Short-chain esters play a significant role in the food industry as flavor and aroma constituents. Candida antarctica lipase B (CALB) is one of the most effective catalysts for organic synthesis. We constructed a CALB-displaying yeast whole-cell biocatalyst and applied it to esterification from caproic acid and ethanol. CALB was fused with the alpha-agglutinin C-terminal and the signal peptide of Glucoamylase in pICAS, a yeast surface display vector, to construct plasmid pICAS-CALB. An extremely Asn-rich linker, named celAL was inserted in the Xho I of pICAS-CALB to construct plasmid pICAS-celAL-CALB. The fused gene was under the control of GAPDH promoter. After incubated at 30 degrees C for 96 h the lipase hydrolytic activity of the yeast whole cells reached a plateau, 26.26 u/(g x dry cell). In nonaqeous media, the yield of 98.0% ethyl hexanoate was obtained after 24 h esterification from caproic acid and ethanol (the molar ratio of caproic acid : ethanol = 1 : 1.25) using lyophilized CALB displaying yeast whole cells.
Biocatalysis ; Candida ; enzymology ; Caproates ; metabolism ; Cloning, Molecular ; Fungal Proteins ; Genetic Engineering ; Lipase ; biosynthesis ; genetics ; Saccharomyces cerevisiae ; genetics ; metabolism

Polyhydroxyalkanoates (PHAs), as a novel class of biopolymer, are attracting more attention due to their diverse material properties and environment-independent biodegradability. Here we report the preparation of PHA exhibiting efficient antibacterial activity by embedding Nisin, a food additive generally recognized as safe, into poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx), a type of PHA with high biocompatibility. We first prepared Nisin-containing PHBHHx films using solvent casting method. Confocal laser scanning microscopy analysis showed that a well-mixed integrated structure of the films with an even distribution of the Nisin particles in the PHBHHx matrices. Then the antimicrobial activity of PHBHHx/Nisin films against Micrococcus luteus was quantified on agar plate by measuring the size of inhibition zone. Cultivation in liquid media further confirmed the releasing of Nisin from the films and the long-time antibacterial activity. Results showed that the threshold of Nisin concentration for long-time and effective inhibition against bacteria growth is 25 μg/g. These results altogether establish a technological foundation for the application of PHA in biomedicine and food industry.
3-Hydroxybutyric Acid ; chemistry ; Anti-Bacterial Agents ; chemistry ; Caproates ; chemistry ; Micrococcus luteus ; drug effects ; Nisin ; chemistry ; Polyhydroxyalkanoates ; chemistry

OBJECTIVETo observe the proliferation and differentiation capacities of mouse induced pluripotent stem cells (miPSCs) cultured in 3-hydroxybutyrate-co-3-hydroxyhexanoate(PHBHHx) three-dimensional films three-dimension films for the purpose of finding a suitable polymeric biomaterials for forming myocardial patches.

METHODSmiPSCs were recovered, passaged, cultured and identified, then miPSCs divided into the experimental and control groups. MiPSCs in the experimental group were cultured with PHBHHx three-dimension films. MiPSCs in the control group were cultured with traditional culture dish. Stem cell culture medium or differential medium were added to miPSCs to detecte cell vitality by CCK-8 after 72 hours or to measure the cTnT expression of miPSCs through immunofluorescence or the cTnT expression quantity through flow cytometry after 15 days.

RESULTSCell activity assay showed that the absorbance values were 0.836 ± 0.038 in the experimental group, 0.312 ± 0.004 in the control group (P<0.05). Scanning electron microscope (SEM) observation showed that miPSCs grew well on the PHBHHx dimensional films with normal shape. Immunofluorescence results demonstrated positive cTnT expression in both groups and flow cytometry measured cTnT expression was (60.32 ± 1.76)% in the experimental group and (47.54 ± 1.46)% in the control group (P<0.05).

CONCLUSIONSmiPSCs can survive, proliferate and differentiate on PHBHHx dimensional films. miPSCs proliferation and differentiation capacities are significantly higher in PHBHHx three-dimensional films culture compared with the traditional cell culture.

3-Hydroxybutyric Acid ; Animals ; Biocompatible Materials ; Caproates ; Cell Culture Techniques ; Cell Differentiation ; Induced Pluripotent Stem Cells ; Mice

The current study was conducted in order to investigate bone formation using matrigel and angiogenic factors with HA and poly epsilon-caprolactone (HA/PCL) in a rat calvarial defect model. Calvarial defect formation was surgically created in Sprague Dawley rats (n=36). Rats in the control group (CD group, n=6) did not receive a graft. The HA/PCL scaffold was grafted with matrigel (M-HA/PCL group, n=6) or without matrigel (HA/PCL group, n=6); and 100 ng of vascular endothelial growth factor with HA/PCL scaffold containing matrigel (VEGF100 group, n=6), 100 ng (PDGF100 group, n=6) and 300 ng (PDGF300 group, n=6) of PDGF with HA/PCL scaffold containing matrigel were grafted in calvarial defects, respectively. Four weeks after surgery, bone formation was evaluated with micro computed tomography (micro CT) scanning, and histologically. According to the results, bone mineral density was significantly increased in the VEGF100, PDGF100, and PDGF300 groups compared to the HA/PCL group, in which angiogenic factors were not applied. In histological evaluation, more new bone formation around scaffolds was observed in the PDGF100 and the PDGF300 groups, compared with the VEGF100 group. Thus, the results indicate that HA/PCL containing matrigel with VEGF and PDGF is an effective grafting material for enhancement of bone formation in critical-sized bone defects. Especially, due to its price and capacity for bone formation, PDGF may be more effective than VEGF.
Angiogenesis Inducing Agents* ; Animals ; Bone Density ; Caproates ; Collagen ; Drug Combinations ; Lactones ; Laminin ; Osteogenesis ; Proteoglycans ; Rats* ; Rats, Sprague-Dawley ; Transplants ; Vascular Endothelial Growth Factor A

OBJECTIVETo prepare a biodegradable drug-eluting stent in myocardium channel and evaluate its effect on myocardium channel after transmyocardial revascularization (TMR).

METHODSA biodegradable drug-eluting stent was prepared using poly (epsilon-caprolactone) (PCL), bovine serum albumin (BSA), and poly (D, L-lactide-co-glycolide) (PLGA) as material of stent, model protein drug, and drug carrier respectively. The amount of BSA in stent and in vitro released BSA of stent were determined by the Coomassie brilliant blue assay. The mechanical strength of stent was tested by universal material testing machines. The material and structure of stent was characterized by nuclear magnetic resonance spectroscopy. The effect of stent on myocardium channel after TMR was evaluated in vivo by a standard animal model of chronic myocardial ischemia in miniswine.

RESULTSThe stent could carry 13.1 microg BSA per mg of stent and the stent could release about 95% of BSA after 30 days. The stent diminished 80% of initial scale under the stress of 1.7 Mpa. It also kept the myocardium channel patency after TMR.

CONCLUSIONSA biodegradable drug-eluting stent in myocardium channel was successfully prepared. It can sustain the pressure from the heart and achieve the controlled release of drug. The stent can ensure the myocardium channel patency after TMR.

Animals ; Biocompatible Materials ; chemistry ; Blood Vessel Prosthesis ; Caproates ; chemistry ; Cardiac Surgical Procedures ; Disease Models, Animal ; Drug Delivery Systems ; instrumentation ; Heart ; drug effects ; Humans ; Lactones ; chemistry ; Myocardial Ischemia ; drug therapy ; surgery ; Myocardial Revascularization ; instrumentation ; Random Allocation ; Swine ; Swine, Miniature

Copolyesters consisting of 3-hydroxybutyrate (3HB) and 3-hydroxyhexanoate (3HHx) (PHBHHx), a new type of biodegradable material, are receiving considerable attentions recently. The material properties are strongly related to the 3HHx fraction of PHBHHx. As the 3HHx fraction increase, crystallinity and melting point of PHBHHx decrease, flexibility and tractility increase. PHBHHx of different 3HHx fraction can meet different demands of commercial application and research. Aeromonas are the best studied PHBHHx-producing strains. Recent studies have been focused on optimizations of fermentative culture media and culture conditions for low-cost and efficient fermentative production. Aliphatic substrates such as long-chain fatty acid and soybean oil were used in the PHBHHx fermentation as the sole carbon source and energy source. Two-stage fermentation method was also developed for more efficient PHBHHx production. While studies on Aeromonas hydrophila revealed that the monomer composition of PHBHHx could not easily be regulated by fermentative process engineering methods such as changing substrates and fermentative conditions because precursors involved in the PHBHHx synthesis were all from the beta-oxidation pathway. In this study, phbA gene encoding beta-ketothiolase and phbB gene encoding acetoacetyl-CoA reductase were introduced into a PHBHHx-producing strain Aeromonas hydrophila 4AK4 so as to provide a new 3HB precursors synthesis way. phbA gene encodes beta-ketothiolase which can catalyze two acetyl-CoA to form acetoacetyl-CoA; phbB gene encodes acetoacetyl-CoA reductase catalyzing acetoacetly-CoA into 3HB-CoA which is the precursor of 3HB. The introduced novel 3-hydroxybutyrate precursor synthesis pathway allowed the recombinant strain to use unrelated carbon source such as gluconate to provide 3HB precursors for PHBHHx synthesis. Shake-flask experiments were carried out to produce PHBHHx of controllable monomer composition and fermentations in 5 L fermentor were also proceeded for confirmation of these result in large-scale culture. In flask culture, it was possible to reduce the 3HHx mol fraction in PHBHHx from 15 % in the wild type to 3% - 12% in the recombinant by simply changing the ratio of gluconate to lauric acid in the culture media. When lauric acid was used as the sole carbon source, 51.5 g/L Cell Dry Weight (CDW) containing 62 % PHBHHx with 9.7 % 3HHx mol fraction was obtained in 56 hours of fermentation in a 5 liter fermentor. When co-substrates of sodium gluconate and lauric acid (1:1) were used as carbon sources, 32.8 g/L CDW containing 52 % PHBHHx with 6.7% 3HHx mol fraction was obtained in 48 hours of fermentation. These results showed the possibility for fermentative production of PHBHHx with controllable monomer composition.
3-Hydroxybutyric Acid ; metabolism ; Acetyl-CoA C-Acyltransferase ; genetics ; metabolism ; Aeromonas hydrophila ; enzymology ; genetics ; metabolism ; Alcohol Oxidoreductases ; genetics ; metabolism ; Bacterial Proteins ; genetics ; metabolism ; Biotechnology ; methods ; Caproates ; metabolism ; Fermentation ; genetics ; physiology ; Lauric Acids ; metabolism

To study the attachment, proliferation and differentiation of neural stem cells (NSCs) on surface modified PHBHHx films and to establish the theory of PHBHHx application in NSCs-based brain tissue engineering. PHBHHx film was fabricated by a solution-casting method, and the morphology of the film was observed under scanning electron microscopy(SEM). The films were treated by NaOH or lipase, then the surface hydrophilic property was characterized using water contact angle measurement. NSCs were isolated from the cerebral cortex of rat embryos on embryonic day 14.5, and cultured on surface treated PHBHHx films. The morphology of NSCs attached on the film was visualized under SEM, and the survival and differentiation of NSCs were observed through immunocytochemical staining. Compared with the untreated PHBHHx films, the water contact angle of NaOH or lipase treated PHBHHx films decreased dramatically, and the number of NSCs attached significantly increased. NSCs survived well on treated PHBHHx films and differentiated into neurons and glial cells. The amelioration of hydrophilic property of PHBHHx film improved its biocompatibility with NSCs. PHBHHx can serve as a novel CNS tissue engineering biomaterial applied for NSCs transplantation, brain repairing and regeneration.
3-Hydroxybutyric Acid ; chemistry ; Animals ; Caproates ; chemistry ; Cell Adhesion ; physiology ; Cell Differentiation ; drug effects ; Cell Proliferation ; Cells, Cultured ; Cerebral Cortex ; cytology ; Coated Materials, Biocompatible ; chemistry ; Embryonic Stem Cells ; cytology ; Female ; Neural Stem Cells ; cytology ; Rats ; Surface Properties ; Tissue Engineering

Poly (3-hydroxybutyrate-co-3-hydroxyhexanoate, PHBHHx) has superior mechanical and biocompatibility that may enable it to meet cardiovascular tissue engineering applications. We developed hybrid materials based on decellularized xenogenic vascular scaffolds that were coated with PHBHHx to investigate the intravascular biocompatibility. The hybrid patches were implanted in the rabbit abdominal aorta (hybrid patch, n = 12). Only decellularized xenogenic vascular scaffolds were implanted without coating as control (uncoated patch, n = 12). The patches were explanted and examined histologically, and biochemically at 1, 4 and 12 weeks after the surgery. The hybrid patches maintained original shapes, covered by confluent layer of cells and had less calcification than uncoated control. The results indicated that PHBHHx coating reduced calcification, promoted the repopulation of hybrid patch with recipients cells. In conclusion, PHBHHx showed remarkable intravascular biocompatibility and would benefit endothelization which would be a useful candidate for lumen of cardiovascular tissue engineering.
3-Hydroxybutyric Acid ; chemistry ; Animals ; Aorta, Abdominal ; surgery ; Caproates ; chemistry ; Cell Adhesion ; Coated Materials, Biocompatible ; chemistry ; pharmacology ; Goats ; Humans ; Implants, Experimental ; Pulmonary Artery ; cytology ; drug effects ; Rabbits ; Surface Properties ; Tissue Engineering ; Tissue Scaffolds

BACKGROUNDA major shortcoming in tissue engineered blood vessels (TEBVs) is the lack of healthy and easily attainable smooth muscle cells (SMCs). Smooth muscle progenitor cells (SPCs), especially from peripheral blood, may offer an alternative cell source for tissue engineering involving a less invasive harvesting technique.

METHODSSPCs were isolated from 5-ml fresh rat peripheral blood by density-gradient centrifugation and cultured for 3 weeks in endothelial growth medium-2-MV (EGM-2-MV) medium containing platelet-derived growth factor-BB (PDGF BB). Before seeded on the synthesized scaffold, SPC-derived smooth muscle outgrowth cell (SOC) phenotypes were assessed by immuno-fluorescent staining, Western blot analysis, and reverse transcription polymerase chain reaction (RT-PCR). The cells were seeded onto the silk fibroin-modified poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (SF-PHBHHx) scaffolds by 6x10(4) cells/cm2 and cultured under the static condition for 3 weeks. The growth and proliferation of the seeded cells on the scaffold were analyzed by 3-(4,5-dimethylthiazol-2-yl)-diphenyltetrazolium bromide (MTT) assay, scanning electron microscope (SEM), and 4,6-diamidino-2-phenylindole (DAPI) staining.

RESULTSSOCs displayed specific "hill and valley" morphology, expressed the specific markers of the SMC lineage: smooth muscle (SM) alpha-actin, calponin and smooth muscle myosin heavy chain (SM MHC) at protein and messenger ribonucleic acid (mRNA) levels. RT-PCR results demonstrate that SOCs also expressed smooth muscle protein 22alpha (SM22alpha), a contractile protein, and extracellular matrix components elastin and matrix Gla protein (MGP), as well as vascular endothelial growth factor (VEGF). After seeded on the SF-PHBHHx scaffold, the cells showed excellent metabolic activity and proliferation.

CONCLUSIONSPCs isolated from peripheral blood can be differentiated into the SMCs in vitro and have an impressive growth potential in the biodegradable synthesized scaffold. Thus, SPCs may be a promising cell source for constructing TEBVs.

3-Hydroxybutyric Acid ; chemistry ; Animals ; Blood Vessels ; cytology ; Caproates ; chemistry ; Cell Adhesion ; Cell Differentiation ; Cell Proliferation ; Immunophenotyping ; Microscopy, Electron, Scanning ; Muscle, Smooth, Vascular ; cytology ; Myocytes, Smooth Muscle ; cytology ; RNA, Messenger ; analysis ; Rats ; Tissue Engineering ; Vascular Endothelial Growth Factor A ; genetics

No abstract available.
Drug Monitoring ; Mycophenolic Acid