Development of the Low-Temperature Inducible System for Recombinant Protein Production in Escherichia coli Nissle 1917.

The pET system is commonly used for producing foreign proteins in Escherichia coli, but its reliance on the costly and metabolically demanding inducer IPTG limits its industrial use. This study engineered a low-temperature inducible system (LTIS) in E. coli Nissle 1917 (EcN) by combining the T7 expression system with the thermal inducible mechanism CI857-λPRPL to generate the new LTIS strain, ENL7P. The strain ENL7P-sfGFP-Km underwent overnight culture at 37 °C for 14-16 h, followed by subculturing at 30 °C for 24 h. This resulted in a notable 5.53-fold increase in the sfGFP induction rate when the strain was cultivated under 37-30 °C conditions. Moreover, gene expression was induced using a two-stage strategy. Initially, the strain was cultured overnight at 39 °C for 14-16 h, followed by a subculture at 30 °C for 6 h, and finally, another subculture at 30 °C for 24 h. This cultivation strategy led to an impressive 158.37-fold induction rate for sfGFP. Similar effects could be achieved through utilization of the LTIS system for inducing the production of thermophilic trehalose synthase from Thermus antranikianii (TaTS). The results of this study proved that the LTIS system has the potential for industrial applications.

Effects of Chitosan on Chondrocytes Derived from Human Nasal Septal Cartilage.

BACKGROUND/AIM: The aim of this study was to evaluate the influence of chitosan on the growth of nasal septal chondrocytes (NSCs). The final goal was to establish a novel methodology to enhance nasal septal cartilage regeneration. MATERIALS AND METHODS: Human NSCs were isolated and their morphology was examined using Alcian blue staining and observed by light microscopy. The isolated NSCs were grown with various concentrations of chitosan and the expression of COL2A1 was investigated. RESULTS: NSCs were successfully isolated from nasal septal cartilage. Co-culture with 0.2% of chitosan greatly enhanced proliferation of NSCs compared to control cells. However, 0.5% of chitosan was harmful to NSCs, resulting in cell detachment from the culture plate. Furthermore, the addition of 0.2% chitosan significantly improved the expression of COL2A1 in NSCs. CONCLUSION: To our knowledge, this is the first report to demonstrate that chitosan could effectively guide the attachment and growth of human NSCs. Chitosan appears to be a promising additive for NSC culture, which sets the stage for studying tissue regeneration in nasal septal cartilage deficiency, rhinoplasty, and craniofacial reconstruction.

Integration of Multiple Phage Attachment Sites System to Create the Chromosomal T7 System for Protein Production in Escherichia coli Nissle 1917.

Escherichia coli Nissle 1917 (EcN) is a probiotic used to treat gastrointestinal diseases. The probiotic and endotoxin-free characteristics of EcN support its potential to be developed into a microbial expression system. With this aim, in this study, the powerful T7 expression system was constructed in the cryptic plasmid-free EcN (EcNP) to generate the T7 expression host ENL6P. The concept of multiple copies of gene expression cassettes regulated by the chromosomal T7 promoter was promoted due to plasmid instability issues with protein production in ENL6P. The integration of multiple phage attachment sites (IMPACT) system, which combined Cre-lox72, CRIM, and lambda red recombinase systems, was designed to simplify the manipulation and achieve the multiple φ80 bacterial attachment sites (attB) in ENL6P to generate the new strain ENL6PP4 with four φ80 attB sites. The strain can simultaneously integrate four copies of gene expression cassettes in the chromosome to produce recombinant proteins. The IMPACT systems incorporated several tools in gene editing to rapidly achieve more robust and stable microbial strains for research and various industrial applications.

Robust State/Fault Estimation and Fault-Tolerant Control in Discrete-Time T-S Fuzzy Systems: An Embedded Smoothing Signal Model Approach.

This study investigates a simple design method of the robust state/fault estimation and fault-tolerant control (FTC) of discrete-time Takagi-Sugeno (T-S) fuzzy systems. To avoid the corruption of the fault signal on state estimation, a novel smoothing signal model of fault signal is embedded in the T-S fuzzy model for the robust H∞ state/fault estimation of the discrete-time nonlinear system with external disturbance by the traditional fuzzy observer. When the component and sensor faults are generated from different fault sources, two smoothing signal models for component and sensor faults are both embedded in the T-S fuzzy system for robust state/fault estimation. Since the nonsingular smoothing signal model and T-S fuzzy model are augmented together for signal reconstruction, the traditional fuzzy Luenberger-type observer can be employed to robustly estimate state/fault signal simultaneously from the H∞ estimation perspective. By utilizing the estimated state and fault signal, a traditional H∞ observer-based controller is also introduced for the FTC with powerful disturbance attenuation capability of the effect caused by the smoothing model error and external disturbance. Moreover, the robust H∞ observer-based FTC design is transformed into a linear matrix inequality (LMI) -constrained optimization problem by the proposed two-step design procedure. With the help of LMI TOOLBOX in MATLAB, we can easily design the fuzzy Luenberger-type observer for efficient robust H∞ state/fault estimation and solve the H∞ observer-based FTC design problem of discrete nonlinear systems. Two simulation examples are given to validate the performance of state/fault estimation and FTC of the proposed methods.

Design and In Vivo Verification of a CMOS Bone-Guided Cochlear Implant Microsystem.

OBJECTIVE: To develop and verify a CMOS bone-guided cochlear implant (BGCI) microsystem with electrodes placed on the bone surface of the cochlea and the outside of round window for treating high-frequency hearing loss. METHODS: The BGCI microsystem consists of an external unit and an implanted unit. The external system-on-chip is designed to process acoustic signals through an acquisition circuit and an acoustic DSP processor to generate stimulation patterns and commands that are transmitted to the implanted unit through a 13.56 MHz wireless power and bidirectional data telemetry. In the wireless power telemetry, a voltage doubler/tripler (2X/3X) active rectifier is used to enhance the power conversion efficiency and generate 2 and 3 V output voltages. In the wireless data telemetry, phase-locked loop based binary phase-shift keying and load-shift keying modulators/demodulators are adopted for the downlink and uplink data through high-Q coils, respectively. The implanted chip with four-channel high-voltage-tolerant stimulator generates biphasic stimulation currents up to 800 µA. RESULTS: Electrical tests on the fabricated BGCI microsystem have been performed to verify the chip functions. The in vivo animal tests in guinea pigs have shown the evoked third wave of electrically evoked auditory brainstem response waveforms. It is verified that auditory nerves can be successfully stimulated and acoustic hearing can be partially preserved. CONCLUSION AND SIGNIFICANCE: Different from traditional cochlear implants, the proposed BGCI microsystem is less invasive, preserves partially acoustic hearing, and provides an effective alternative for treating high-frequency hearing loss.

Genetic screen in Drosophila muscle identifies autophagy-mediated T-tubule remodeling and a Rab2 role in autophagy.

Transverse (T)-tubules make-up a specialized network of tubulated muscle cell membranes involved in excitation-contraction coupling for power of contraction. Little is known about how T-tubules maintain highly organized structures and contacts throughout the contractile system despite the ongoing muscle remodeling that occurs with muscle atrophy, damage and aging. We uncovered an essential role for autophagy in T-tubule remodeling with genetic screens of a developmentally regulated remodeling program in Drosophila abdominal muscles. Here, we show that autophagy is both upregulated with and required for progression through T-tubule disassembly stages. Along with known mediators of autophagosome-lysosome fusion, our screens uncovered an unexpected shared role for Rab2 with a broadly conserved function in autophagic clearance. Rab2 localizes to autophagosomes and binds to HOPS complex members, suggesting a direct role in autophagosome tethering/fusion. Together, the high membrane flux with muscle remodeling permits unprecedented analysis both of T-tubule dynamics and fundamental trafficking mechanisms.

Quantitative observation of focused-ultrasound-induced vascular leakage and deformation via fluorescein angiography and optical coherence tomography.

Focused ultrasound (FUS) is a recently discovered noninvasive technique for local and temporal enhancement of vascular permeability, which facilitates drug delivery from the vessels into the surrounding tissue. However, exposure to FUS at a high intensity may cause permanent damage. To investigate the effects of the FUS treatment on blood vessels, we propose to use fluorescein angiography (FA) and optical coherence tomography (OCT) for real-time observation of the diffusion of fluorescence dye from blood vessels and to evaluate the morphological changes of the vessels in vivo. With time-resolved FA imaging, the relationship between the exposed power and the improved permeability of the vessels can be assessed according to the enhancement of the fluorescent intensity due to the dye leakage. Furthermore, the variation of the time-resolved fluorescent intensities can be used to identify the occurrence of dye leakage. In contrast, OCT can be implemented for the reconstruction of tissue microstructures. To quantitatively evaluate the morphological changes of the vessels after the FUS exposure with OCT, a new algorithm was proposed to estimate the vessel area based on the comparison of backscattering properties resulting from the tissue and vascular structures. Results showed that the vessel area increased as the exposed power increased, and the area became significantly larger at a higher FUS exposure power of 10 W. In conclusion, integrated FA and OCT observation can be potentially effective for monitoring the outcome and investigating the effects of FUS treatment.

Clinical correlation of nonalcoholic fatty liver disease in a Chinese taxi drivers population in Taiwan: Experience at a teaching hospital.

BACKGROUND: To explore any gender-related differences in the prevalence of conditions-associated with non-alcoholic fatty liver disease (NAFLD) among Taiwanese taxi drivers in Taipei, Taiwan. METHODS: We studied 1635 healthy taxi drivers (1541 males and 94 females) who volunteered for physical check-ups in 2006. Blood samples and ultrasound fatty liver sonography results were collected. RESULTS: The prevalence of NAFLD was 66.4% and revealed no statistically significant decrease with increasing age (p = 0.58). Males exhibited a greater prevalence of NAFLD than did females (67.5% vs 47.9%, p < 0.0001). Gender-related differences for associated factors were found. For males, hypertension, hyperuricemia, higher AST, higher ALT, hypertriglyceridemia, and higher fasting plasma glucose were significantly related to NAFLD. These conditions were not sigfinicantly related to NAFLD in females. CONCLUSION: Several gender-related differences were noted for NAFLD among Taiwanese taxi drivers.