Multiorgan microfluidic platform with breathable lung chamber for inhalation or intravenous drug screening and development

Efficient and economical delivery of pharmaceuticals to patients is critical for effective therapy. Here we describe a multiorgan (lung, liver, and breast cancer) microphysiological system (“Body-on-a-Chip”) designed to mimic both inhalation therapy and/or intravenous therapy using curcumin as a model drug. This system is “pumpless” and self-contained using a rocker platform for fluid (blood surrogate) bidirectional recirculation. Our lung chamber is constructed to maintain an air-liquid interface and contained a “breathable” component that was designed to mimic breathing by simulating gas exchange, contraction and expansion of the “lung” using a reciprocating pump. Three cell lines were used: A549 for the lung, HepG2 C3A for the liver, and MDA MB231 for breast cancer. All cell lines were maintained with high viability (>85%) in the device for at least 48 hr. Curcumin is used to treat breast cancer and this allowed us to compare inhalation delivery versus intravenous delivery of the drug in terms of effectiveness and potentially toxicity. Inhalation therapy could be potentially applied at home by the patient while intravenous therapy would need to be applied in a clinical setting. Inhalation therapy would be more economical and allow more frequent dosing with a potentially lower level of drug. For 24 hr exposure to 2.5 and 25 µM curcumin in the flow device the effect on lung and liver viability was small to insignificant, while there was a significant decrease in viability of the breast cancer (to 69% at 2.5 µM and 51% at 25 µM). Intravenous delivery also selectively decreased breast cancer viability (to 88% at 2.5 µM and 79% at 25 µM) but was less effective than inhalation therapy. The response in the static device controls was significantly reduced from that with recirculation demonstrating the effect of flow. These results demonstrate for the first time the feasibility of constructing a multiorgan microphysiological system with recirculating flow that incorporates a “breathable” lung module that maintains an air-liquid interface.     

Preparation of Triacylglycerol Molecular Species by Interesterification Using Endocellular Lipase in n-Hexane

The interesterification of triacylglycerol with fatty acid was done to prepare triacylglycerol molecular species. Optimum operating conditions for the interesterification using a 1,3-positional specific endocellular lipase from Rhizopus japonicus NR400 in a batch system were investigated. The reaction was done at 40°C for 5 hr in the following system: Trioleoylglycerol-palmitic acid = 1:3.5 (mol/mol), 10 ml n-hexane/g trioleoylglycerol, and 2500 units of enzyme/g trioleoylglycerol. Under these conditions, the content of palmitoyl groups in 1,3-positions of triacylglycerol was about 60 mol%. Additional interesterification (2-cycle reaction) using palmitic acid and the novel triacylglycerol prepared by one-step interesterification (1-cycle reaction) resulted in a preparation of highly pure 1,3-dipalmitoyl-2-oleoylglycerol.     

微生物学实验模块“蛋白酶产生菌的筛选、培养和选育”的教改研究与实践

围绕"蛋白酶产生菌的筛选、培养和选育"主线,设计了一个由5个相互关联、前后衔接、形成一体的实验组成的综合性研究型实验模块,并进行教学实践。它们是:培养基的配制、分装与灭菌;产蛋白酶细菌的分离与纯化;产蛋白酶细菌的培养发酵与条件优化;产蛋白酶细菌的鉴定;产蛋白酶细菌的紫外诱变育种。     

Sas20 is a highly flexible starch-binding protein in the Ruminococcus bromii cell-surface amylosome

Ruminococcus bromii is a keystone species in the human gut that has the rare ability to degrade dietary resistant starch (RS). This bacterium secretes a suite of starch-active proteins that work together within larger complexes called amylosomes that allow R. bromii to bind and degrade RS. Starch adherence system protein 20 (Sas20) is one of the more abundant proteins assembled within amylosomes, but little could be predicted about its molecular features based on amino acid sequence. Here, we performed a structure–function analysis of Sas20 and determined that it features two discrete starch-binding domains separated by a flexible linker. We show that Sas20 domain 1 contains an N-terminal β-sandwich followed by a cluster of α-helices, and the nonreducing end of maltooligosaccharides can be captured between these structural features. Furthermore, the crystal structure of a close homolog of Sas20 domain 2 revealed a unique bilobed starch-binding groove that targets the helical α1,4-linked glycan chains found in amorphous regions of amylopectin and crystalline regions of amylose. Affinity PAGE and isothermal titration calorimetry demonstrated that both domains bind maltoheptaose and soluble starch with relatively high affinity (K_d ≤ 20 μM) but exhibit limited or no binding to cyclodextrins. Finally, small-angle X-ray scattering analysis of the individual and combined domains support that these structures are highly flexible, which may allow the protein to adopt conformations that enhance its starch-targeting efficiency. Taken together, we conclude that Sas20 binds distinct features within the starch granule, facilitating the ability of R. bromii to hydrolyze dietary RS.     

SEASONAL MODULE DYNAMICS OF TURBINARIA TRIQUETRA (FUCALES,PHAEOPHYCEAE) IN THE SOUTHERN RED SEA1

Module dynamics in the fucoid alga Turbinaria triquetra (J. Agardh) Kützing were studied on a shallow reef flat in the southern Red Sea. Seasonal patterns in thallus density and size were determined, and the initiation, growth, reproduction, and shedding of modules were studied using a tagging approach. The effects of module density and module/thallus size on module initiation, growth, reproduction, and shedding were analyzed, and the occurrence of intraspecific competition among modules was examined. Seasonal variation occurred mainly at the modular level. There was a restricted period of new module formation in the cooler season, followed by fast growth and reproduction, massive shedding of modules from the end of the cooler season onward, and strongly reduced biomass in summer. There was no evidence of suppressed growth in small modules due to intraspecific competition. Module density and thallus/module size had opposite effects on elongation rates. High module densities enhanced maximum elongation rates (fastest‐growing module per thallus), resulting in longer thalli. On the other hand, elongation rates decreased and tissue loss increased with increasing module length. Reproduction had no clear effect on elongation rates, indicating that there was no direct trade‐off between reproduction and growth. The apparent size‐dependence of reproduction was due to delayed fertility in young modules. Module initiation and shedding were independent of module density. Shedding was also independent of module size and reproductive status. We conclude that seasonal changes in the environment affect module initiation, growth, reproduction, and shedding, whereas density and size‐dependent processes mainly affect growth rates.     

The structural basis of alpha-glucan recognition by a family 41 carbohydrate-binding module from Thermotoga maritima

Starch recognition by carbohydrate-binding modules (CBMs) is important for the activity of starch-degrading enzymes. The N-terminal family 41 CBM, TmCBM41 (from pullulanase PulA secreted by Thermotoga maritima) was shown to have alpha-glucan binding activity with specificity for alpha-1,4-glucans but was able to tolerate the alpha-1,6-linkages found roughly every three or four glucose units in pullulan. Using X-ray crystallography, the structures were solved for TmCBM41 in an uncomplexed form and in complex with maltotetraose and 6(3)-alpha-D-glucosyl-maltotriose (GM3). Ligand binding was facilitated by stacking interactions between the alpha-faces of the glucose residues and two tryptophan side-chains in the two main subsites of the carbohydrate-binding site. Overall, this mode of starch binding is quite well conserved by other starch-binding modules. The structure in complex with GM3 revealed a third binding subsite with the flexibility to accommodate an alpha-1,4- or an alpha-1,6-linked glucose.     

Plasticity of the TSG-6 HA-binding loop and mobility in the TSG-6-HA complex revealed by NMR and X-ray crystallography

Tumour necrosis factor-stimulated gene-6 (TSG-6) is a glycosaminoglycan-binding protein expressed during inflammatory and inflammation-like processes. Previously NMR structures were calculated for the Link module of TSG-6 (Link_TSG6) in its free state and when bound to an octasaccharide of hyaluronan (HA(8)). Heparin was found to compete for HA binding even though it interacts at a site that is distinct from the HA-binding surface. Here we present crystallography data on the free protein, and (15)N NMR relaxation data for the uncomplexed and HA(8)-bound forms of Link_TSG6. Although the Link module is comparatively rigid overall, the free protein shows a high degree of mobility in the beta4/beta5 loop and at the Cys47-Cys68 disulfide bond, both of which are regions involved in HA binding. When bound to HA(8), this dynamic behaviour is dampened, but not eliminated, suggesting a degree of dynamic matching between the protein and sugar that may decrease the entropic penalty of complex formation. A further highly dynamic residue is Lys54, which is distant from the HA-binding site, but was previously shown to be involved in heparin binding. When HA is bound, Lys54 becomes less mobile, providing evidence for an allosteric effect linking the HA and heparin-binding sites. A mechanism is suggested involving the beta2-strand and alpha2-helix. The crystal structure of free Link_TSG6 contains five molecules in the asymmetric unit that are highly similar to the NMR structure and support the dynamic behaviour seen near the HA-binding site: they show little or no electron density for the beta4/beta5 loop and display multiple conformations for the Cys47-Cys68 disulfide bond. The crystal structures were used in docking calculations with heparin. An extended interface between a Link_TSG6 dimer and heparin 11-mer was identified that is in excellent agreement with previous mutagenesis and calorimetric data, providing the basis for further investigation of this interaction.