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Hepatocellular carcinoma (HCC) ranks among the most prevalent cancers worldwide, highlighting the urgent need for improved diagnostic and therapeutic methodologies. The standard treatment regimen generally involves surgical intervention followed by systemic therapies; however, the median survival rates for patients remain unsatisfactory. Chimeric antigen receptor (CAR) T-cell therapy has emerged as a pivotal advancement in cancer treatment. Both clinical and preclinical studies emphasize the notable efficacy of CAR T cells in targeting HCC. Various molecules, such as GPC3, c-Met, and NKG2D, show significant promise as potential immunotherapeutic targets in liver cancer. Despite this, employing CAR T cells to treat solid tumors like HCC poses considerable challenges within the discipline. Numerous innovations have significant potential to enhance the efficacy of CAR T-cell therapy for HCC, including improvements in T cell trafficking, strategies to counteract the immunosuppressive tumor microenvironment, and enhanced safety protocols. Ongoing efforts to discover therapeutic targets for CAR T cells highlight the need for the development of more practical manufacturing strategies for CAR-modified cells. This review synthesizes recent findings and clinical advancements in the use of CAR T-cell therapies for HCC treatment. We elucidate the therapeutic benefits of CAR T cells in HCC and identify the primary barriers to their broader application. Our analysis aims to provide a comprehensive overview of the current status and future prospects of CAR T-cell immunotherapy for HCC.

Dielectric barrier discharges (DBDs) provide a promising technology for generating non-equilibrium cold plasmas at atmospheric pressure. For both application-focused and fundamental research, it is important to explore the discharge mode transition and electron heating mechanism to enable effective independent tuning of key plasma parameters in a DBD system. In this work, we report numerical studies of the effects of single-frequency excitation on atmospheric argon DBDs, which are carried out in the medium driving frequency (MF) range from 50 kHz to 5 MHz by using a one-dimensional hydrodynamics coupling model. The spatio-temporal evolution of particle density associated with the discharge mode transition and electron dynamic behavior has been investigated. By tuning different components of a single frequency, we observe the electron heating behaviors of the individual modes and mode transitions from the Townsend discharge to the glow discharge in the low frequency to the Ω mode and the hybrid mode in the medium frequency to the α-mode and the γ-mode in the radio frequency. The physical analysis is understood based on these fundamental insights into the plasma physics.

Manipulating electromagnetic (EM) waves by plasma–dielectric–plasma (PDP) waveguides or plasma array structures presents significant potential in microwave signal processing, such as filtering, signal delay, and EM enhancement or shielding. Owing to the simple structure and easy fabrication, the waveguide with a tooth-shaped resonator has been a strong candidate as a filtering device. Based on our previous work focusing on U-shaped filtering excited by PDP waveguides with a double-teeth structure, in this work, the formation mechanism of a U-shape filtering spectrum is systematically explored by transmission line theory (TLT) with proper field distributions. The results indicate that the U-shape spectrum consists of boundary edges and a filtering stopband. The boundary edges are attributed to Fano-type resonance, and the enhanced destructive interference from double teeth is responsible for the stopband. Such an approach shows a specific and clear mechanism for the generated U-shaped spectrum. In addition, the theoretical analysis of double teeth without Fano-type resonances is rigorously demonstrated using TLT, which significantly contributes to bandwidth modulation of stopband filtering in theory. These results contribute to the understanding of the formation mechanism of a U-shaped spectrum from a gap plasmon waveguide (such as PDP or metal–insulator–metal (MIM)) with tooth-shaped resonators, offering a feasible direction for the optimization of filtering properties, as well as offering significant parameters for subsequent experimental design.

A small rock shotcrete robot is applied in the deep tunnel dynamic disaster physical simulation system in order to simulate the final operation process of the tunnel project. The robot’s spraying operation and control include many kind of factors. In this paper, the main parameters affecting the spraying operation are analyzed by establishing the spraying model and coating growth model. For matching reasonably the spraying parameters, the matching equation of spraying quality and spraying gun parameter model and spraying feed speed has been derived. Based on the matching equation, the spraying gun structure and robot control system are designed, and the spraying distance and spraying gun movement velocity including horizontal and vertical driving have been controlled better. On the basis of theoretical analysis, a small rock hole shotcrete robot prototype has been developed and the spraying experiments have been finished. The experimental results showed that the model and equation established can guided the spraying quality control. With the increase of the injection elevation angle, the length of the long and short axes of the elliptic coating becomes larger, and the average thickness of the coating center is between 2.23 and 2.34 mm, which meets the design index of the injection robot. The rock hole shotcrete robot meet the design requirements.

Abstract The pooling layer in convolutional neural networks plays a crucial role in reducing spatial dimensions, and improving computational efficiency. However, standard pooling operations such as max pooling or average pooling are not suitable for all applications and data types. Therefore, developing custom pooling layers that can adaptively learn and extract relevant features from specific datasets is of great significance. In this paper, we propose a novel approach to design and implement customizable pooling layers to enhance feature extraction capabilities in CNNs. The proposed T-Max-Avg pooling layer incorporates a threshold parameter T, which selects the K highest interacting pixels as specified, allowing it to control whether the output features of the input data are based on the maximum values or weighted averages. By learning the optimal pooling strategy during training, our custom pooling layer can effectively capture and represent discriminative information in the input data, thereby improving classification performance. Experimental results show that the proposed T-Max-Avg pooling layer achieves good performance on three different datasets. When compared to LeNet-5 model with average pooling, max pooling, and Avg-TopK methods, the T-Max-Avg pooling method achieves the highest accuracy on CIFAR-10, CIFAR-100, and MNIST datasets.

Abstract Background Mutations in TP53 gene is considered a main driver of hepatocellular carcinoma (HCC). While TP53 mutations are the leading cause of p53 dysfunction, their occurrence rates may drop to approximately 10% in cohorts without hepatitis B virus and aflatoxin exposure. This observation suggests that the deactivation of wild-type p53 (p53wt) may be a critical factor in the majority of HCC cases. However, the mechanism undermining p53wt activity in the liver remains unclear. Methods Microarray analysis and luciferase assay were utilized to confirm target associations. Gain- and/or loss-of-function methods were employed to assess alterations in signaling pathways. Protein interactions were analyzed by molecular immunological methods and further visualized by confocal microscopy. Bioinformatic analysis was performed to analyze clinical significance. Tumor xenograft nude mice were used to validate the findings in vivo. Results Our study highlights the oncogenic role of Rictor, a key component of the mammalian target of rapamycin complex 2 (mTORC2), in hepatocytes. Rictor exerts its oncogenic function by binding to p53wt and subsequently blocking p53wt activity based on p53 status, requiring the involvement of mTOR. Moreover, we observed a dynamic nucleocytoplasmic distribution pattern of Rictor, characterized by its translocation from the nucleus (in precancerous lesions) to the cytoplasm (in HCCs) during malignant transformation. Notably, Rictor is directly targeted by the liver-enriched microRNA miR-192, and the disruption of the miR-192-Rictor-p53-miR-192 signaling axis was consistently observed in both human and rat HCC models. Clinical analysis associated lower miR-192/higher Rictor with shorter overall survival and more advanced clinical stages (P

Purposes Electrochemically switched ion permselective (ESIP) is a novel ion selective membrane separation technology, which has been applied for the efficient, continuous and selective separation of low-concentration target ions. However, the ion transfer in the ESIP membrane is disobedient to the Nernst-Planck theory, and the development of mass transfer model of ESIP contributes to the understanding of ESIP separation process. Methods In the ESIP membrane separation system, a modified Nernst-Planck model was proposed to study the mass transfer behavior in the dynamic field coupled with the double pulse potential on the membrane and the cell voltage on the electrode compartment. According to Donnan equilibrium and electroneutrality hypothesis, the steady state model of ion transfer in ESIP membrane separation process was established. The effects of current density, membrane thickness and ionic active site concentration on ion concentration and resistance distribution in membrane separation system were investigated by numerical simulation. Findings The results show that in the diffusion layer of feed solution and the membrane phase, the membrane thickness and the ionic active site concentration have great influence on the ion concentration distribution, and reducing the membrane thickness and increasing the ionic active site concentration are the main ways to improve the ion mass transfer. Under the influence of high current density, thin membrane and ionic active site concentration, resistance in the diffusion layer of feed solution plays a dominant role in the overall resistance, and the ion mass transfer rate can be increased by increasing the flow rate or decreasing the chamber thickness. Conclusions At low current density and thick membrane, the resistance of Donnan layer plays a dominant role, and the ion mass transfer rate is improved by increasing the ionic active sites of the membrane.

As the number of electric vehicles (EVs) increases, massive numbers of EVs have started to gather in commercial parking lots to charge and discharge, which may significantly impact the operation of the grid. There may also be a deviation in the departure time of charged and discharged EVs in commercial parking lots. This deviation can lead to insufficient battery energy when the EVs leave the parking lot. This study uses the simulation software AnyLogic to build a commercial parking lot multi-agent simulation model, and the agent-based model can fully reflect the autonomy of individual EVs. Based on this simulation model, we propose an EV scheduling algorithm. The algorithm contains two main agents. The first is the power distribution center agent (PDCA), which is used to coordinate the energy output of photovoltaic (PV), energy storage system (ESS), and distribution station (DS) to solve the problem of grid overload. The second is the scheduling center agent (SCA), which is used to solve the insufficient battery energy problem due to EVs’ random departures. The SCA includes two stages. In the first stage, a priority scheduling algorithm is proposed to emphasize the fairness of EV charging. In the second stage, a genetic algorithm is used to accurately determine the time interval between charging and discharging to ensure the maximum benefit of EV owner. Finally, simulation experiments are conducted in AnyLogic, and the results demonstrate the superiority of the algorithm over the classical algorithm.

Abstract Background Hepatocellular carcinoma (HCC) is one of the most prevalent cancers in the world, with a high likelihood of metastasis and a dismal prognosis. The reprogramming of glucose metabolism is critical in the development of HCC. The Warburg effect has recently been confirmed to occur in a variety of cancers, including HCC. However, little is known about the molecular biological mechanisms underlying the Warburg effect in HCC cells. In this study, we sought to better understand how methyltransferase 5, N6‐adenosine (METTL5) controls the development of HCC and the Warburg effect. Methods In the current study, quantitative real‐time polymerase chain reaction and Western blotting were used to detect the expression of METTL5 in HCC tissues and cell lines. Several different cell models and animal models were established to determine the role of METTL5 in glucose metabolism reprogramming and the underlying molecular mechanism of HCC. Glutathione‐S‐transferase pulldown, coimmunoprecipitation, RNA sequencing, non‐targeted metabolomics, polysome profiling, and luciferase reporter assays were performed to investigate the molecular mechanisms of METTL5 in HCC cells. Results We discovered that METTL5 drove glucose metabolic reprogramming to promote the proliferation and metastasis of HCC. Mechanistically, upregulation of METTL5 promoted c‐Myc stability and thus activated its downstream glycolytic genes lactate dehydrogenase A (LDHA), enolase 1 (ENO1), triosephosphate isomerase 1 (TPI1), solute carrier family 2 member 1 (SLC2A1), and pyruvate kinase M2 (PKM2). The c‐Box and ubiquitin binding domain (UBA) regions of ubiquitin specific peptidase 5 (USP5) binded to c‐Myc protein and inhibited K48‐linked polyubiquitination of c‐Myc. Further study revealed that METTL5 controled the USP5 translation process, which in turn regulated the ubiquitination of c‐Myc. Furthermore, we identified cAMP responsive element binding protein 1 (CREB1)/P300 as a critical transcriptional regulator of METTL5 that promoted the transcription of METTL5 in HCC. In patient‐derived tumor xenograft (PDX) models, adenovirus‐mediated knockout of METTL5 had a good antitumor effect and prolonged the survival of PDX‐bearing mice. Conclusions These findings point to a novel mechanism by which CREB1/P300‐METTL5‐USP5‐c‐Myc controls abnormal glucose metabolism and promotes tumor growth, suggesting that METTL5 is a potential therapeutic target and prognostic biomarker for HCC.

Ti-based alloys fabricated by wire and arc additive manufacturing (WAAM) exhibit continuous grain boundaries of α phase (GB-α), which easily initiate cracks and provide a continuous crack propagation path. Herein, an activating flux of CaF2 particles was introduced into WAAM-fabricated Ti–6Al–4V alloy to tailor the microstructure for crack buffering (i.e., inhibit smooth propagation of cracks). The results showed that the addition of CaF2 particles disrupted the continuous GB-α through the dowel-like lamellar α phase (DL-α), which was primary α lamella deeply embedded in the adjacent prior-β grains. This microstructure imparted an optimal combination of strain and strength even in the presence of pores in the WAAM-fabricated Ti–6Al–4V alloy. The enhanced mechanical properties can be attributed to uniform plastic deformations and tendency to transgranular fracture of tensile specimens. DL-α relieved stress concentration at the grain boundary and guided cracks into the prior-β grain interior (i.e., avoided inter-crystalline fracture), thereby promoting the crack buffering effect. The study can provide theory guidance and data support for improving the mechanical performance of WAAM-fabricated Ti–6Al–4V alloy.

A fixed-time adaptive guaranteed performance tracking control is investigated for a category of nonholonomic mobile robots (NMRs) under asymmetric state constraints. For the sake of favorable transient and steady-state properties of the system, a prescribed performance function (PPF) is introduced and a transform function is further constructed. Based on the backstepping technique, an asymmetric barrier Lyapunov function is formulated to ensure the tracking errors converge within a human-specified time. On the foundation of this, the occupation of communication channel is effectively reduced by assigning an event-triggered mechanism (ETM) with relative threshold to the process of controller design. By utilizing the proposed control strategy, the NMR is capable of implementing the enemy dislodging mission while the enemy can always be caught by the NMR and the collision would never be presented. Finally, two simulation experiments are given to verify the effectiveness of the proposed scheme.

A guaranteed performance event-triggered adaptive consensus control is established for uncertain multiagent systems under time-varying actuator faults. To eliminate the impact caused by actuator faults, an adaptive neural network compensation strategy is developed. Simultaneously, by implementing the asymmetric barrier Lyapunov function and transform function, a prescribed time consensus control with guaranteed performance, is constructed. Furthermore, to reduce the frequency of information transmission, an adjustable switching event-triggered control (ASETC) is proposed by using a modified hyperbolic tangent function. It combines the advantage of the relative threshold strategies and the characteristics of the hyperbolic tangent function, giving better flexibility in saving network resources and guaranteeing system performance. By applying the constructed control method, systems with prescribed performance consensus in a prescribed time are achievable while limited network resources and unknown time-varying faults are present. Some simulation examples implemented in MATLAB (R2022a) are given to demonstrate the above results.

Electric ships have been developed in recent years to reduce greenhouse gas emissions. In this system, inverters are the key equipment for the permanent-magnet synchronous motor (PMSM) drive system. The cascaded insulated-gated bipolar transistor (IGBT)-based H-bridge inverter is one of the most attractive multilevel topologies for modern electric ship applications. Usually, the fault-tolerant control strategy is designed to keep the ship in operation for a certain period. However, the fault-tolerant control strategy with hardware redundancy is expensive and slow in response. In addition, after fault-tolerant control, the ship’s PMSM may experience shock and overheating, and IGBT life is reduced due to uneven switching frequency distribution. Therefore, a stratified reconfiguration carrier disposition Sinusoidal Pulse Width Modulation (SPWM) fault-tolerant control strategy is proposed. The proposed strategy can achieve fault tolerance without any extra hardware. A reconfiguration carrier is applied to improve the fundamental amplitude of inverter output voltage to maintain the operation of the ship’s PMSM. In addition, the available states of faulty H-bridge are fully used to contribute to the output. These can improve the life of IGBTs by reducing and balancing the power loss of each H-bridge. The principles of the proposed strategy are described in detail in this study. Taking a cascaded H-bridge seven-level inverter as an example, simulation and experimental results verify that the proposed strategy, in general, has a potential future application on electric ships.

Abstract Background Enhanced de novo lipogenesis is essential for hepatocellular carcinoma (HCC). Abnormally high cullin‐associated and neddylation‐dissociated 1 (CAND1) expression is associated with poor clinical prognosis in HCC. The SKP1‐Cullin‐1‐F‐box (SCF) complex consists of the SKP1, Cullin‐1 and F‐box proteins (FBPs) and performs multiple functions including adipogenesis. SCF complex was modulated by CAND1, but Whether and how the CAND1 promotes HCC by regulating SCF complex and lipogenesis are unknown. Methods HCC samples were used to analyze the correlations between CAND1 expression and clinicopathological characteristics such as survival and prognosis. The in vitro functions of CAND1, FBXO11 and heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNPA2B1) were measured by cell proliferation, colony formation and migration assays. The in vivo functions were tested in multiple mouse liver cancer models including patient‐derived xenograft (PDX), cell line‐derived xenograft and AKT/NRASV12‐induced primary liver cancer models. Injections of adeno‐associated virus targeting CAND1 (AAV‐shCAND1) were performed to evaluate the therapeutic efficacy of targeting CAND1. RNA‐Seq and lipidomic assays followed by serial biochemical experiments including mass spectrometry, immunoprecipitation and GST pull‐down were performed to dissect the underlying mechanisms. Results CAND1 promoted the expression of lipid synthesis genes by disrupting SCF complex assembly and lipid accumulation. Furthermore, we identified hnRNPA2B1 as a novel F‐box protein 11 (FBXO11)‐binding partner. FBXO11 directly bound to hnRNPA2B1 and promoted hnRNPA2B1 ubiquitination and subsequent degradation. Our evaluations of the therapeutic efficacy of AAV‐shCAND1 injections confirmed that targeting the CAND1‐SCFFBXO11‐hnRNPA2B1A signalling axis was therapeutically effective. CAND1 downregulation significantly reduced the tumour burden in a primary mouse liver cancer model and a PDX model. Conclusions Our results highlight that CAND1 is associated with poor prognosis in HCC and regulates lipid metabolic reprogramming by dissociating the SCF complex. Targeting the CAND1‐SCFFBXO11‐hnRNPA2B1 axis may be a novel strategy for HCC treatment.

Abstract Despite of advances in treatment options, hepatocellular carcinoma (HCC) remains nearly incurable and has been recognized as the third leading cause of cancer-related deaths worldwide. As a deubiquitinating enzyme, the antitumor effect of ubiquitin-specific peptidase 53 (USP53) has been demonstrated on few malignancies. In this study, we investigated the potential antitumor role of USP53 in HCC. The results showed that USP53 was downregulated in HCC tissues as well as in HCC cell lines using both in silico data as well as patient samples. Furthermore, the ectopic expression of USP53 inhibited the proliferation, migration and invasion, and induced the apoptosis of HCC cells. Co-immunoprecipitation (CO-IP) assay and mass spectrometry (MS) combined with the gene set enrichment analysis (GSEA) identified cytochrome c (CYCS) as an interacting partner of USP53. USP53 overexpression increased the stability of CYCS in HCC cells following cycloheximide treatment. Finally, the overexpression of CYCS compensated for the decreased apoptotic rates in cells with USP53 knocked down, suggesting that USP53 induced the apoptosis in HCC cells through the deubiquitination of CYCS. To summarize, we identified USP53 as a tumor suppressor as well as a therapeutic target in HCC, providing novel insights into its pivotal role in cell apoptosis.

In this work, we report an approach to control electron dynamics and metastable species generation and enhance the density of atmospheric pressure non-equilibrium plasmas by using dual-frequency excitation sources. The atmospheric dielectric barrier discharge (DBD) of an α-mode radio frequency (RF = 5 MHz) discharge controlled by a low-frequency (LF = 50 kHz) bias is studied based on a one-dimensional (1D) fluid model. Results show that the variation in amplitude ratio of RF and LF modulates the electron dynamic process, resulting in different spatial distributions of electron and metastable particle densities. Moreover, it is further shown that the electron density is substantially increased when the LF component voltage amplitude is larger than 300 V for the initial setting. The discharge process is characterized by fast Fourier transform of the spatio-temporal evolution of the electron power absorption and discharge current. As the LF is applied, three-wave interactions induced by LF and RF coupling are clearly observed, where the sum and beat frequencies between LF and RF are increased, which results in a substantial increase in the electron density. On the other hand, the high RF harmonics, especially for the fundamental and the third harmonic components, are suppressed when increasing the LF component. This work demonstrates that dual-frequency excitation is efficacious to modulate the electron dynamic behaviors and metastable species generation of atmospheric pressure plasma, which can provide a possible approach of optimizing plasma parameters.

Pyrolysis technology has received increasing attention in recent years due to its great potential in the field of low-rank coal clean and efficient conversion. Since pyrolysis reaction is very fast and prone to overreaction, the downer-type reactor is considered as a pyrolyzer due to its unique plug flow reactor characteristics. However, the low solids holdup, which is not beneficial for the fast heat transfer, limits its industrial application. Thus, how to realize high-density operation is crucial to the successful application of the downer reactor. Herein, the definition and strategies of high-density operation in the downer were introduced at first. And then, considering the increasing influence of computational fluid dynamics (CFD) in the fluidization industry, the state-of-the-art progress in downer simulation was reviewed, in which the newly developed drag models for downers were carefully discussed and compared. Also, to help prediction of the pyrolysis behaviors, the widely used pyrolysis kinetic models were systematically summarized. Combined with the potential of the downer in the field of coal pyrolysis, the relevant research progress of hot-state simulation of the downer pyrolyzer were introduced and analyzed. Finally, the suggestions on how to carry out follow-up work were given. It is expected that this review could give a better understanding for designing and optimizing downer pyrolyzer.

In this study, to establish the biomechanical characteristics of commercial vehicle drivers’ muscles and bones while operating the three pedals, a driver pedal-operation simulator was built, and the real-life situation was reconstructed in OpenSim 3.3 software. We set up three seat heights to investigate the drivers’ lower limbs, and the research proceeded in two parts: experiment and simulation. Chinese adult males in the 95th percentile were selected as the research participants. In the experiment, Delsys wireless surface electromyography (EMG) sensors were used to collect the EMG signals of the four main muscle groups of the lower limbs when the drivers operated the three pedals. Then, we analyzed the muscle activation and the degree of muscle fatigue. The simulation was based on OpenSim software to analyze the driver’s lower limb joint angles and joint torque. The results show that the activation of the hamstrings, gastrocnemius, and rectus femoris muscles were higher in the four muscle groups. In respect of torque, in most cases, hip joint torque > knee joint torque > ankle joint torque. The knee joint angles were the largest, and the ankle joint angles changed the most. The experimental results provide a reference for improving drivers’ handling comfort in commercial vehicles and provide theoretical bases for cab design and layout optimization.

Cu and other elements in waste acid from copper smelting flue gas sulphuric acid system have high extraction value. In this paper aiming at the precipitation reaction process of Cu2+ with S2O32-, through experiments, thermodynamic calculation, and product characterization, the precipitation reaction way of Cu2+ was systematically investigated and analyzed. The kinetic parameters of the main reaction and the side reaction in the vulcanization system were determined experimentally to clarify the law of factors which affect the reaction rate. The experimental results show that: S2O32- and Cu2+ directly participate in the reaction to produce CuS, and the reaction process is accompanied by the disproportionation side reaction of S2O32- and H+. The reaction order of S2O32-+Cu2+ is higher than that of S2O32-+H+, and the reaction activation energy of the former is greater than that of the latter. Therefore, increasing the concentration of the vulcanizing agent and increasing the reaction temperature can significantly increase the main reaction selectivity. When the reaction temperature is 80 ℃, the molar ratio of S2O32- to Cu2+ is 2∶1, and the reaction time is 120 min, the removal rate of Cu2+ reaches 99.45%.

OsSUT genes have been demonstrated to be relevant for diverse biological processes in rice. In this study, we identified the close relationship between a 31-bp insertion in a 5' untranslated region (5' UTR) of the OsSUT3 gene and higher OsSUT3 expression in rice panicles by qRT-PCR and transgenic research. Statistically significant results (P < 0.01) were found for this 31-bp insertions/deletions (indels) in the rice pollen development and other panicle traits, such as the pollen number, pollen fertility, seeding rate, and grain length. An evolution analysis showed that the proportion of the 31-bp insertion significantly increases in rice domestication. Therefore, the 31-bp Indel could be considered as a convenient molecular marker to screen more pollen and better panicle traits in rice breeding.

Walnut shell (WS) was used as carbon source and KOH as activator. A series of activated carbon with different pore structures were produced by changing the mass ratio of KOH to carbonized precursor from walnut shell (KOH/carbonized precursor ratio), so as to reveal the relationship between the pore structure and the electrochemical performance. The result shows that the specific surface area, total pore volume, and percentage of mesoporous volume of activated carbon increased obviously with rising KOH/carbonized precursor ratio. Both the initial discharge specific capacity and the scaling performance of the composite cathode increased of first and then decreased slightly with the increase of specific surface area, total pore volume, and percentage of mesoporous volume. When the KOH/carbonized precursor ratio was 6∶1, WSAC-6/S with large specific surface area (3 913 m2/g), total pore volume (2.26 cm3/g), and percentage of mesoporous volume (59.4%) showed high initial specific capacity (1 397 mAh/g) and stable charge/discharge performance (869 mAh/g after 100 cycles at 0.2 C). Especially, at 0.5 C, it still exhibited excellent electrochemical performance, and the specific discharge capacity maintained at 707 mAh/g after 200 cycles.

Mo-Bi-S composite supported on carbon nanotubes (CNTs) was synthesized by hydrothermal method(Mo-Bi-S@CNTs). The effects of preparation temperature on the morphology, structure, and CO2 electrocatalytic performance of the catalysts were investigated. The results show that the Mo-Bi-S nanoparticles synthesized at 200 ℃ were smaller than others, about 10~15 nm in size and distributed uniformly on CNTs. At the same time, they showed better electrocatalytic performance in [NH2-emim]BF4-H2O (ω=40%) electrolyte. The Faraday conversion efficiency of methanol was as high as 63.0% over Mo-Bi-S@CNTs-200 ℃ at the potential -0.3 V vs. SCE. After 6 hours of continuous operation, the current density of the cell remained stable of about -13.5 mA/cm2. In summary, Mo-Bi-S@CNTs-200 ℃ was a kind of electrocatalytic CO2 reduction catalyst with high catalytic activity, methanol selectivity, and stability.

Objective: Emerging studies suggest that long non-coding RNAs (lncRNAs) play crucial roles in hepatocellular carcinoma (HCC). A rapidly increasing number of studies have shown that metabolic changes including lipid metabolic reprogramming play a significant role in the progression of HCC. But it remains to be elucidated how lncRNAs affect tumor cell metabolism. Methods: Through analysis and screening of The Cancer Genome Atlas-Liver Hepatocellular Carcinoma (TCGA-LIHC) dataset, we found a novel lncRNA RP11-386G11.10 was overexpressed, related to prognosis, conserved and non-protein-coding in HCC and related to poor prognosis. Then, CCK-8, colony formation, Transwell invasion, wound healing assays were performed and nude mouse subcutaneous tumour formation and lung metastasis models were established to explore the effect of RP11-386G11.10 on HCC tumour growth and metastasis. Chromatography-mass spectrometry (GC-MS) and Nile red staining detected the effect of RP11-386G11.10 on lipid metabolism in HCC. Mechanistically, we clarified the RP11-386G11.10/miR-345-3p/HNRNPU signalling pathway through dual luciferase reporter, RNA immunoprecipitation (RIP) and chromatin immunoprecipitation (ChIP) assays and identified ZBTB7A as a transcription factor of RP11-386G11.10. Results: RP11-386G11.10 was overexpressed in HCC and positively correlated with tumour size, TNM stage, and poor prognosis in HCC patients. RP11-386G11.10 promoted the proliferation and metastasis of HCC cells in vitro and in vivo. Mechanistically, RP11-386G11.10 acted as a competing endogenous RNA (ceRNA) for miR-345-3p to regulate the expression of HNRNPU and its downstream lipogenic enzymes, leading to lipid accumulation in HCC cells and promoting their growth and metastasis. In addition, we identified ZBTB7A as a transcription factor of RP11-386G11.10. Moreover, HNRNPU promoted the expression of ZBTB7A in HCC cells, thereby increasing the transcriptional activity of RP11-386G11.10, and forming a positive feedback loop, ultimately leading continuous lipid accumulation, growth and metastasis in HCC cells. Conclusions: Our results indicated that the lncRNA RP11-386G11.10 was a novel oncogenic lncRNA that was strongly correlated with the poor prognosis of HCC. The ZBTB7A-RP11-386G11.10-HNRNPU positive feedback loop promoted the progression of HCC by regulating lipid anabolism. RP11-386G11.10 may become a new diagnostic and prognostic biomarker and therapy target for HCC.

Abstract Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide. However, there still remains a lack of effective diagnostic and therapeutic targets for this disease. Increasing evidence demonstrates that RNA modifications play an important role in the progression of HCC, but the role of the N7-methylguanosine (m7G) methylation modification in HCC has not been properly evaluated. Thus, the goal of the present study was to investigate the function and mechanism of the m7G methyltransferase WD repeat domain 4 (WDR4) in HCC as well as its clinical relevance and potential value. We first verified the high expression of WDR4 in HCC and observed that upregulated WDR4 expression increased the m7G methylation level in HCC. WDR4 promoted HCC cell proliferation by inducing the G2/M cell cycle transition and inhibiting apoptosis in addition to enhancing metastasis and sorafenib resistance through epithelial-mesenchymal transition (EMT). Furthermore, we observed that c-MYC (MYC) can activate WDR4 transcription and that WDR4 promotes CCNB1 mRNA stability and translation to enhance HCC progression. Mechanistically, we determined that WDR4 enhances CCNB1 translation by promoting the binding of EIF2A to CCNB1 mRNA. Furthermore, CCNB1 was observed to promote PI3K and AKT phosphorylation in HCC and reduce P53 protein expression by promoting P53 ubiquitination. In summary, we elucidated the MYC/WDR4/CCNB1 signalling pathway and its impact on PI3K/AKT and P53. Furthermore, the result showed that the m7G methyltransferase WDR4 is a tumour promoter in the development and progression of HCC and may act as a candidate therapeutic target in HCC treatment.

Grid-connected inverters are widely used to integrate energy into the grid in renewable energy applications. However, the inverter usually has a high probability of failure due to a large number of semiconductor devices. In addition, especially in the field of marine renewable energy, the humid environment in offshore and coastal areas may make the inverter more prone to failure, which reduces system reliability. Therefore, a serial fault-tolerant topology based on a sustainable reconfiguration is proposed. The proposed topology can be reconfigured continuously by making full use of healthy devices, which can ensure the output voltage capability and improve the possibility of uninterrupted performance as much as possible. In addition, the cooperative modulation signal is selected for the recombined topology to output the desired voltage, which ensures the quality of the power generated by marine renewable energy. Principles of topology reconfiguration and modulation signal selection is described in detail. Simulation and experimental results verify the validation of the proposed method on the seven-level grid-connected inverter.

A real-time tuna detection network on mobile devices is a common tool for accurate tuna catch statistics. However, most object detection models have multiple parameters, and normal mobile devices have difficulties in satisfying real-time detection. Based on YOLOv3, this paper proposes a Tuna-YOLO, which is a lightweight object detection network for mobile devices. Firstly, following a comparison of the performance of various lightweight backbone networks, the MobileNet v3 was used as a backbone structure to reduce the number of parameters and calculations. Secondly, the SENET module was replaced with a CBAM attention module to further improve the feature extraction ability of tuna. Then, the knowledge distillation was used to make the Tuna-YOLO detect more accurate. We created a small dataset by deframing electronic surveillance video of fishing boats and labeled the data. After data annotation on the dataset, the K-means algorithm was used to get nine better anchor boxes on the basis of label information, which was used to improve the detection precision. In addition, we compared the detection performance of the Tuna-YOLO and three versions of YOLO v5-6.1 s/m/l after image enhancement. The results show that the Tuna-YOLO reduces the parameters of YOLOv3 from 234.74 MB to 88.45 MB, increases detection precision from 93.33% to 95.83%, and increases the calculation speed from 10.12 fps to 15.23 fps. The performance of the Tuna-YOLO is better than three versions of YOLO v5-6.1 s/m/l. Tuna-YOLO provides a basis for subsequent deployment of algorithms to mobile devices and real-time catch statistics.

Nowadays, depression has become a common mental disorder with high morbidity and mortality. Due to the limitations of traditional interview-based depression detection, it has become an urgent problem to realize objective, convenient and fast detection. This study is to explore ubiquitous methods of depression detection based on combination learning and functional networks, using sleep physiological data. Sleep physiological data were collected using a portable physiological data instrument, and then preprocess and extract several related features. We applied combination learning to discover the best sleep stage, the optimal features subset, and the most effective classifier, which are hidden behind physiological features, to detect depression. Physiological features in the optimal feature subset based on Euclidean distance are mapped to nodes to construct the functional network. The optimal feature subset was combined with the functional network attributes as the input of the most effective classifier to get the ultimate performance of depression detection. Controlled trials based on ubiquitous sleep physiological data were conducted on different genders. Experiments show that the best results for male and female were derived from slow wave sleep (SWS) and rapid eye movement (REM), with performances of 92.21% and 94.56%, AUC of 0.944 and 0.971, respectively. Thus, our study may provide an effective and ubiquitous method for detect depression.

Abstract Introduction Multidomain intervention approaches have emerged as a potential strategy to reduce dementia risk. We sought to describe the baseline assessment approaches, health conditions, and risk profiles for brain aging of participants in the randomized controlled Multimodal INterventions to delay Dementia and disability in rural China (MIND‐China). Methods MIND‐China engaged residents who were ≥60 years of age and living in rural communities in the western Shandong province. In March to September 2018, all participants underwent the core module assessments via face‐to‐face interviews, clinical examinations, neuropsychological testings, and laboratory tests. Specific modules of examination were performed for sub‐samples, including brain magnetic resonance imaging scans, genetic and blood biochemical markers, actigraphy testing, cardiopulmonary coupling analysis for sleep quality and disturbances, audiometric testing, and optical coherence tomography examination. We performed descriptive analysis. Results In total, 5765 participants (74.9% of all eligible residents) undertook the baseline assessments. The mean age was 70.9 years (standard deviation, 5.9), 57.2% were women, 40.6% were illiterate, and 88.3% were farmers. The overall prevalence of common chronic diseases was 67.2% for hypertension, 23.4% for dyslipidemia, 23.5% for heart disease, 14.4% for diabetes mellitus, and 5.4% for dementia. The prevalence rates of hypertension, diabetes mellitus, dyslipidemia, obesity, heart disease, depressive symptoms, and dementia were higher in women than in men (P

In order to obtain more children’s physical training information and improve the accuracy of children’s physical training monitoring, a multisensor-based children’s physical training information collection, analysis, and monitoring system is proposed. In the process of physical training and sports training, people’s physical training information collection is directly related to the level and effectiveness of physical training. With the combination of multisensor concept and sports training information collection, it can collect the key index data of sports mobilization in real time with the help of multiple sensors and information technology. Taking children’s physical training as the object, this paper designs a multisensor physical training data information acquisition terminal, collects different training characteristic data with the help of multisensor equipment, and then comprehensively analyzes and monitors the physical information with the help of certain fusion technology, so as to construct a human posture recognition algorithm based on children’s physical training information acquisition. Support vector machine and decision tree are used to classify children’s different physical exercise states, and a relatively perfect algorithm architecture of human posture recognition is constructed. The results show that for two decision trees, each decision tree is trained with a total of 675 groups of data, and a total of 342 groups of data are verified and pruned. The two decision trees take 7.17 s and 7.32 s to complete the training process, respectively. It can be seen that when the number of training groups is equal, the training time of the two placement methods is close, so it can be considered that the two placement methods have little effect on the training speed of decision tree. The experimental data show that the design of children’s physical training monitoring system in this paper has a certain market value.

Energy absorption of electromagnetic (EM) waves in collisional dusty plasmas is attracting much attention from researchers due to its applications in many fields, such as plasma antennas, blackout research during reentry, plasma stealth technology, and the design of microwave transmission in plasmas. In this paper, the propagation characteristics, in particular, the energy absorption effects of EM waves in partially ionized unmagnetized collisional dusty plasmas, are investigated. The results show that the effective energy absorption of EM waves is mainly due to the interaction between electrons and waves, and the plasmas with proper collisional frequencies can be used as a broadband frequency EM wave absorption medium with moderate absorption efficiency. Quantitative analyses of the characteristics of the EM waves that propagate in the collisional dusty plasmas are also included in this study.

Sleep staging has attracted significant attention as a critical step in auxiliary diagnosis of sleep disease. To avoid subjectivity of doctor’s manual sleep staging, and to realize scientific management of massive physiological data, an ontology-based decision support tool is proposed. The tool implements an automated procedure for sleep staging using dual-channel electroencephalogram (EEG) signals. First of all, it encodes EEG features, sleep-related concepts and other contextual information to “EEG-Sleep ontology”. Secondly, a rule-set is constructed based on a data mining technique. Finally, the first two steps are processed in a reasoning engine which is automatically assign each 30 s epoch (segment) sleep stage to one of five possible sleep stages: WA, NREM1, NREM2, SWS and REM. The rule set is obtained using EEG data taken from the Sleep-EDF database [EXPANDED] according to the random forest algorithm (RF), we prove that the performance of the proposed method with 89.12% accuracy, and 0.81 Kappa statistics is superior to other algorithms such as Bayesian network, C4.5, support vector machine, and multilayer perceptron. Additionally, our proposed approach improved performance when compared to other studies using a small subset of the Sleep-EDF database [EXPANDED].

Background/Aims: Liver is a vital organ and retains its regeneration capability throughout adulthood, which requires contributions from different cell populations, including liver precursors and intrahepatic stem cells. To overcome the mortality of hepatic progenitors (iHPs) in vitro, we aim to establish reversibly immortalized hepatic progenitor cells from mouse embryonic liver. Methods and Results: Using retroviral system to stably express SV40 T antigen flanked with Cre/LoxP sites, we establish a repertoire of iHP clones with varied differentiation potential. The iHP cells maintain long-term proliferative activity and express varied levels of progenitor markers (Pou5f1/Oct4 and Dlk) and hepatocyte markers (AFP, Alb and ApoB). Five representative iHP clones express hepatic/pancreatic transcription factors HNF3α/Foxa1, HNF3β/Foxa2, and HNF4α/MODY1. Dexamethasone is shown to promote the expression of hepatocyte markers AFP and TAT, along with ICG-uptake and glycogen storage functions in the iHP clones. Cre-mediated removal of SV40 T antigen reverses the proliferative activity of iHP cells. When iHP cells are subcutaneously implanted in athymic nude mice, no tumor formation is observed for up to 8 weeks. Conclusions: We demonstrate that the established iHP cells are stable, reversible, and non-tumorigenic hepatic progenitor-like cells, which should be valuable for studying liver organogenesis, metabolic regulations, and hepatic lineage-specific differentiation.

The poor discharge and charge capacities, and the cycle performance of current Li–air batteries represent critical obstacles to their practical application. The fabrication of an integrated structural air electrode with stable parallel micropore channels and excellent electrocatalytic activity is an efficient strategy for solving these problems. Herein, a novel approach involving the synthesis of nitrogen-doped carbon nanotube (N-CNT) arrays on a carbon paper substrate with a conductive carbon-black layer for use as the air electrode is presented. This design achieves faster oxygen, lithium ion, and electron transfer, which allows higher oxygen reduction/evolution reaction activities. As a result, the N-CNT arrays (N/C = 1:20) deliver distinctly higher discharge and charge capacities, 2203 and 186 mAh g−1, than those of active carbons with capacities of 497 and 71 mAh g−1 at 0.05 mA cm−2, respectively. A theoretical analysis of the experimental results shows that the parallel micropore channels of the air electrode decrease oxygen diffusion resistance and lithium ion transfer resistance, enhancing the discharge and charge capacities and cycle performance of Li–air batteries. Additionally, the N-CNT arrays with a high pyridinic nitrogen content can decompose the lithium peroxide product and recover the electrode morphology, thereby further improving the discharge–charge performance of Li–air batteries.

Despite an increased interest in sustainable energy conversion systems, there have been limited studies investigating the electrocatalytic reaction mechanism of methanol oxidation on Ni-based amorphous materials in alkaline media. A thorough understanding of such mechanisms would aid in the development of amorphous catalytic materials for methanol oxidation reactions. In the present work, amorphous Ni-B and Ni-B-Co nanoparticles were prepared by a simple chemical reduction, and their electrocatalytic properties were investigated by cyclic voltammetry measurements. The diffusion coefficients (D0) for Ni-B, Ni-B-Co0.02, Ni-B-Co0.05, and Ni-B-Co0.1 nanoparticles were calculated to be 1.28 × 10−9, 2.35 × 10−9, 4.48 × 10−9 and 2.67 × 10−9 cm2 s−1, respectively. The reaction order of methanol in the studied transformation was approximately 0.5 for all studied catalysts, whereas the reaction order of the hydroxide ion was nearly 1. The activation energy (Ea) values of the reaction were also calculated for the Ni-B and Ni-B-Co nanoparticle systems. Based on our kinetic studies, a mechanism for the methanol oxidation reaction was proposed which involved formation of an electrocatalytic layer on the surface of amorphous Ni−B and Ni-B-Co nanoparticles. And methanol and hydroxide ions could diffuse freely through this three-dimensional porous conductive layer.

The aim of the current study is to identify the potential biomarkers involved in Hepatocellular carcinoma (HCC) carcinogenesis. A comparative proteomics approach was utilized to identify the differentially expressed proteins in the serum of 10 HCC patients and 10 controls. A total of 12 significantly altered proteins were identified by mass spectrometry. Of the 12 proteins identified, HSP90 was one of the most significantly altered proteins and its over-expression in the serum of 20 HCC patients was confirmed using ELISA analysis. The observations suggest that HSP90 might be a potential biomarker for early diagnosis, prognosis, and monitoring in the therapy of HCC. This work demonstrates that a comprehensive strategy of proteomic identification combined with further validation should be adopted in the field of cancer biomarker discovery.

Cartilage tissue engineering holds great promise for treating cartilaginous pathologies including degenerative disorders and traumatic injuries. Effective cartilage regeneration requires an optimal combination of biomaterial scaffolds, chondrogenic seed cells, and biofactors. Obtaining sufficient chondrocytes remains a major challenge due to the limited proliferative capability of primary chondrocytes. Here we investigate if reversibly immortalized mouse articular chondrocytes (iMACs) acquire long-term proliferative capability while retaining the chondrogenic phenotype. Primary mouse articular chondrocytes (MACs) can be efficiently immortalized with a retroviral vector-expressing SV40 large T antigen flanked with Cre/loxP sites. iMACs exhibit long-term proliferation in culture, although the immortalization phenotype can be reversed by Cre recombinase. iMACs express the chondrocyte markers Col2a1 and aggrecan and produce chondroid matrix in micromass culture. iMACs form subcutaneous cartilaginous masses in athymic mice. Histologic analysis and chondroid matrix staining demonstrate that iMACs can survive, proliferate, and produce chondroid matrix. The chondrogenic growth factor BMP2 promotes iMACs to produce more mature chondroid matrix resembling mature articular cartilage. Taken together, our results demonstrate that iMACs acquire long-term proliferative capability without losing the intrinsic chondrogenic features of MACs. Thus, iMACs provide a valuable cellular platform to optimize biomaterial scaffolds for cartilage regeneration, to identify biofactors that promote the proliferation and differentiation of chondrogenic progenitors, and to elucidate the molecular mechanisms underlying chondrogenesis.

To compare cardiopulmonary resuscitation (CPR) quality between manual CPR and miniaturized chest compressor (MCC) CPR. To improve CPR quality through evaluating the quality of our clinical work of resuscitation by real-time video recording system.The study was a retrospective observational study of adult patients who experienced CPR at the emergency department of Shanghai Tenth People's Hospital from March 2013 to August 2014. All the performance of CPR were checked back by the record of "digital real-time video recording system". Average chest compression rate, actual chest compression rate, the percentage of hands-off period, time lag from patient arrival to chest compression, time lag from patient arrival to manual ventilation, time lag from patient arrival to first IV establish were compared. Causes of chest compression hands-off time were also studied.112 cases of resuscitation attempts were obtained. Average chest compression rate was over 100 compression per minute (cpm) in the majority of cases. However, indicators such as percentage of hands-off periods, time lag from patient arrival to the first manual ventilation and time lag from patient arrival to the first IV establish seemed to be worse in the manual CPR group compared to MCC CPR group. The saving of operators change time seemed to counteract the time spent on MCC equipment. Indicators such as percentage of hands-off periods, time lag between patient arrival to the first chest compression, time lag between patient arrival to the first manual ventilation and time lag from patient arrival to the first IV establish may influence the survival.Our CPR quality remained to be improved. MCC may have a potentially positive role in CPR.

The advent of fluorescence-based quantitative real-time PCR (qPCR) has revolutionized the quantification of gene expression analysis in many fields, including life sciences, agriculture, forensic science, molecular diagnostics, and medicine. While SYBR Green-based qPCR is the most commonly-used platform due to its inexpensive nature and robust chemistry, quantifying the expression of genes with low abundance or RNA samples extracted from highly restricted or limited sources can be challenging because the detection sensitivity of SYBR Green-based qPCR is limited. Here, we develop a novel and effective touchdown qPCR (TqPCR) protocol by incorporating a 4-cycle touchdown stage prior to the quantification amplification stage. Using the same cDNA templates, we find that TqPCR can reduce the average Cq values for Gapdh, Rps13, and Hprt1 reference genes by 4.45, 5.47, and 4.94 cycles, respectively, when compared with conventional qPCR; the overall average Cq value reduction for the three reference genes together is 4.95. We further find that TqPCR can improve PCR amplification efficiency and thus increase detection sensitivity. When the quantification of Wnt3A-induced target gene expression in mesenchymal stem cells is analyzed, we find that, while both conventional qPCR and TqPCR can detect the up-regulation of the relatively abundant target Axin2, only TqPCR can detect the up-regulation of the lowly-expressed targets Oct4 and Gbx2. Finally, we demonstrate that the MRQ2 and MRQ3 primer pairs derived from mouse reference gene Tbp can be used to validate the RNA/cDNA integrity of qPCR samples. Taken together, our results strongly suggest that TqPCR may increase detection sensitivity and PCR amplification efficiency. Overall, TqPCR should be advantageous over conventional qPCR in expression quantification, especially when the transcripts of interest are lowly expressed, and/or the availability of total RNA is highly restricted or limited.

The deuterium excess in summer monsoon precipitation, determined from isotopic measurements(δ18O and Keywords: deuterium excess, relative humidity, origin of precipitation

PurposeTo evaluate the value of DWI in detecting the lesions of pre- and post-radiofrequency ablation (RFA) of the rabbit liver VX2 tumors.Materials and methodsTwenty-two New Zealand White rabbits were tested. The protocol was approved by the Committee on the Ethics of Animal Experiments. Twenty separate tumor fragments were implanted into the livers of 20 rabbits, the liver was exposed by performing midline laparotomy. 3.0T MR DWI (b = 0, 200, 400, 600, 800,1000 s/mm2) were performed 14-21 days after tumor implantation (mean, 17 days) in the 18 tumor-bearing animals. Then RFA was performed in the 18 tumor-bearing animals and in the two healthy animals. 3.0T MR DWI was performed 7-10 days after RFA (mean, 8 days). Pathology exam was performed immediately after the completion of post- RFA MR imaging. Analyzing the features of MRI and ADC values in the pre- and post- RFA lesions of the VX2 tumors, and histopathologic results were compared with imaging findings.ResultsThe difference of ADC value between viable tumor and normal liver parenchyma was significant (PConclusions3.0T MR DWI can be used to follow up the progress of the RFA lesion, it is useful in detecting different tissues after RFA, and it is valuable in the further clinical research.

Multipotent mesenchymal stem cells (MSCs) can undergo self-renewal and give rise to multi-lineages under given differentiation cues. It is frequently desirable to achieve a stable and high level of transgene expression in MSCs in order to elucidate possible molecular mechanisms through which MSC self-renewal and lineage commitment are regulated. Retroviral or lentiviral vector-mediated gene expression in MSCs usually decreases over time. Here, we choose to use the piggyBac transposon system and conduct a systematic comparison of six commonly-used constitutive promoters for their abilities to drive RFP or firefly luciferase expression in somatic HEK-293 cells and MSC iMEF cells. The analyzed promoters include three viral promoters (CMV, CMV-IVS, and SV40), one housekeeping gene promoter (UbC), and two composite promoters of viral and housekeeping gene promoters (hEFH and CAG-hEFH). CMV-derived promoters are shown to drive the highest transgene expression in HEK-293 cells, which is however significantly reduced in MSCs. Conversely, the composite promoter hEFH exhibits the highest transgene expression in MSCs whereas its promoter activity is modest in HEK-293 cells. The reduced transgene expression driven by CMV promoters in MSCs may be at least in part caused by DNA methylation, or to a lesser extent histone deacetlyation. However, the hEFH promoter is not significantly affected by these epigenetic modifications. Taken together, our results demonstrate that the hEFH composite promoter may be an ideal promoter to drive long-term and high level transgene expression using the piggyBac transposon vector in progenitor cells such as MSCs.

Three-dimensional organoids have been recently established from various tissue-specific progenitors (such as intestinal stem cells), induced pluripotent stem cells, or embryonic stem cells. These cultured self-sustaining stem cell-based organoids may become valuable systems to study the roles of tissue-specific stem cells in tissue genesis and disease development. It is thus conceivable that effective genetic manipulations in such organoids may allow us to reconstruct disease processes and/or develop novel therapeutics. Recombinant adenoviruses are one of the most commonly used viral vectors for in vitro and in vivo gene deliveries. In this study, we investigate if adenoviruses can be used to effectively deliver transgenes into the cultured "mini-gut" organoids derived from intestinal stem cells. Using adenoviral vectors that express fluorescent proteins, we demonstrate that adenoviruses can effectively deliver transgenes into the cultured 3-D "mini-gut" organoids. The transgene expression can last at least 10 days in the cultured organoids. As a proof-of-principle experiment, we demonstrate that adenovirus-mediated noggin expression effectively support the survival and self-renewal of mini-gut organoids, while adenovirus-mediated expression of BMP4 inhibits the self-sustainability and proliferation of the organoids. Thus, our results strongly suggest that adenovirus vectors can be explored as effective gene delivery vehicles to introduce genetic manipulations in 3-D organoids.

RNA interference (RNAi) denotes sequence-specific mRNA degradation induced by short interfering double-stranded RNA (siRNA) and has become a revolutionary tool for functional annotation of mammalian genes, as well as for development of novel therapeutics. The practical applications of RNAi are usually achieved by expressing short hairpin RNAs (shRNAs) or siRNAs in cells. However, a major technical challenge is to simultaneously express multiple siRNAs to silence one or more genes. We previously developed pSOS system, in which siRNA duplexes are made from oligo templates driven by opposing U6 and H1 promoters. While effective, it is not equipped to express multiple siRNAs in a single vector. Gibson DNA Assembly (GDA) is an in vitro recombination system that has the capacity to assemble multiple overlapping DNA molecules in a single isothermal step. Here, we developed a GDA-based pSOK assembly system for constructing single vectors that express multiple siRNA sites. The assembly fragments were generated by PCR amplifications from the U6-H1 template vector pB2B. GDA assembly specificity was conferred by the overlapping unique siRNA sequences of insert fragments. To prove the technical feasibility, we constructed pSOK vectors that contain four siRNA sites and three siRNA sites targeting human and mouse β-catenin, respectively. The assembly reactions were efficient, and candidate clones were readily identified by PCR screening. Multiple β-catenin siRNAs effectively silenced endogenous β-catenin expression, inhibited Wnt3A-induced β-catenin/Tcf4 reporter activity and expression of Wnt/β-catenin downstream genes. Silencing β-catenin in mesenchymal stem cells inhibited Wnt3A-induced early osteogenic differentiation and significantly diminished synergistic osteogenic activity between BMP9 and Wnt3A in vitro and in vivo. These findings demonstrate that the GDA-based pSOK system has been proven simplistic, effective and versatile for simultaneous expression of multiple siRNAs. Thus, the reported pSOK system should be a valuable tool for gene function studies and development of novel therapeutics.

Mesenchymal stem cells (MSCs) are multipotent progenitors, which can undergo self-renewal and give rise to multi-lineages. A great deal of attentions have been paid to their potential use in regenerative medicine as potential therapeutic genes can be introduced into MSCs. Genetic manipulations in MSCs requires effective gene deliveries. Recombinant adenoviruses are widely used gene transfer vectors. We have found that although MSCs can be infected in vitro by adenoviruses, high virus titers are needed to achieve high efficiency. Here, we investigate if the commonly-used cationic polymer Polybrene can potentiate adenovirus-mediated transgene delivery into MSCs, such as C2C12 cells and iMEFs. Using the AdRFP adenovirus, we find that AdRFP transduction efficiency is significantly increased by Polybrene in a dose-dependent fashion peaking at 8 μg/ml in C2C12 and iMEFs cells. Quantitative luciferase assay reveals that Polybrene significantly enhances AdFLuc-mediated luciferase activity in C2C12 and iMEFs at as low as 4 μg/ml and 2 μg/ml, respectively. FACS analysis indicates that Polybrene (at 4 μg/ml) increases the percentage of RFP-positive cells by approximately 430 folds in AdRFP-transduced iMEFs, suggesting Polybrene may increase adenovirus infection efficiency. Furthermore, Polybrene can enhance AdBMP9-induced osteogenic differentiation of MSCs as early osteogenic marker alkaline phosphatase activity can be increased more than 73 folds by Polybrene (4 μg/ml) in AdBMP9-transduced iMEFs. No cytotoxicity was observed in C2C12 and iMEFs at Polybrene up to 40 μg/ml, which is about 10-fold higher than the effective concentration required to enhance adenovirus transduction in MSCs. Taken together, our results demonstrate that Polybrene should be routinely used as a safe, effective and inexpensive augmenting agent for adenovirus-mediated gene transfer in MSCs, as well as other types of mammalian cells.

Mouse embryonic fibroblasts (MEFs) are mesenchymal stem cell (MSC)-like multipotent progenitor cells and can undergo self-renewal and differentiate into to multiple lineages, including bone, cartilage and adipose. Primary MEFs have limited life span in culture, which thus hampers MEFs' basic research and translational applications. To overcome this challenge, we investigate if piggyBac transposon-mediated expression of SV40 T antigen can effectively immortalize mouse MEFs and that the immortalized MEFs can maintain long-term cell proliferation without compromising their multipotency. Using the piggyBac vector MPH86 which expresses SV40 T antigen flanked with flippase (FLP) recognition target (FRT) sites, we demonstrate that mouse embryonic fibroblasts (MEFs) can be efficiently immortalized. The immortalized MEFs (piMEFs) exhibit an enhanced proliferative activity and maintain long-term cell proliferation, which can be reversed by FLP recombinase. The piMEFs express most MEF markers and retain multipotency as they can differentiate into osteogenic, chondrogenic and adipogenic lineages upon BMP9 stimulation in vitro. Stem cell implantation studies indicate that piMEFs can form bone, cartilage and adipose tissues upon BMP9 stimulation, whereas FLP-mediated removal of SV40 T antigen diminishes the ability of piMEFs to differentiate into these lineages, possibly due to the reduced expansion of progenitor populations. Our results demonstrate that piggyBac transposon-mediated expression of SV40 T can effectively immortalize MEFs and that the reversibly immortalized piMEFs not only maintain long-term cell proliferation but also retain their multipotency. Thus, the high transposition efficiency and the potential footprint-free natures may render piggyBac transposition an effective and safe strategy to immortalize progenitor cells isolated from limited tissue supplies, which is essential for basic and translational studies.

Adiponectin, the adipose-derived hormone, plays an important role in the suppression of metabolic disorders that can result in type 2 diabetes, obesity, and atherosclerosis. It has been shown that up-regulation of adiponectin or adiponectin receptor has a number of therapeutic benefits. Given that it is hard to convert the full size adiponectin protein into a viable drug, adiponectin receptor agonists could be designed or identified using high-throughput screening. Here, we report on the development of a two-step screening process to identify adiponectin agonists. First step, we developed a high throughput screening assay based on fluorescence polarization to identify adiponectin ligands. The fluorescence polarization assay reported here could be adapted to screening against larger small molecular compound libraries. A natural product library containing 10,000 compounds was screened and 9 hits were selected for validation. These compounds have been taken for the second-step in vitro tests to confirm their agonistic activity. The most active adiponectin receptor 1 agonists are matairesinol, arctiin, (-)-arctigenin and gramine. The most active adiponectin receptor 2 agonists are parthenolide, taxifoliol, deoxyschizandrin, and syringin. These compounds may be useful drug candidates for hypoadiponectin related diseases.