight hours after induction, the bleed volume was quantified with micro computed tomography(micro-CT) and recombinant FVIII treatment initiated. On Day 8, inflammation in the knees was characterized by fluorescence molecular tomography. On Day 14, knee pathology was characterized by micro-CT and histopathology. In a second study, contrast agent was injected into the knee of wild-type(WT) mice, followed by histopathological evaluation on Day 14.Results: The average joint bleed volume before treatment was 3.9 mm3. The inflammation-related fluorescent intensities in the injured knees were significantly increased on Day 8. The injured knees had significantly increased synovitis scores, vessel counts, and areas of hemosiderin compared to un-injured knees. However, no cartilage-or bone pathology was observed. The bleed volume before initiation of treatment correlated with the degree of synovitis and was associated with high fluorescent intensity on Day 8. In F8-KO and WT mice, persistence of contrast agent in the joint elicited morphological changes.Conclusion: When applying a delayed on-demand treatment regimen to hemophilic mice subjected to an induced knee hemarthrosis, the degree of histopathological changes on Day 14 reflected the bleed volume prior to initiation of treatment.

Comparison of Two Chemically-Induced Colitis-Models in Adult Zebrafish, Using Optical Projection Tomography and Novel Transcriptional Markers

Crohn’s disease and ulcerative colitis—inflammatory bowel diseases (IBD)—are chronic conditions with an inadequately understood pathogenesis. Employing a set of novel molecular markers in a gene expression assay (qPCR), we have used adult zebrafish to investigate two acute inflammatory models, induced by the haptenizing agents oxazolone and TNBS. In addition, goblet cell dynamics in the scales and intestine and 5-HT (serotonin) in intestinal tissues were investigated through optical projection tomography. Gene expression studies revealed a distinct and significant upregulation of proinflammatory cytokines, acute-phase reactants and metalloprotease 9 in both chemical models, primarily after 72 hours. In comparison, transcription factors and cytokines associated with Th1 and Th17 (Crohn’s) and Th2 (ulcerative colitis) were mainly not affected in this acute setting. However, elevated transcript levels were detected in Foxp3, IL-10 and T-bet, which are linked with tolerance and Tregs in mammals. Goblet cells in scales were depleted in both chemical models and in the intestine of oxazolone-treated fish. A marked 5-HT signal was noted in intestinal tissue of some chemically treated zebrafish. In conclusion, a distinct acute inflammatory reaction was induced in both chemical models. Further, oxazolone and TNBS did not result in clear-cut Th2 and Th1/Th17 pathway signaling at this early timepoint, but the applied molecular tools may provide further insight to the IBD pathogenesis and translational value of the IBD zebrafish model.

A structural and data-driven approach to engineering a plant cytochrome P450 enzyme

Functional manipulation of biosynthetic enzymes such as cytochrome P450 s(or P450 s) has attracted great interest in metabolic engineering of plant natural products.Cucurbitacins and mogrosides are plant triterpenoids that share the same backbone but display contrasting bioactivities.This structural and functional diversity of the two metabolites can be manipulated by engineering P450 s.However,the functional redesign of P450 s through directed evolution(DE) or structure-guided protein engineering is time consuming and challenging,often because of a lack of high-throughput screening methods and crystal structures of P450 s.In this study,we used an integrated approach combining computational protein design,evolutionary information,and experimental data-driven optimization to alter the substrate specificity of a multifunctional P450(CYP87 D20)from cucumber.After three rounds of iterative design and evaluation of 96 protein variants,CYP87 D20,which is involved in the cucurbitacin C biosynthetic pathway,was successfully transformed into a P450 mono-oxygenase that performs a single specific hydroxylation at C11 of cucurbitadienol.This integrated P450-engineering approach can be further applied to create a de novo pathway to produce mogrol,the precursor of the natural sweetener mogroside,or to alter the structural diversity of plant triterpenoids by functionally manipulating other P450 s.

The upregulated intestinal folate transporters direct the uptake of ligand-modified nanoparticles for enhanced oral insulin delivery

Transporters are traditionally considered to transport small molecules rather than large-sized nanoparticles due to their small pores.In this study,we demonstrate that the upregulated intestinal transporter(PCFT),which reaches a maximum of 12.3-fold expression in the intestinal epithelial cells of diabetic rats,mediates the uptake of the folic acid-grafted nanoparticles(FNP).Specifically,the upregulated PCFT could exert its function to mediate the endocytosis of FNP and efficiently stimulate the traverse of FNP across enterocytes by the lysosome-evading pathway,Golgi-targeting pathway and basolateral exocytosis,featuring a high oral insulin bioavailability of 14.4%in the diabetic rats.Conversely,in cells with relatively low PCFT expression,the positive surface charge contributes to the cellular uptake of FNP,and FNP are mainly degraded in the lysosomes.Overall,we emphasize that the upregulated intestinal transporters could direct the uptake of ligand-modified nanoparticles by mediating the endocytosis and intracellular trafficking of ligand-modified nanoparticles via the transporter-mediated pathway.This study may also theoretically provide insightful guidelines for the rational design of transporter-targeted nanoparticles to achieve efficient drug delivery in diverse diseases.

New insights into substrate folding preference of plant OSCs

Sterols and triterpenes are structurally diverse bioactive molecules generated through cyclization of linear 2,3-oxidosqualene. Based on carbocationic intermediates generated during initial substrate preorganization step, oxidosqualene cyclases （OSCs） are roughly segregated into protosteryl cation group that mainly catalyzes tetracyclic products and dammarenyl cation group which mostly generates pentacyclic products. However, in contrast to well-studied cascade of ring-forming reactions, little is known about the mechanism underlying the initial sub- strate folding process. Previously, we have identified a cucurbitadienol synthase （Bi） and its null allele bi （C393Y） from cucumber. By integration of homology modeling, residue coevolution and site-directed mutagenesis, we discover that four covarying amino acids including C393 constitute a dynamic domain that may be involved in substrate folding process for Bi. We also reveal a group of co-conserved residues that closely associated with the segregation of plant OSCs. These residues may act col- laboratively in choice of specific substrate folding inter- mediate for OSCs. Thus, engineer plant OSCs from into five-ringed producer. our findings open a door to four-ringed skeleton catalysts

Synergistic optimization framework for the process synthesis and design of biorefineries

The conceptual process design of novel bioprocesses in biorefinery setups is an important task,which remains yet challenging due to several limitations.We propose a novel framework incorporating superstructure optimization and simulation-based optimization synergistically.In this context,several approaches for superstructure optimization based on different surrogate models can be deployed.By means of a case study,the framework is introduced and validated,and the different superstructure optimization approaches are benchmarked.The results indicate that even though surrogate-based optimization approaches alleviate the underlying computational issues,there remains a potential issue regarding their validation.The development of appropriate surrogate models,comprising the selection of surrogate type,sampling type,and size for training and cross-validation sets,are essential factors.Regarding this aspect,satisfactory validation metrics do not ensure a successful outcome from its embedded use in an optimization problem.Furthermore,the framework’s synergistic effects by sequentially performing superstructure optimization to determine candidate process topologies and simulationbased optimization to consolidate the process design under uncertainty offer an alternative and promising approach.These findings invite for a critical assessment of surrogatebased optimization approaches and point out the necessity of benchmarking to ensure consistency and quality of optimized solutions.

手术期间输注血小板和红细胞对肝移植手术后生存率的影响

背景手术期间输入红细胞与原位肝移植患者的不良预后有关。实验性研究已经证实血小板可导致肝脏再灌注损伤，但是输注同种异体血小板对患者预后的影响尚未见详细的研究。本研究中，我们评估了不同血液制品对原位肝移植患者预后的影响。方法对1989年到2004年之间行第1次原位肝移植的433例患者与预后有关的29个变量，包括血液制品输注等进行了研究。各项数据采用单因素和多因素逐步Cox比例风险模型法进行了统计分析，并对血小板输注采用校正倾向评分分析法以控制使用血液制品中的选择性偏差。结果输注血液制品的移植患者比例从1989～1996年的100％下降到1997～2004年的74％。在单因素和多因素分析中，移植的指征、输注血小板和红细胞是预测移植患者1年生存率最重要的因素。这些危险因素与终末肝病分型评分和garnofsky评分等已被公认的肝脏疾病指数无关。输注血小板和红细胞呈剂量依赖性影响1年生存率，输注每单位血小板的危险比为1．377（P=0．01），输注每单位红细胞的危险比为1．057（P=0．001）。经校正倾向性评分分析证实，血小板输注对生存率有负面影响。结论本项回顾性研究表明，除红细胞外，血小板输注也是影响原位肝移植术后存活的独立危险因素。这些研究结果对肝移植受体的临床液体输注具有重要意义。