The effect of the controlled release of nerve growth factor from collagen gel on the efficiency of neural cell culture.

In this study, we tested the hypothesis that the amount of nerve growth factor (NGF) required for pheochromocytoma (PC12) cell culture can be dramatically reduced by controlled release of NGF from a collagen gel coating on the culture surface. Cells were cultured on collagen gels loaded with various amounts of NGF. As a control, PC12 cells were cultured on collagen gels with daily addition of various amounts of NGF to the culture medium. After an initial 12h burst, NGF was steadily released from the gels for 4 days. Apoptotic activity and cell viability were determined using terminal uridine nick end labeling and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, respectively. Neuronal differentiation was determined using immunocytochemistry and Western blot analysis. Compared to 100 ng NGF daily addition (300 ng over 3 days), 1 ng NGF daily addition showed dramatically decreased cell viability and neuronal differentiation and increased apoptotic activity. In contrast, collagen gels loaded with 10 ng NGF yielded cell viability, apoptotic activity, and neuronal differentiation similar to those of culture with 100 ng NGF daily addition. Our method reduced the amount of NGF required for PC12 cell culture to 1/3th of that used in daily addition without affecting cell viability, apoptosis, or differentiation. This method could economize large-scale culture of stem cells by reducing the amount of costly growth factors needed.

Porous poly(lactic-co-glycolic acid) microsphere as cell culture substrate and cell transplantation vehicle for adipose tissue engineering.

Tissue engineering often requires ex vivo cell expansion to obtain a large number of transplantable cells. However, the trypsinization process used to harvest ex vivo expanded cells for transplantation interrupts interactions between cultured cells and their extracellular matrices, facilitating apoptosis and consequently limiting the therapeutic efficacy of the transplanted cells. In the present study, open macroporous poly(lactic-co-glycolic acid) (PLGA) microspheres were used as a cell culture substrate to expand human adipose-derived stromal cells (ASCs) ex vivo and as a cell transplantation vehicle for adipose tissue engineering, thus avoiding the trypsinization necessary for transplantation of ex vivo expanded cells. Human ASCs cultured on macroporous PLGA microspheres in stirred suspension bioreactors expanded 3.8-fold over 7 days and differentiated into an adipogenic lineage. The apoptotic activity of ASCs cultured on microspheres was significantly lower than that of trypsinized ASCs. ASCs cultured on microspheres survived much better than trypsinized ASCs upon transplantation. The implantation of ASCs cultured on microspheres resulted in much more extensive adipose tissue formation than the implantation of ASCs cultured on plates, trypsinized, and subsequently mixed with microspheres. Ex vivo cell expansion and transplantation using this system would improve the therapeutic efficacy of cells over the current methods used for tissue engineering.

Biodegradation of gaseous styrene by Brevibacillus sp. using a novel agitating biotrickling filter.

A novel biofilter with an agitator to control excessive biomass accumulation, the agitating biotrickling filter (ABTF) system, was developed for treatment of gaseous styrene using Brevibacillus sp. as the sole microorganism the ABTF exhibited an elimination capacity of 3 kg styrene m(-3) day(-1). After 110 days, the biodegradation efficiency decreased because of the clogging. The excess biomass was effectively removed by agitation. After the first agitation step, 42.4 g biomass was eliminated, and the removal efficiency increased from 60% to 95%. Stable operation of the ABTF was achieved by controlling the biomass accumulation via the agitation of the filter bed.

Enantioconvergent bioconversion of p-chlorostyrene oxide to (R)-p-chlorophenyl-1,2-ethandiol by the bacterial epoxide hydrolase of Caulobacter crescentus.

The enantioselective hydrolysis of eight racemic styrene oxide derivatives has been investigated by using the recombinant cell containing epoxide hydrolase (EH) of Caulobacter crescentus. Some styrene oxide derivatives were hydrolyzed via enantioconvergent manner so that enantiopure diol products could be prepared with a 100% theoretical yield. The recombinant cell containing C. crescentus EH exhibited an ability to hydrolyze racemic p-chlorostyrene oxide the most enantioconvergently, thus affording the formation of the corresponding (R)-diol with enantiomeric excess (ee) as high as 95% and a 72% yield in preparative-scale (16.8 g/l) bioconversion.

Enhanced heterologous production of desosaminyl macrolides and their hydroxylated derivatives by overexpression of the pikD regulatory gene in Streptomyces venezuelae.

To elevate the production level of heterologous polyketide in Streptomyces venezuelae, an additional copy of the positive regulatory gene pikD was introduced into the pikromycin (Pik) polyketide synthase (PKS) deletion mutant of S. venezuelae ATCC 15439 expressing tylosin PKS genes. The resulting mutant strain showed enhanced production of both tylactone (TL) and desosaminyl tylactone (DesTL) of 2.7- and 17.1-fold, respectively. The notable increase in DesTL production strongly suggested that PikD upregulates the expression of the desosamine (des) biosynthetic gene cluster. In addition, two hydroxylated forms of DesTL were newly detected from the extract of this mutant. These hydroxylated forms presumably resulted from a PikD-dependent increase in expression of the pikC gene that encodes P450 hydroxylase. Gene expression analysis by reverse transcriptase PCR and bioconversion experiments of 10-deoxymethynolide, narbonolide, and TL into the corresponding desosaminyl macrolides indicated that PikD is a positive regulator of the des and pikC genes, as well as the Pik PKS genes. These results demonstrate the role of PikD as a pathway-specific positive regulator of the entire Pik biosynthetic pathway and its usefulness in the development of a host-vector system for efficient heterologous production of desosaminyl macrolides and novel hydroxylated compounds.

Production and characterization of monoclonal antibody to botulinum neurotoxin type B light chain by phage display.

A monoclonal antibody to the light chain of botulinum neurotoxin type B (BoNT/B) was generated and its protective activity was evaluated in vivo. A chimeric rabbit/human Fab library was generated using bone marrow and spleen cDNAs of rabbits immunized with the BoNT/B light chain, and three monoclonal antibodies specific to the catalytic domain of BoNT/B were isolated. One of these clones, BCXRH1, was specific to a conformation-dependent epitope, and partially neutralized the BoNT/B complex in vivo.

Combined therapy with human cord blood cell transplantation and basic fibroblast growth factor delivery for treatment of myocardial infarction.

BACKGROUND: Transplanting cord blood-derived cells has been shown to augment neovascularization in ischaemic tissue. AIM: To test whether sustained delivery of basic fibroblast growth factor (bFGF) enhances the efficacy of angiogenic cord blood mononuclear cell (CBMNC) transplantation therapy in treating myocardial infarction. METHODS: Three weeks after myocardial infarction, Sprague-Dawley rats were randomised to either injection of medium only (control), CBMNC transplantation, sustained bFGF delivery, or combined CBMNC transplantation and sustained bFGF delivery. Six weeks after treatment, tissue formation, neovascularization, and apoptotic activity in the infarct regions were evaluated by histology and immunohistochemistry. Left ventricular (LV) dimensions and function were evaluated by magnetic resonance imaging. RESULTS: Combined bFGF delivery and CBMNC transplantation significantly enhanced neovascularization in the ischaemic myocardium, as compared with either therapy alone. The enhanced neovascularization was likely due to increased VEGF and bFGF expression. The combined therapy also exhibited a reduced infarct area and apoptosis in the ischaemic myocardium, as compared with either individual therapy. The combined therapy did not attenuate LV dilation or increase ejection fraction significantly over either individual therapy. CONCLUSION: This study demonstrates that sustained bFGF delivery enhances the angiogenic efficacy of CBMNC transplantation in rat myocardial infarction models.

Bioconversion of 12-, 14-, and 16-membered ring aglycones to glycosylated macrolides in an engineered strain of Streptomyces venezuelae.

To develop a system for combinatorial biosynthesis of glycosylated macrolides, Streptomyces venezuelae was genetically manipulated to be deficient in the production of its macrolide antibiotics by deletion of the entire biosynthetic gene cluster encoding the pikromycin polyketide synthases and desosamine biosynthetic enzymes. Two engineered deoxysugar biosynthetic pathways for the biosynthesis of thymidine diphosphate (TDP)-D-quinovose or TDP-D-olivose in conjunction with the glycosyltransferase-auxiliary protein pair DesVII/DesVIII derived from S. venezuelae were expressed in the mutant strain. Feeding the representative 12-, 14-, and 16-membered ring macrolactones including 10-deoxymethynolide, narbonolide, and tylactone, respectively, to each mutant strain capable of producing TDP-D-quinovose or TDP-D-olivose resulted in the successful production of the corresponding quinovose- and olivose-glycosylated macrolides. In mutant strains where the DesVII/DesVIII glycosyltransferase-auxiliary protein pair was replaced by TylMII/TylMIII derived from Streptomyces fradiae, quinovosyl and olivosyl tylactone were produced; however, neither glycosylated 10-deoxymethynolide nor narbonolide were generated, suggesting that the glycosyltransferase TylMII has more stringent substrate specificity toward its aglycones than DesVII. These results demonstrate successful generation of structurally diverse hybrid macrolides using a S. venezuelae in vivo system and provide further insight into the substrate flexibility of glycosyltransferases.

Engineered adipose tissue formation enhanced by basic fibroblast growth factor and a mechanically stable environment.

Engineered adipose tissue can be used in plastic and reconstructive surgery to augment soft tissue lost due to mastectomy or lumpectomy. The three-dimensional space provided by a scaffold capable of withstanding in vivo compressive forces and neovascularization may promote engineered adipose tissue formation. The objective of this study was to determine whether voluminous adipose tissue can be engineered by combining a mechanically stable environment with basic fibroblast growth factor (bFGF). Mechanical support structures, fabricated from biodegradable synthetic polymers, were placed into subcutaneous pockets of athymic mice. Human preadipocytes, containing fibrin matrix, with (group 1) or without (group 2) bFGF were injected into the space created by the support structures. Additionally, human preadipocytes containing fibrin matrix, with (group 3) or without (group 4) bFGF, were injected into subcutaneous spaces without support structures. Six weeks after implantation, the original implant volume was approximately maintained in groups 1 and 2, whereas groups 3 and 4 showed significant implant shrinkage. Adipogenesis and angiogenesis were more extensive in the group 1 than any other group. The fraction of human nuclear antigen-positive adipocytes in the implant was highest in group 1. Mouse adipocyte-specific genes were also expressed in the implants, again at the highest levels in group 1. Implanted preadipocyte apoptosis was significantly reduced in the groups treated with bFGF (groups 1 and 3) as opposed to those without (groups 2 and 4). This study demonstrates that combining a mechanically stable environment with bFGF can promote voluminous adipose tissue regeneration. This adipogenesis was likely promoted by the mechanically stable three-dimensional space, enhanced neovascularization, implanted cell survival, and host adipogenic cell migration. The method described in this study could be useful to augment adipose tissue used in plastic and reconstructive surgery.

An economical and highly productive cell-free protein synthesis system utilizing fructose-1,6-bisphosphate as an energy source.

In this study, we describe the development of a cost effective and highly productive cell-free protein synthesis system derived from Escherichia coli. Through the use of an optimal energy source and cell extract, approximately 1.3mg/mL of protein was generated from a single batch reaction at greatly reduced reagent costs. Compared to previously reported systems, the described method yields approximately 14-fold higher productivity per unit reagent cost making this cell-free synthesis technique a promising alternative for more efficient protein production.

Basic fibroblast growth factor promotes bone marrow stromal cell transplantation-mediated neural regeneration in traumatic brain injury.

The current study was designed to evaluate the effects of basic fibroblast growth factor (bFGF) on human BMSC (hBMSC) transplantation-mediated neural regeneration in traumatic brain injury (TBI). Fibrin gel was used as a delivery vehicle to release bFGF locally in the TBI sites in a controlled manner. To test this hypothesis, hBMSCs suspended in fibrin gel containing bFGF were transplanted to rat TBI sites. Transplantation of hBMSCs suspended in fibrin gel without bFGF served as a control. hBMSC transplantation and bFGF treatment showed enhanced neural tissue regeneration than that of the control. The infarction volume and apoptotic activity of the transplanted hBMSCs were significantly decreased, and functional outcomes were significantly improved in the hBMSC transplantation and bFGF treatment group than in the control group. This study demonstrates that bFGF significantly enhances histological and functional recovery when used in hBMSC transplantation therapy in TBI.

The behavior of neural stem cells on biodegradable synthetic polymers.

The biocompatibility of polymer scaffolds as neural stem cell transplantation matrices has not yet been studied extensively. In this study, we evaluated the biocompatibility of various biodegradable polymers for neural stem cells. The biocompatibility tests were performed by culturing hippocampal progenitor cells (HiB5) on films of poly(lactic-co-glycolic acid) (PLGA), poly(L-lactide-co-epsilon-caprolactone) (PLCL) and poly(L-lactic acid) (PLLA) or in the presence of extracts from these polymers. Specifically, the viability, mitochondrial metabolic activity, proliferation, apoptosis and neurite out-growth of HiB5 cells were examined in biocompatibility tests. Among the tested polymers, PLGA performed best with respect to cell viability, mitochondrial metabolic activity and apoptotic activity. Compared to the other polymers, PLLA showed the worst results in all categories evaluated. PLGA also showed favorable results for neurite out-growth of HiB5 cells. The results of this study demonstrate the promising biocompatibility of PLGA as a scaffold for neural stem cell transplantation for nerve regeneration.

Additive effect of endothelial progenitor cell mobilization and bone marrow mononuclear cell transplantation on angiogenesis in mouse ischemic limbs.

The methods of therapeutic angiogenesis include endothelial progenitor cell (EPC) mobilization with cytokines [e.g., granulocyte colony-stimulating factor (G-CSF)] and bone marrow mononuclear cell (BMMNC) transplantation. Combined angiogenic therapies may be superior to a single angiogenic therapy for the treatment of limb ischemia. Therefore, we investigated whether the angiogenic efficacy of a combination of two angiogenic strategies is superior to either strategy alone. One day after the surgical induction of hindlimb ischemia, mice were randomized to receive either no treatment, EPC mobilization with G-CSF administration, BMMNC transplantation using a fibrin matrix, or a combination of EPC mobilization with BMMNC transplantation using a fibrin matrix. EPC mobilization with G-CSF or BMMNC transplantation using a fibrin matrix significantly increased the microvessel density compared with no treatment. Importantly, a combination of EPC mobilization with BMMNC transplantation using a fibrin matrix further increased the densities of microvessels and BrdU-positive capillaries compared to either strategy alone. Basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) expression was higher in the EPC mobilization with G-CSF or BMMNC transplantation group than in the no treatment group. The combination therapy of EPC mobilization with G-CSF and BMMNC transplantation resulted in more extensive expression of bFGF and VEGF than the single therapy of either EPC mobilization with G-CSF treatment or BMMNC transplantation. This study demonstrates that the combination therapy of BMMNC transplantation and EPC mobilization potentiates the angiogenic efficacy of either single therapy in mouse limb ischemia models.

Prolonged cell-free protein synthesis using dual energy sources: Combined use of creatine phosphate and glucose for the efficient supply of ATP and retarded accumulation of phosphate.

The accumulation of inorganic phosphate inhibits protein synthesis in cell-free protein synthesis reactions that are energized by high-energy-phosphate-containing compounds. This study developed a new scheme for supplying energy using dual energy sources to enhance the regeneration of ATP and lower the rate of phosphate accumulation. In the proposed scheme, where creatine phosphate (CP) and glucose were simultaneously used as the energy sources, the phosphate released from the CP was subsequently used in the glycolytic pathway for the utilization of the glucose, which enhanced the ATP supply and reduced the rate of inorganic phosphate accumulation. When tested against different proteins, the developed method produced 2-3 times more protein than the conventional ATP regeneration methods using single energy sources.

Generation of functional dopamine neurons from neural precursor cells isolated from the subventricular zone and white matter of the adult rat brain using Nurr1 overexpression.

Neural precursor (NP) cells from adult mammalian brains can be isolated, expanded in vitro, and potentially used as cell replacement source material for treatment of intractable brain disorders. Reduced ethical concerns, lack of teratoma formation, and possible ex vivo autologous transplantation are critical advantages to using adult NP donor cells over cells from fetal brain tissue or embryonic stem cells. However, the usage of adult NP cells is limited by the ability to induce specific neurochemical phenotypes in these cells. Here, we demonstrate induction of a dopaminergic phenotype in NP cells isolated from the subventricular zone (SVZ) and white matter of rodent adult brains using overexpression of the nuclear receptor Nurr1 in vitro. Forced expression of Nurr1, a transcriptional factor specific to midbrain dopamine (DA) neuron development, caused in the adult cells an acquisition of the DA neurotransmitter phenotype and sufficient differentiation toward morphologically, phenotypically, and ultrastructurally mature DA neurons. Co-expression of neurogenic factor Mash1 and treatment with neurogenic cytokines brain-derived neurotrophic factor and neurotrophin-3 greatly enhanced Nurr1-induced DA neuron yield. The Nurr1-induced DA neurons demonstrated in vitro presynaptic DA neuronal functionality, releasing DA neurotransmitter in response to depolarization stimuli and specific DA reuptake. Furthermore, Nurr1-engineered adult SVZ NP cells survived, integrated, and differentiated into DA neurons in vivo that can reverse the behavioral deficit in the host striatum of parkinsonian rats. These findings open the possibility for the use of precursor cells from adult brains as a cell source for neuronal replacement treatment of Parkinson disease. Disclosure of potential conflicts of interest is found at the end of this article.

A neutralizable epitope is induced on HGF upon its interaction with its receptor cMet.

A new conformational neutralizable epitope is created on heptocyte growth factor (HGF), when it interacts with its receptor, cMet. By immunizing rabbits with HGF-cMet complex, we successfully generated a monoclonal antibody (SFN68) that inhibits HGF-cMet interaction, and blocks the biological function mediated by HGF. To define the epitope, we screened out an epitope-mimicking peptide, KSLSRHDHIHHH, from a phage display of combinatorial peptide library. In molecular mimicry this peptide bound to cMet and inhibited HGF-cMet interaction. No humoral response was induced to this epitope-mimicking peptide when immunization was done with HGF alone.

Development and mathematical modeling of a two-stage reactor system for trichloroethylene degradation using Methylosinus trichosporium OB3b.

A two-stage reactor system was developed for the continuous degradation of gas-phase trichloroethylene (TCE). Methylosinus trichosporium OB3b was immobilized on activated carbon in a TCE degradation reactor, trickling biofilter (TBF). The TBF was coupled with a continuous stirred tank reactor (CSTR) to allow recirculation of microbial cells from/to the TBF for the reactivation of inactivated cells during TCE degradation. The mass transfer aspect of the TBF was analyzed, and mass transfer coefficient of 3.9 h(-1) was estimated. The loss of soluble methane monooxygenase (sMMO) activity was modeled based on a material balance on the CSTR and TBF, and transformation capacity (T (c)) was determined to be 20.2 micromol mg(-1). Maximum TCE degradation rate of 525 mg 1(-1) d(-1) was obtained and reactor has been stably operated for more than 270 days.

Functional analysis of desVIII homologues involved in glycosylation of macrolide antibiotics by interspecies complementation.

The DesVIII is an auxiliary protein which enhances the transfer of TDP-d-desosamine catalyzed by DesVII glycosyltransferase in the biosynthesis of macrolide antibiotics, neomethymycin, methymycin and pikromycin, in Streptomyces venezuelae ATCC 15439. Homologues of the desVIII gene are present in a number of aminosugar-containing antibiotic biosynthetic gene clusters including eryCII from the erythromycin producer Saccharopolyspora erythraea, oleP1 from the oleandomycin producer Streptomyces antibioticus, dnrQ from the doxorubicin producer Streptomyces peucetius, and tylMIII from the tylosin producer Streptomyces fradiae. In order to gain further insight into the function of these DesVIII homologues, interspecies complementation experiments were carried out by expressing each gene in a desVIII deletion mutant strain of S. venezuelae. Complementation by expressing EryCII, OleP1, and DnrQ in this mutant strain restored the production of glycosylated macrolides to an approximate level of 66%, 26% and 26%, respectively, compared to self-complementation by DesVIII. However, expression of TylMIII did not restore the antibiotic production. These results suggest that the DesVIII homologues (except for TylMIII) can functionally replace the native DesVIII for glycosylation to proceed in vivo and their functions are similar in acting as glycosyltransferase auxiliary proteins. The requirement of glycosyltransferase auxiliary protein seems to be more widespread in polyketide biosynthetic pathways than previously known.

A poly(lactide-co-glycolide)/hydroxyapatite composite scaffold with enhanced osteoconductivity.

Biodegradable polymer/ceramic scaffolds can overcome the limitations of conventional ceramic bone substitutes. However, the conventional methods of polymer/ceramic scaffold fabrication often use organic solvents, which might be harmful to cells or tissues. Moreover, scaffolds fabricated with the conventional methods have limited ceramic exposure on the scaffold surface since the polymer solution envelopes the ceramic particles during the fabrication process. In this study, we developed a novel fabrication method for the efficient exposure of ceramic onto the scaffold surface, which would enhance the osteoconductivity and wettability of the scaffold. Poly(D,L-lactide-co-glycolide)/nanohydroxyapatite (PLGA/HA) scaffolds were fabricated by the gas foaming and particulate leaching (GF/PL) method without the use of organic solvents. Selective staining of ceramic particles indicated that HA nanoparticles exposed to the scaffold surface were observed more abundantly in the GF/PL scaffold than in the conventional solvent casting and particulate leaching (SC/PL) scaffold. Both types of scaffolds were implanted to critical size defects in rat skulls for 8 weeks. The GF/PL scaffolds exhibited significantly enhanced bone regeneration when compared with the SC/PL scaffolds. Histological analyses and microcomputed tomography of the regenerated tissues showed that bone formation was more extensive on the GF/PL scaffolds than on the SC/PL scaffolds. Compared with the SC/PL scaffolds, the enhanced bone formation on the GF/PL scaffolds may result from the higher exposure of HA nanoparticles to the scaffold surface. These results show that the biodegradable polymer/ceramic composite scaffolds fabricated with the novel GF/PL method can enhance bone regeneration compared with those fabricated with the conventional SC/PL method.

Use of signal sequences as an in situ removable sequence element to stimulate protein synthesis in cell-free extracts.

This study developed a method to boost the expression of recombinant proteins in a cell-free protein synthesis system without leaving additional amino acid residues. It was found that the nucleotide sequences of the signal peptides serve as an efficient downstream box to stimulate protein synthesis when they were fused upstream of the target genes. The extent of stimulation was critically affected by the identity of the second codons of the signal sequences. Moreover, the yield of the synthesized protein was enhanced by as much as 10 times in the presence of an optimal second codon. The signal peptides were in situ cleaved and the target proteins were produced in their native sizes by carrying out the cell-free synthesis reactions in the presence of Triton X-100, most likely through the activation of signal peptidase in the S30 extract. The amplification of the template DNA and the addition of the signal sequences were accomplished by PCR. Hence, elevated levels of recombinant proteins were generated within several hours.