High cell-density expression system: a novel method for extracellular production of difficult-to-express proteins

Yeast's extracellular expression provides a cost-efficient means of producing industrially useful recombinant proteins. However, depending on the protein to be expressed, the production results in a poor yield, which is occasionally accompanied with loss of the expression plasmid and hence hampered growth of the host in the inducing medium. Here we propose an alternative approach, high cell-density expression, to improve the yield of a certain range of so-called difficult-to-express proteins. In this expression system, recombinant yeast cells resting in stationary phase (OD(660)=3-4) are suspended in a small aliquot of inducing medium to form a high cell-density culture (e.g., OD(660)=15). When applied to the yeast strains harboring Lentinula edodes laccase (Lcc1 or Lcc4) expressing plasmids, the high cell-density system allowed the host cells to synthesize elevated amounts of the laccase which resulted in >1000- to 6000-fold higher yield than those synthesized in a classical growth-associated manner. The resting cells required aerobic agitation for the maximum production. The production system also worked for other foreign enzymes but not for beta-galactosidase from Aspergillus oryzae or Escherichia coli, likely suggesting an involvement of chaperons that act on a certain range of secretory proteins.     

Cobalt limitation of growth and mercury methylation in sulfate-reducing bacteria

Sulfate-reducing bacteria (SRB) have been identified as the primary organisms responsible for monomethylmercury (MeHg) production in aquatic environments, but little is known of the physiologyand biochemistry of mercury(Hg) methylation. Corrinoid compounds have been implicated in enzymatic Hg methylation, although recent experiments with a vitamin B12 inhibitor indicated that incomplete-oxidizing SRB likely do not use a corrinoid-enzyme for Hg methylation, whereas experiments with complete-oxidizing SRB were inconclusive due to overall growth limitation. Here we explore the role of corrinoid-containing methyltransferases, which contain a cobalt-reactive center, in Hg methylation. To this end, we performed cobalt-limitation experiments on two SRB strains: Desulfococcus multivorans, a complete-oxidizer that uses the acetyl-CoA pathway for major carbon metabolism, and Desulfovibrio africanus, an incomplete-oxidizer that does not contain the acetyl-CoA pathway. Cultures of D. multivorans grown with no direct addition of Co or B12 became cobalt-limited and produced 3 times less MeHg per cell than control cultures. Differences in growth rate and Hg bioavailability do not account for this large decrease in MeHg production upon Co limitation. In contrast, the growth and Hg methylation rates of D. africanus cultures remained nearly constant regardless of the inorganic cobalt and vitamin B12 concentrations in the medium. These results are consistent with mercury being methylated by different pathways in the two strains: catalyzed by a B12-containing methyltransferase in D. multivorans and a B12-independent methyltransferase in D. africanus. If complete-oxidizing SRB like D. multivorans account for the bulk of MeHg production in coastal sediments as reported, the ambient Co concentration and speciation may control the rate of Hg methylation.     

Role of the temperature distribution on the PN junction behaviour in the electro‐thermal simulation

Electro‐thermal simulations of a PIN‐diode based on the finite‐element method, show a non‐uniform temperature distribution inside the device during switching transients. Hence, the implicit assumption of a uniform temperature distribution when coupling an analytical electrical model and a thermal model yields inaccurate electro‐thermal behaviour of the PIN‐diode so far. The idea of including non‐uniform temperature distribution into power semiconductor device models is not new, as accurate electro‐thermal simulations are required for designing compact power electronic systems (as IC or MCM). Instead of using a one‐dimensional finite difference or element method, the bond graphs and the hydrodynamic method are utilized to build an electro‐thermal model of the PIN‐diode. The results obtained by this original technique are compared with those obtained by a commercial finite‐element simulator. The results are similar but the computation effort of the proposed technique is a fraction of that required by finite‐element simulators. Moreover, the proposed technique may be applied easily to other power semiconductor devices. Copyright © 2004 John Wiley & Sons, Ltd.     

Rho/ROCK Pathway and Noncoding RNAs: Implications in Ischemic Stroke and Spinal Cord Injury

Ischemic strokes (IS) and spinal cord injuries (SCI) are major causes of disability. RhoA is a small GTPase protein that activates a downstream effector, ROCK. The up-regulation of the RhoA/ROCK pathway contributes to neuronal apoptosis, neuroinflammation, blood-brain barrier dysfunction, astrogliosis, and axon growth inhibition in IS and SCI. Noncoding RNAs (ncRNAs), such as microRNAs (miRNAs) and long noncoding RNAs (lncRNAs), were previously considered to be non-functional. However, they have attracted much attention because they play an essential role in regulating gene expression in physiological and pathological conditions. There is growing evidence that ROCK inhibitors, such as fasudil and VX-210, can reduce injury in IS and SCI in animal models and clinical trials. Recently, it has been reported that miRNAs are decreased in IS and SCI, while lncRNAs are increased. Inhibiting the Rho/ROCK pathway with miRNAs alleviates apoptosis, neuroinflammation, oxidative stress, and axon growth inhibition in IS and SCI. Further studies are required to explore the significance of ncRNAs in IS and SCI and to establish new strategies for preventing and treating these devastating diseases.     

Platinum on Carbon‐Catalyzed Precise Reduction Control of Trichloromethyl to Geminal‐Dichloromethyl Groups

Geminal‐dichloromethyl derivatives could be efficiently synthesized by the highly chemoselective platinum on carbon‐catalyzed mono‐dechlorination of trichloromethyl substrates in dimethylacetamide (DMA) as a specific solvent at 25 °C under a hydrogen atmosphere.     

Functional Graphene for Peritumoral Brain Microenvironment Modulation Therapy in Glioblastoma

Peritumoral brain invasion is the main target to cure glioblastoma. Chemoradiotherapy and targeted therapies fail to combat peritumoral relapse. Brain inaccessibility and tumor heterogeneity explain this failure, combined with overlooking the peritumor microenvironment. Reduce graphene oxide (rGO) provides a unique opportunity to modulate the local brain microenvironment. Multimodal graphene impacts are reported on glioblastoma cells in vitro but fail when translated in vivo because of low diffusion. This issue is solved by developing a new rGO formulation involving ultramixing during the functionalization with polyethyleneimine (PEI) leading to the formation of highly water-stable rGO-PEI. Wide mice brain diffusion and biocompatibility are demonstrated. Using an invasive GL261 model, an anti-invasive effect is observed. A major unexpected modification of the peritumoral area is also observed with the neutralization of gliosis. In vitro, mechanistic investigations are performed using primary astrocytes and cytokine array. The result suggests that direct contact of rGO-PEI_UT neutralizes astrogliosis, decreasing several proinflammatory cytokines that would explain a bystander tumor anti-invasive effect. rGO also significantly downregulates several proinvasive/protumoral cytokines at the tumor cell level. The results open the way to a new microenvironment anti-invasive nanotherapy using a new graphene nanomaterial that is optimized for in vivo brain delivery.     

Toward Engineered Biosynthesis of Drugs in Human Cells

Biosynthetic genes are not only responsible for the formation of bioactive substances but also suited for other applications including gene therapy. To test the feasibility of human cells producing antibiotics in situ when provided with a heterologous biosynthetic gene, we focused on cytochrome P450, the class of enzymes important in conferring bioactivity to natural product precursors. We selected Fma-P450 that plays a central role in the fumagillin antimicrobial biosynthesis in Aspergillus fumigatus to examine fungal metabolite production by HeLa cells that express fma-P450 heterologously. Here we show that HeLa cells harboring fma-P450 can biosynthesize 5-hydroxyl-β-trans-bergamoten and cytotoxic 5-epi-demethoxyfumagillol when supplemented with the nontoxic precursor β-trans-bergamotene. While the production level was insufficient to effect cell death, we demonstrate that programming human cells to autogenerate antibiotics by introducing a heterologous biosynthetic gene is feasible.     

Near-Infrared-Triggered On-Demand Controlled Release of Adeno-Associated Virus from Alginate Hydrogel Microbeads with Heat Transducer for Gene Therapy

Gene therapy using adeno-associated virus (AAV) has potential as a radical treatment modality for genetic diseases such as sensorineural deafness. To establish clinical applications, it is necessary to avoid immune response to AAV by controlled release system of AAV. Here, a near-infrared (NIR)-triggered on-demand AAV release system using alginate hydrogel microbeads with a heat transducer is proposed. By using a centrifuge-based microdroplet shooting device, the microbeads encapsulating AAV with Fe₃O₄ microparticles (Fe₃O₄-MPs) as a heat transducer are fabricated. Fe₃O₄-MPs generated heat by NIR enhanced the diffusion speed of the AAV, resulting in the AAV being released from the microbeads. By irradiating the microbeads encapsulating fluorescent polystyrene nanoparticles (FP-NPs) (viral model) with NIR, the fluorescence intensity decreased only for FP-NPs with a diameter of 20 nm and not for 100 or 200 nm, confirming that this system can release virus with a diameter of several tens of nanometers. By irradiating NIR to the AAV-encapsulating microbeads with Fe₃O₄-MPs, the AAV is released on demand, and gene transfection to cells by AAV is confirmed without loss of viral activity. The NIR-triggered AAV release system proposed in this study increases the number of alternatives for the method of drug release in gene therapy.