Development of tissue culture system with automated pulsation and Kalman filter control for an artificial artery model.

Tissue-engineered arterial vessels have been used as substitutes for unnecessary animal experiments to evaluate the pharmacokinetics of drugs targeting various arteriopathies caused by structural or physiological arterial defects. An arterial tissue culture system was established to simulate the mechanical characteristics of a heart-beating pump and to do online feedback control of lactate and glucose concentrations. The mechanically controlled flow pump mimicked the heart pumping inside a tissue-engineered artery composed of muscle and endothelial cells within a nanofibrous scaffold. After monitoring the pH of the culture medium online, lactate and glucose were estimated using the Kalman filter algorithm, and the set-point online control was operated to maintain glucose for artery tissue engineering. The composition of the artificial artery was confirmed by immunofluorescence staining, and its mechanical characteristics were examined. The online automated system successfully demonstrated its applicability as a standardized process for arterial tissue culture to replace animal arterial experiments.

Differential spectroscopic analysis of size-dependent phycobilisome from Spirulina maxima.

C-phycocyanin (C-Pc), a photosynthetic pigment for use as a fluorescent indicator or in pharmaceutical, food, and cosmetic products, exists in a phycobilisome complex with allophycocyanin (APC), phycoerythrin (PE), and linker polypeptides. This heterogeneity makes it difficult to quantify phycobilisome composition in an ultraviolet-visible (UV-vis) spectrum. In this study, derivative analysis of UV-vis spectra was successfully applied to display the distinct wavelengths at which C-Pc, APC, and PE have maximal peaks. In all samples, C-Pc of the largest portion had a "zero-crossing" first order, APC did not have a zero-crossing first order, and PE did not have first derivative for zero crossing or local minimum from the 500 and 700 nm, respectively. The results show that derivative analyses coupled with signal smoothing can be applied to elucidate the composition of phycobilisome under various conditions including purification and environment.

A systematic correlation analysis of carotenoids, chlorophyll, non-pigmented cell mass, and cell number for the blueprint of Dunaliella salina culture in a photobioreactor.

Microalgal carotenoids are attractive health ingredients, but their production should be optimized to improve cost-effectiveness. Understanding cellular physiology centered on carotenoid synthesis is the prerequisite for this work. Therefore, systematic correlation analyses were conducted among chlorophyll, carotenoids, non-pigmented cell mass, and cell number of Dunaliella salina in a specified condition over a relatively long culture time. First, an integrated correlation was performed: a temporal profile of the carotenoids was correlated with those of other factors, including chlorophyll, non-pigmented cell mass, and cell number. Pearson and Spearman correlation analyses were performed to identify linearity and monotonicity of the correlation, respectively, and then cross-correlation was executed to determine if the correlation had a time lag. Second, to understand the cellular potential of metabolism, the procedure was repeated to provide a data set composed of the specific synthesis rates of the factors or growth rate, which additionally provided kinetic correlations among the constituting components of the cell, excluding the effect of cell number. This systematic approach could generate a blueprint model that is composed of only what it needs, which could make it possible to efficiently control and optimize the process.

Biological activities of an engineered tautomycetin analogue via disruption of tmcR-encoding hydroxylase in Streptomyces sp. CK4412.

Tautomycetin (TMC), originally isolated from Streptomyces griseochromogenes, has been reported to possess biological functions including T cell-specific immunosuppressive and anticancer activities through a mechanism of differential inhibition of protein phosphatases such as PP1, PP2A, and SHP2. Independently isolated Streptomyces sp. CK4412 was also reported to produce a structurally identical TMC compound. Previously, we isolated and characterized the entire TMC biosynthetic gene cluster from Streptomyces sp. CK4412. In silico database comparison revealed a 1,359-bp tmcR as a putative bacterial Cytochrome P450 hydroxylase gene in the TMC biosynthetic gene cluster. Through targeted gene disruption and complementation, the tmcR mutant was confirmed to produce a C5-deoxy-TMC, the same analogue produced by the S. griseochromogenes ttnI mutant, implying that TmcR behaves as a regiospecific C5-oxygenase in the TMC biosynthetic pathway in Streptomyces sp. CK4412. In particular, the C5-deoxy-TMC from the tmcR mutant exhibited 3.2-fold higher inhibition activity toward SHP2 with significantly reduced inhibition activities toward PP1, and human Vero and lung cancer cells. These results suggested that C5 regiospecific modification of the TMC polyketide moiety may result in a drug development target for use in preferentially enhancing immunosuppressive activity while minimizing its undesirable biological activities.

The dependence of enhanced lysosomal activity on the cellular aging of bovine aortic endothelial cells.

Lysosomes are a cell organelle type processing antimicrobial activity. Here, we investigate the lysosomal activity in a primary cell line, bovine aortic endothelial cells (BAECs), during cellular aging, based on the antimicrobial activity of lysosomes isolated from BAECs at cell passages 4, 6, 8, and 10. Cytochemical analysis of lysosomes with LysoTracker reagent revealed the number of lysosome-like organelles surrounding the nucleus initially increased drastically in the BAECs and continued increasing gradually until passage 10. The lysosomes isolated from each successive passage of BAECs exhibited increased antimicrobial activity against Escherichia coli, and, in addition, an age-dependent increase in lysosome intensity coincided with increased lysosomal antimicrobial activity.

Proline reduces the binding of transcriptional regulator ArgR to upstream of argB in Corynebacterium glutamicum.

In this study, the ArgR-binding sites on the arg operon Corynbebacterium glutamicum were characterized by in vivo chromatin immunoprecipitation (ChIP). In addition, the ArgR-binding affinity in the presence of glutamate, proline, or arginine was examined to get further information on expression control. The ChIP assay showed that the ArgR protein binds specifically to the upstream regions of argC, argB, argF, and argG. Upon proline supplementation, ArgR-binding affinity was significantly reduced upstream of argB, resulting in increased ornithine production. In contrast, there was no change in the binding affinity of ArgR to the upstream regions of argC, argF, argG, or argB following the addition of glutamate and arginine. These results suggest that the upstream region of argB on the arg operon plays an important role in interacting with ArgR under proline-supplemented conditions and that proline causes an increase in the endogenous level of ornithine by reducing the binding affinity of ArgR to the upstream region of argB.

Kinetic and thermodynamic analyses of adhesion of a peptide, Trp-Lys-Tyr-Met-Val-D-Met (WKYMVm), and human formyl peptide receptor (hFPR).

The kinetic and thermodynamic properties of a peptide-receptor interaction was investigated by measuring the adhesion force in the reaction via atomic force microscopy (AFM). Trp-Lys-Tyr-Met-Val-D-Met (WKYMVm), considered as a model system in the present study, is a potent neutrophil chemo-attractant. Since being identified as an agonist for formyl peptide receptor (FPR), WKYMVm's high affinity to FPR has been verified through investigation of its kinetic and physiological behaviors via conventional methods. However, there have been no reports on the adhesion force of WKYMVm-FPR. In this research, we measured the adhesion force of WKYMVm-FPR using AFM. Kinetic parameters obtained from the relationship between the adhesion force and loading rate were used to characterize the thermodynamic properties of WKYMVm-hFPR binding.

Compartmental culture of embryonic stem cell-derived neurons in microfluidic devices for use in axonal biology.

Axonal pathology has been clearly implicated in neurodegenerative diseases making the compartmental culture of neurons a useful research tool. Primary neurons have already been cultured in compartmental microfluidic devices but their derivation from an animal is a time-consuming and difficult work and has a limit in their sources. Embryonic stem cell (ESC)-derived neurons (ESC_Ns) overcome this limit, since ESCs can be renewed without limit and can be differentiated into ESC_Ns by robust and reproducible protocols. In this research, ESC_Ns were derived from mouse ESCs in compartmental microfluidic devices, and their axons were isolated from the somal cell bodies. Once embryoid bodies (EBs) were localized in the microfluidic culture chamber, ESC_Ns spread out from the EBs and occupied the cell culture chamber. Their axons traversed the microchannels and finally were isolated from the somata, providing an arrangement comparable to dissociated primary neurons. This ESC_N compartmental microfluidic culture system not only offers a substitute for the primary neuron counterpart system but also makes it possible to make comparisons between the two systems.

Shear stress effect on transfection of neurons cultured in microfluidic devices.

Non-invasive genetic manipulation in primary neurons is important in many areas of neuroscience research. Although highly efficient transfections can be performed using viral methods those procedures come with many drawbacks concerning safety issues. Compared to viral methods, non-viral transfection methods have significantly lower transfection rate which limited its use in neuroscience research. This paper describes a novel microfluidic device that was used to investigate the effect of shear stress on transfection efficiency of lipoplex (DNA entangled with liposome) to primary neurons. The device can be used to simultaneously generate regions with multiple shear stress levels using a single device. This device is compatible with cells growing on a monolayer on a conventional tissue culture Petri dish. When exposed to shear stress, post-mitotic primary rat cortical neurons' transfection rate increased by up to 3-fold when compared to static conventional method. Similar effect was observed with mitotic neuronal cell line NIE-115 where upto 45% transfection efficiency was achieved with the aid of shear stress. Through this research, we demonstrated the efficiency of the reversibly binding microfluidic device in executing transfection experiments and corroborated the fact that shear stress is a new parameter to improve non-viral transfection to cells.

Author Correction: Artificial controlled model of blood circulation system for adhesive evaluation.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

A Novel Thrombolytic and Anticoagulant Serine Protease from Polychaeta, Diopatra sugokai.

Ischemic stroke can result from blockage of blood vessels, forming fibrin clots in the body and causing irreparable brain damage. Remedial thrombolytic agents or anticoagulants have been studied; however, because the FDA-approved tissue plasminogen activator has low efficacy and side effects, it is necessary to develop safer and more effective treatment candidates. This study aimed at assessing the fibrinolytic and anticoagulation features of a novel serine protease extracted and purified from Diopatra sugokai, a polychaeta that inhabits tidal flats. The purified serine protease was obtained through ammonium sulfate precipitation, affinity chromatography, and ion-exchange chromatography. Its molecular size was identified via SDS-PAGE. To characterize its enzymatic activities, the protease activity at various pH and temperatures, and in the presence of various inhibitors, was measured via azocasein assay. Its fibrinolytic activity and anticoagulant effect were assessed by fibrin zymography, fibrin plate assay, and fibrinogenolytic activity assays. The novel 38 kDa serine protease had strong indirect thrombolytic activity rather than direct activity over broad pH (4-10) and temperature (37°C-70°C) ranges. In addition, the novel serine protease exhibited anticoagulant activity by degrading the α-, ß-, and γ-chains of fibrinogen. In addition, it did not produce cytotoxicity in endothelial cells. Therefore, this newly isolated serine protease is worthy of further investigation as a novel alkaline serine protease for thrombolytic therapy against brain ischemia.

Development of coaxial alginate-PCL nanofibrous dressing for controlled release of Spirulina extract.

Spirulina has widely been highlighted as a source of bioactive material that can be impregnated into dressing materials. The aim of this study was to widen the application fields of Spirulina extract-containing nanofiber, which has been suggested as an attractive dressing material in several previous studies. The bioactivity release pattern, water absorbance, and mechanical strength must be controllable. Spirulina extract was physically impregnated inside a nanofiber without significant chemical bonding to polycaprolactone or alginate polymers. This led to an initial burst and continual release of bioactive molecules from the nanofiber. By altering the concentration of Spirulina extract, mechanical strength and water absorbance were controllable. In addition, the dressing patch showed no cytotoxicity towards human epithelial cells, not causing skin-irritation. This indicates that the coaxially fabricated patch is a controllable dressing material that can be customized to have a specific mechanical strength, water absorbance, and bioactive release pattern, making it suitable for wide applications.

Chitosan-coated C-phycocyanin Liposome for Extending the Neuroprotective Time Window Against Ischemic Brain Stroke.

The time window for neuroprotection during ischemic brain stroke is short, and hence, development of neuroprotectants is critical to extend this time window. This study sought to verify if muco-adhesive chitosan coating improves the neuroprotective potential of the pre-proven C-Phycocyanin-pertaining liposome (C-Pc liposome). The use of chitosan-coated liposomes extended the neuroprotective time window by 6 h after occlusion, and further improved the neuroprotection efficiency of the C-Pc liposome in a rat Middle Cerebral Artery Occlusion (MCAO) model. Beneficial changes in mRNA expressions of antioxidants, inflammatory cytokines and glia scar proteoglycans were evident in the C-Pc liposomes. In addition, in the cultured astrocytes, the chitosan- coated C-Pc liposome expressed anti-oxidative activity without cytotoxicity.

Artificial controlled model of blood circulation system for adhesive evaluation.

Since there are several casualties due to uncontrolled bleeding resulting from simple injury to surgery, effective styptic or vessel adhesives are important; however, their development is limited by the lack of standardized systems to evaluate potential compounds. The current study outlines the development of an aorta styptic evaluation system, comprising of decellularized swine aorta tissue and a heart pump-mimicking system. Although the cells in the swine aorta were removed, the structural stability of the aorta was sustained due to the maintenance of the extracellular matrix. Using a control adhesive, Cyanoacrylate, the developed model was found to have similar adhesive efficacy to intact aorta. The circulatory-mimicking system was designed to mimic the beat rate and strength of blood-flow from the heart, which was necessary to evaluate the adherent efficacy. The decellularized aorta improves instabilities of intact tissues, which occurs on account of storage and origin, thereby allowing for a more standardized system. The system was able to simulate several symptoms of circulation, according to patient age and health, by adjusting pumping frequency and intensity. Therefore, this system can be used as a standardized evaluation system for screening adhesives. Further, it would also evaluate other medical devices, such as stent or medications.

Nasal Delivery of Antioxidants by Cholesterol-incorporated Liposomes Extends the Neuroprotective Time Window in Cerebral Ischemia.

Antioxidants have the potential to prevent cerebral ischemia-reperfusion (IR)-associated secondary damage induced by reactive oxygen species (ROS); however, the short therapeutic time window of IR is a considerable obstacle. Nano-sized nasal delivery systems provide an effective means of delivering drugs through the BBB, but few such systems have been developed to extend the treatment time window in IR. In this work, a nanosized nasal delivery system for antioxidants was found to have the potential to extend the neuroprotective time window. The authors chose to use the antioxidant C-phycocyanin (C-Pc) to design a neuroprotective liposome with a long life, controllable release, and high neuronal uptake rate. Liposomes formulated with various cholesterol to phospholipid ratios were assessed thermodynamically, kinetically, and biologically. Thermodynamically stable, monodispersive, and release-controllable C-Pc liposomes were more effectively taken up by Neuro2a cells than free C-Pc and were biocompatible, maintaining the anti-oxidative properties of C-Pc. When optimal C-Pc liposomes were administered to middle cerebral artery occlusion (MCAO) rats 2 h after onset, infarct sizes were smaller and behavioral activities improved compared with the same metrics in free C-Pc-treated rats. Liposomal delivery still reduced infarct sizes and improved behavioral activity 6 h after onset, whereas free C-Pc did not.

Development of a Spirulina Extract/Alginate-Imbedded PCL Nanofibrous Cosmetic Patch.

Cosmetic patches have recently been developed as skin products for personal care owing to rapid advances in the technology of delivery of active ingredients, moisture, and adhesiveness to skin. Alginate and Spirulina are typical marine resources used in cosmetic products. This research involved the development of a Spirulina extract-impregnated alginate nanofiber cosmetic patch supported by a polycaprolactone (PCL) nanofiber cover (Spi/Alg-PCL NF patch). In addition to the ability of alginate to affect moisture and adhesiveness to skin, the impregnation of Spirulina extract strengthened those abilities as well as its own bioactive effectiveness. All fabrication processing steps were undertaken in aqueous solution. The three components (alginate, Spirulina extract, and PCL) had no detected cytotoxicity in human keratinocyte cell-based examination. In addition, wetting the pre-dried patch on the skin resulted in the Spirulina extract being released within 30 min. The results indicate the excellence of the Spi/Alg-PCL NF patch as a skin-care cosmetic device.

Purification of serine protease from polychaeta, Lumbrineris nipponica, and assessment of its fibrinolytic activity.

Ischemic stroke and cardiovascular disease can occur from blockage of blood vessels by fibrin clots formed naturally in the body. Therapeutic drugs of anticoagulant or thrombolytic agents have been studied; however, various problems have been reported such as side effects and low efficacy. Thus, development of new candidates that are more effective and safe is necessary. The objective of this study is to evaluate fibrinolytic activity, anti-coagulation, and characterization of serine protease purified from Lumbrineris nipponica, polychaeta, for new thrombolytic agents. In the present study, we isolated and identified a new fibrinolytic serine protease from L. nipponica. The N-terminal sequence of the identified serine protease was EAMMDLADQLEQSLN, which is not homologous with any known serine protease. The size of the purified serine protease was 28 kDa, and the protein purification yield was 12.7%. The optimal enzyme activity was observed at 50°C and pH 2.0. A fibrin plate assay confirmed that indirect fibrinolytic activity of the purified serine protease was higher than that of urokinase-PA, whereas direct fibrinolytic activity, which causes bleeding side effects, was relatively low. The serine protease did not induce any cytotoxicity toward the endothelial cell line. In addition, anticoagulant activity was verified by an in vivo DVT animal model system. These results suggest that serine protease purified from L. nipponica has the potential to be an alternative fibrinolytic agent for the treatment of thrombosis and use in various biomedical applications.

Assessment of Neuronal Cell-Based Cytotoxicity of Neurotoxins from an Estuarine Nemertean in the Han River Estuary.

A heteronemertean, Yininemertes pratensis, was collected in Han River Estuary, South Korea. This estuarine nemertean has been known by the local fishermen for harmful effects to the glass eels, juveniles of Japanese eel Anguilla japonica, migrating to fresh water. The present study confirmed the neurotoxic effects of this heteronemertean ribbon worm at the cellular level. Derivative types of neurotoxic tetrodotoxin (TTX), 5,11-dideoxy TTX (m/z 288) and 11-norTTX-6(S)-01 (m/z 305.97), were identified through HPLC and MALDI-TOF MS. However, significant neurotoxicity was confirmed in the fraction containing an undefined molecule corresponding to the 291.1 (m/z) peak, when tested in rat primary astrocytes and dorsal ganglion cells. This study is the first to report neurotoxins of the estuarine nemertean, fairly abundant in the Han River estuary, and suggests the long-term monitoring of population dynamics and surveillance of the toxicity in this river estuary.

Mechanotransduction-Induced Lipid Production System with High Robustness and Controllability for Microalgae.

Microalgae lipids are a promising energy source, but current biochemical methods of lipid-inductions such as nitrogen deprivation have low process robustness and controllability. Recently, use of mechanotransduction based membrane distortion by applying compression stress in a 2D-microsystem was suggested as a way to overcome these limitations of biochemical induction. However, reproduction in large numbers of cells without cell death has been difficult to overcome because compression for direct membrane distortion reduces culture volume and leads to cell death due to nutrient deprivation. In this study, a mechanotransduction-induced lipid production (MDLP) system that redirects elastic microbeads to induce membrane distortion of microalgae with alleviating cell death was developed. This system resulted in accumulation of lipid in as little as 4 hr. Once compressed, porous microbeads absorb media and swell simultaneously while homogeneously inducing compression stress of microalgae. The absorbed media within beads could be supplied to adjacent cells and could minimize cell death from nutrient deficiency. All mechanotransduction was confirmed by measuring upregulation of calcium influx and Mat3 genes. The microbeads ensured robustness and controllability in repeated compression/de-compression processes. Overall, the MDLP system has potential for use as a fundamental biodiesel process that requires robustness and controllability.

Co-stimulation of HaCaT keratinization with mechanical stress and air-exposure using a novel 3D culture device.

Artificial skin or skin equivalents have been used for clinical purpose to skin graft and as substitutes for animal experiments. The culture of cell lines such as HaCaT has the potential to produce large amounts of artificial skin at a low cost. However, there is a limit to keratinization due to the restriction of differentiation in HaCaT. In this study, a culture device that mimics the in vivo keratinization mechanism, co-stimulated by air-exposure and mechanical stimulation, was developed to construct skin equivalents. The device can reconstruct the epidermal morphology, including the cornified layer, similar to its formation in vivo. Under the condition, epidermis was differentiated in the spinous and granular layers. Formation of the stratum corneum is consistent with the mRNA and protein expressions of differentiation markers. The device is the first of its kind to combine air-exposure with mechanical stress to co-stimulate keratinization, which can facilitate the economically viable production of HaCaT-based artificial skin substitutes.