Simple and low-cost microscopy setup for 3D particle field measurement using incoherent illumination and open-source hardware.

The quantification of 3D particle field is of interest for a vast range of fields. While in-line particle holography (PH) can provide high-resolution measurements of particles, it suffers from speckle noise. Plenoptic imaging (PI) is less susceptible to speckle noises, but it involves a trade-off between spatial and angular resolution, rendering images with low resolution. Here, we report a simple microscopy setup with the goals of getting the strengths of both techniques. It is built with off-the-shelf and cost-effective components including a photographic lens, a diaphragm, and a CCD camera. The cost of the microscopy setup is affordable to small labs and individual researchers. The pupil plane of the proposed setup can be mechanically accessible, allowing us to implement pupil plane modulation and increase the depth of field (DOF) without requiring any additional relay lenses. It also allows us to understand the working principle of pupil plane modulation clearly, benefiting microscopy education. It illuminates the sample (particles) using diffuse white light, and thus avoids the problem of speckle noise. It captures multiple perspective images via pupil plane modulation, without requiring trading off angular and spatial resolution. We validate the setup with 2D and 3D particle samples. RESEARCH HIGHLIGHTS: We report a simple and cost-effective microscopy setup with the goals of getting the strengths of plenoptic imaging and in-line particle holography. It is built with off-the-shelf and cost-effective components. The cost of the microscopy setup is affordable to small labs and individual researchers. The pupil plane of the proposed setup can be mechanically accessible, allowing us to implement pupil plane modulation and increase the DOF without requiring any additional relay lenses. It also allows us to understand the working principle of pupil plane modulation clearly, benefiting microscopy education. It illuminates the sample (particles) using diffuse white light, and thus avoids the problem of speckle noise. It captures multiple perspective images via pupil plane modulation, without requiring trading off angular and spatial resolution. We validate the setup with 2D and 3D particle samples.

Mitochondrial Dysfunction in Metabolic Dysfunction Fatty Liver Disease (MAFLD).

Nonalcoholic fatty liver disease (NAFLD) has become an increasingly common disease in Western countries and has become the major cause of liver cirrhosis or hepatocellular carcinoma (HCC) in addition to viral hepatitis in recent decades. Furthermore, studies have shown that NAFLD is inextricably linked to the development of extrahepatic diseases. However, there is currently no effective treatment to cure NAFLD. In addition, in 2020, NAFLD was renamed metabolic dysfunction fatty liver disease (MAFLD) to show that its pathogenesis is closely related to metabolic disorders. Recent studies have reported that the development of MAFLD is inextricably associated with mitochondrial dysfunction in hepatocytes and hepatic stellate cells (HSCs). Simultaneously, mitochondrial stress caused by structural and functional disorders stimulates the occurrence and accumulation of fat and lipo-toxicity in hepatocytes and HSCs. In addition, the interaction between mitochondrial dysfunction and the liver-gut axis has also become a new point during the development of MAFLD. In this review, we summarize the effects of several potential treatment strategies for MAFLD, including antioxidants, reagents, and intestinal microorganisms and metabolites.

An online preparative high-performance liquid chromatography system with enrichment and purification modes for the efficient and systematic separation of Panax notoginseng saponins.

In this study, an online preparative high-performance liquid chromatography (prep-HPLC) system based on the combination of the enrichment and purification modes for the efficient and systematic separation of Panax notoginseng saponins (PNS) was achieved. Five separation columns were used for the first and second separation of target components, eighteen trap columns were used to capture the effluents from the first separation or loading the trapped sample effluents, and a two-position eight-port valve was used to switch between the first and second separations. The conditions for the first and second separation of PNS were simulated and optimized with the online prep-HPLC system. Then, the PNS were separated using optimized chromatographic conditions. Notably, 14 monomer compounds with >90% purity (11 compounds with purity >97%) were simultaneously isolated from PNS using the above self-developed device, and their chemical structures were identified. Moreover, the separation time was less than 33.0 h. After 6 repeated enrichment and purification, the weight of each compound obtained was more than 5.0 mg, with compound 2 weighing over 900 mg. In brief, the self-developed prep-HPLC system, which integrated enrichment and purification, is suitable for the efficient and systematic separation of PNS and has broad application prospects, especially for the separation of complex chemical components in natural products.

Hepatocyte-specific HDAC3 ablation promotes hepatocellular carcinoma in females by suppressing Foxa1/2.

BACKGROUND: Hepatocellular carcinoma (HCC), the most common primary liver cancer, prevails mainly in males and has long been attributed to androgens and higher circumstantial levels of interleukin-6 (IL-6) produced by resident hepatic macrophages. METHODS: Constitutively hepatocyte-specific histone deacetylase 3 (HDAC3)-deficient (HDAC3LCKO) mice and constitutively hepatocyte-specific HDAC3 knockout and systemic IL-6 simultaneously ablated (HDAC3LCKO& IL-6-/-) mice were used in our study to explore the causes of sex differences in HCC. Additionally, we performed human HCC tissues with an IHC score. Correlation analysis and linear regression plots were constructed to reveal the association between HDAC3 and its candidate genes. To further elucidate that HDAC3 controls the expression of Foxa1/2, we knocked down HDAC3 in HUH7 liver cancer cells. RESULTS: We observed a contrary sex disparity, with an earlier onset and higher incidence of HCC in female mice when HDAC3 was selectively ablated in the liver. Loss of HDAC3 led to constant liver injury and the spontaneous development of HCC. Unlike the significant elevation of IL-6 in male mice at a very early age, female mice exhibit stable IL-6 levels, and IL-6 ablation did not eliminate the sex disparity in hepatocarcinogenesis in HDAC3-deficient mice. Oestrogen often protects the liver when combined with oestrogen receptor alpha (ERα); however, ovariectomy in HDAC3-ablated female mice significantly delayed tumourigenesis. The oestrogen-ERα axis can also play a role in tumour promotion in the absence of Foxa1 and Foxa2 in the receptor complex. Loss of HDAC3 profoundly reduced the expression of both Foxa1 and Foxa2 and impaired the binding between Foxa1/2 and ERα. Furthermore, a more frequent HDAC3 decrease accompanied by the simultaneous Foxa1/2 decline was found in female HCC compared to that in male HCC. CONCLUSION: In summary, we reported that loss of HDAC3 reduces Foxa1/2 and thus promotes HCC development in females in an oestrogen-dependent manner.

Facile Recrystallization Process for Tuning the Crystal Morphology and Thermal Safety of Industrial Grade PYX.

In this study, the crystal appearance of industrial grade 2,6-diamino-3,5-dinitropyridine (PYX) was mostly needle-shaped or rod-shaped with an average aspect ratio of 3.47 and roundness of 0.47. According to national military standards, the explosion percentage of impact sensitivity s about 40% and friction sensitivity is about 60%. To improve loading density and pressing safety, the solvent-antisolvent method was used to optimize the crystal morphology, i.e., to reduce the aspect ratio and increase the roundness value. Firstly, the solubility of PYX in DMSO, DMF, and NMP was measured by the static differential weight method, and the solubility model was established. The results showed that the Apelblat equation and Van't Hoff equation could be used to clarify the temperature dependence of PYX solubility in a single solvent. Scanning electron microscopy (SEM) was used to characterize the morphology of the recrystallized samples. After recrystallization, the aspect ratio of the samples decreased from 3.47 to 1.19, and roundness increased from 0.47 to 0.86. The morphology was greatly improved, and the particle size decreased. The structures before and after recrystallization were characterized by infrared spectroscopy (IR). The results showed that no chemical structure changes occurred during recrystallization, and the chemical purity was improved by 0.7%. According to the GJB-772A-97 explosion probability method, the mechanical sensitivity of explosives was characterized. After recrystallization, the impact sensitivity of explosives was significantly reduced from 40% to 12%. A differential scanning calorimeter (DSC) was used to study the thermal decomposition. The thermal decomposition temperature peak of the sample after recrystallization was 5 °C higher than that of the raw PYX. The thermal decomposition kinetic parameters of the samples were calculated by AKTS software, and the thermal decomposition process under isothermal conditions was predicted. The results showed that the activation energy (E) of the samples after recrystallization was higher by 37.9~527.6 kJ/mol than raw PYX, so the thermal stability and safety of the recrystallized samples were improved.

Simple, non-mechanical and automatic calibration approach for axial-scanning microscopy with an electrically tunable lens.

We describe a simple and robust calibration approach for axial-scanning microscopy that realizes axial focus shifts with an electrically tunable lens (ETL). We demonstrate the calibration approach based on a microscope with an ETL placed close to the rear stop of the objective lens. By introducing a target-consisted of repeating lines at one known frequency and placed at a ~45° angle to the imaging path, the calibration method captures multiple images at different ETL currents and calibrates the dependence of the axial focus shift on the ETL current by evaluating the sharpness of the captured images. It calibrates the dependence of the magnification of the microscope on the ETL current by measuring the period of the repeating lines in the captured images. The experimental results show that different from the conventional calibration procedure, the proposed scheme does not involve any mechanical scanning and can simultaneously calibrate the dependence of the axial focus shift and the magnification on the ETL current. This might facilitate imaging studies that require the measurement of fine structures in a 3D volume. We also show the calibration procedure can be used to estimate the radius of a conner-arc sample, fabricated using laser micromachining. We believe that this easy-to-use calibration approach may facilitate use of ETLs for a variety of imaging platforms. It may also provide new insights for the development of novel 3D surface measurement methods. RESEARCH HIGHLIGHTS: The proposed calibration scheme does not involve any mechanical scanning and can simultaneously calibrate the dependence of the axial focus shift and the magnification on the electrically tunable lens (ETL) current. It might facilitate imaging studies that require the measurement of fine structures in a 3D volume, and the use of ETLs for a variety of imaging platforms. It may also provide new insights for the development of novel 3D surface measurement methods.

Increased Small Ubiquitin-like Modifier-Activating Enzyme SAE1 Promotes Hepatocellular Carcinoma by Enhancing mTOR SUMOylation.

SUMOylation, one of the most important posttranslational modifications of proteins, plays an essential role in various biological processes; however, enzymes that control SUMOylation in hepatocellular carcinoma (HCC) are still unclear. Comprehensive exploration of the expression and clinical significance of SUMO enzymes in HCC would be of great value. Here, we obtained the gene expression profile of each small ubiquitin-like modifier (SUMO) protein and the corresponding clinical information from The Cancer Genome Atlas. We found that all SUMO enzymes were significantly increased in HCC tissues compared with that in adjacent nontumorous tissues. We identified a 6-gene prognostic signature, including SAE1, PIAS2, PIAS3, SENP3, SENP5, and UBC9, that could effectively predict the overall survival in patients with HCC. Specifically, SAE1 was the most valuable prognostic indicator. In 282 clinical samples, we found that SAE1 was closely related to the clinicopathologic parameters and prognosis of patients with HCC. In vitro and in vivo studies showed that SAE1 knockdown inhibits the proliferation, migration, and invasion of HCC cells. Mechanistically, we confirmed that SAE1 plays a role in driving HCC progression, which is largely dependent on the SUMOylation of mTOR signaling. In conclusion, our study revealed that the expression of SUMO enzymes, especially SAE1, is highly associated with HCC development and acts as a promising prognostic predictor.

Effects of cactus Opuntia dillenii polysaccharide-based coatings loaded with glutathione on the preservation of freshly cut Chinese water chestnut.

This study explored the effects of coatings based on glutathione-loaded cactus Opuntia dillenii polysaccharide (ODP) on the preservation of freshly cut Chinese water chestnut. Freshly cut Chinese water chestnut samples were treated with one of the three dipping solutions, namely, distilled water (control), 0.4 % glutathione (treatment-1) or 1 % ODP + 0.4 % glutathione (treatment-2) and stored at 3 °C for 10 days. All treatments suppressed respiration rate, weight loss and decreases in firmness and browning and increased soluble solid content and likeness score compared with the control (P < 0.05). In terms of sensory quality, treatment-2 extended the shelf life of the freshly cut Chinese water chestnut at least by 6 days compared with the control group. Results verified that treatment with ODP-based coatings incorporated with glutathione may be a promising method for preserving freshly cut Chinese water chestnut.

Oxidative stress-mediated mitochondrial fission promotes hepatic stellate cell activation via stimulating oxidative phosphorylation.

Previous studies have demonstrated dysregulated mitochondrial dynamics in fibrotic livers and hepatocytes. Little is currently known about how mitochondrial dynamics are involved, nor is it clear how mitochondrial dynamics participate in hepatic stellate cell (HSC) activation. In the present study, we investigated the role of mitochondrial dynamics in HSC activation and the underlying mechanisms. We verified that mitochondrial fission was enhanced in human and mouse fibrotic livers and active HSCs. Moreover, increased mitochondrial fission driven by fis1 overexpression could promote HSC activation. Inhibiting mitochondrial fission using mitochondrial fission inhibitor-1 (Mdivi-1) could inhibit activation and induce apoptosis of active HSCs, indicating that increased mitochondrial fission is essential for HSC activation. Mdivi-1 treatment also induced apoptosis in active HSCs in vivo and thus ameliorated CCl4-induced liver fibrosis. We also found that oxidative phosphorylation (OxPhos) was increased in active HSCs, and OxPhos inhibitors inhibited activation and induced apoptosis in active HSCs. Moreover, increasing mitochondrial fission upregulated OxPhos, while inhibiting mitochondrial fission downregulated OxPhos, suggesting that mitochondrial fission stimulates OxPhos during HSC activation. Next, we found that inhibition of oxidative stress using mitoquinone mesylate (mitoQ) and Tempol inhibited mitochondrial fission and OxPhos and induced apoptosis in active HSCs, suggesting that oxidative stress contributes to excessive mitochondrial fission during HSC activation. In conclusion, our study revealed that oxidative stress contributes to enhanced mitochondrial fission, which triggers OxPhos during HSC activation. Importantly, inhibiting mitochondrial fission has huge prospects for alleviating liver fibrosis by eliminating active HSCs.

Deconstruction of the Optimal Design of Urban Road Interchange Based on the Integration of Smart Transportation and Big Data.

Urban interchange is the core hub connecting various regions, and it is of great significance for alleviating the problem of traffic congestion. In the process of urban interchange design, it is impossible to strictly control the traffic volume, interchange types, and standards by relying on traditional technologies. Smart transportation and big data are emerging technologies based on data, which can provide technical support for design and decision making. Based on this, this paper first uses smart transportation and big data technology to predict the traffic volume of Nancheng New District, so as to calculate the future development trend of the target area. Then, on the basis of traffic volume, the article uses smart transportation and big data technology to optimize the original urban interchange design scheme from the aspects of traffic capacity, safety, economic benefits, and environmental benefits. Finally, the article evaluates the optimized urban interchange scheme by means of comprehensive quantitative indicators and evaluation methods. Experiments show that the traffic capacity of the interchange on the outer ring road optimized by smart transportation and big data has increased to 72.6%, and the environmental coordination has increased from 45.2% to 55.2%. Moreover, the design aesthetics of the urban interchange after optimized design based on smart transportation and big data has increased to 65.9%. In addition, the comprehensive evaluation value of the urban interchange after optimization of smart transportation and big data reached 82.6. This fully shows that the optimal design of urban interchange based on the integration of smart transportation and big data can greatly improve the traffic capacity of urban roads.

Dietary chitosan modulates the growth performance, body composition and nonspecific immunity of juvenile yellow catfish (Pelteobagrus fulvidraco).

This study investigated the effects of feeding different concentrations of chitosan on the growth performance, body composition and non-specific immune function of juvenile yellow catfish (Pelteobagrus fulvidraco). Four kinds of experimental diets were respectively prepared by adding 0 (control group), 5, 10 and 15 g/kg of chitosan to the basal feed and fed to juvenile yellow catfish for 8 weeks. Results show that the body weight gain rate, specific growth rate, survival rate, body protein content, serum superoxide dismutase activity, catalase activity, glutathione peroxidise activity, lysozyme activity and disease resistance ability against Aeromonas hydrophila of the experimental group with chitosan added to its diet were significantly higher than those of the control group optimally by 36.22 %, 14.37 %, 9.46 %, 8.97 %, 50.89 %, 33.15 %, 21.52 %, 40.80 %, 41.09 %, and 79.71 %, respectively (P < 0.05). No significant differences in feed efficiency among all groups (P > 0.05) were observed. The optimum dose of dietary chitosan required for the maximum growth of juvenile yellow catfish was 8.95 g/kg. Therefore, adding an appropriate amount of chitosan (8.95 g/kg) to the feed of yellow catfish can significantly improve its growth performance, ameliorate body composition and enhance its non-specific immunity.

Measurement and Destruction of Porcine Endogenous Retrovirus in the Chinese Bama Minipig.

Porcine hepatocytes are widely used in bioartificial liver (BAL) systems for the treatment of liver failure, and Chinese Bama minipigs (BMPs) are extensively used for animal experiments in the field of medicine in China. The genome of porcine endogenous retroviruses (PERVs) has not yet been accurately quantified, posing a threat to their clinical application because they act as a source of cells. In this study, we used genome sequence data from BMPs to predict PERV copies and their distribution. We validated and quantified the identified PERV copies and subtypes across different BMP individuals and tissues using quantitative real-time polymerase chain reaction and droplet digital polymerase chain reaction, respectively, and found that the BMP genome contains only 11 to 21 PERV copies. Notably, they lack the C subtype, which is a relatively safe cell source. Moreover, we applied CRISPR/Cas9 technology to knock out the pol fragment of PERVs in primary renal fibroblasts (PRFs) from BMPs and obtain PERV-destructed cells. Overall, our results lay a foundation for obtaining PERV-destructed BMPs as a safe source of hepatocytes for BALs for future applications.

Fabrication of Size-Controllable and Arrangement-Orderly HepG2 Spheroids for Drug Screening via Decellularized Liver Matrix-Derived Micropattern Array Chips.

Three-dimensional (3D) culture via micropattern arrays to generate cellular spheroids seems a promising in vitro biomimetic system for liver tissue engineering applications, such as drug screening. Recently, organ-derived decellularized extracellular matrix emerges as arguably the most biomimetic bioink. Herein, decellularized liver matrix (DLM)-derived micropattern array chips were developed to fabricate size-controllable and arrangement-orderly HepG2 spheroids for drug screening. The porcine DLM was obtained by the removal of cellular components and then ground into powder, followed by enzymolysis. DLM as a coating substrate was compared with collagen type I (Col I) and Matrigel in terms of biological performance for enhancing cell adhesion, proliferation, and functions. Subsequently, we used poly(dimethylsiloxane) (PDMS) to adsorb DLM as the bioink to fabricate micropattern array chips. The optimal shape and size of micropattern were determined by evaluating the morphology, viability, and functions of HepG2 3D cellular aggregates. In addition, drug-susceptibility testing (paclitaxel, doxorubicin HCl, and disulfiram) was performed on this novel platform. The DLM provided the tissue-specific microenvironment that provided suitable supports for HepG2 cells, compared to Col I and Matrigel. A circular micropattern with a diameter of 100 µm was the optimal processing parameter to rapidly fabricate large-scale, size-controllable, and arrangement-orderly HepG2 cellular aggregates with 3D spheroid's shape and high cell viability. Drug screening testing showed that the effect of a drug could be directly demonstrated on-chip by confocal microscopy measuring the viability of spheroids. We provide a novel platform for the large-scale generation of HepG2 spheroids with uniform size and arrangement, thus bringing convenience, reducing error, and increasing reproducibility for a rapid drug discovery by fluorescence quantitative analysis. This methodology may be possible to apply in advancing personalized medicine and drug discovery.

Rapamycin facilitates differentiation of regulatory T cells via enhancement of oxidative phosphorylation.

We explored the interplay between energy metabolism and the impact of rapamycin (Rapa) on regulatory T cell (Treg) differentiation. Naïve CD4+ T cells were stimulated under Treg-polarizing conditions with or without Rapa. Rapa promoted Treg induction, as the expression of Foxp3 and Treg phenotypic markers were enhanced. Rapa disrupts glycolysis while favoring mitochondrial metabolism in induced Tregs (iTregs). Metabolic profiling showed reduced glycolytic metabolites in Rapa-treated iTregs, in line with the downregulation of glucose uptake and the expression of glycolytic enzymes. Conversely, Rapa increased the ratios of ATP/ADP and ATP/AMP, the production of mitochondrial ATP, and the expression of ATP5A. Treatment with oxidative phosphorylation inhibitors suppressed Foxp3 expression in Rapa-treated cells. Moreover, Rapa decreased oleic acid and palmitoleic acid levels and increased l-carnitine and acetylcarnitine levels and CPT1A expression in iTregs, indicative of augmented fatty acid oxidation. In conclusion, Rapa induces metabolic reprogramming in Tregs, affecting their differentiation.

A single nucleotide mutation drastically increases the expression of tumor-homing NGR-TNFα in the E. coli M15-pQE30 system by improving gene transcription.

Due to their potent immune stimulation, tumor necrosis factor alpha (TNFα) variants with tumor-homing activity are attractive as novel antitumor drugs. The promising antitumor effect of NGR-TNFα in clinical trials triggered extensive interest in developing novel tumor-homing TNFα variants in recent years. Owing to its promising antitumor effect, NGR-TNFα is usually used as a control for newly developed tumor-homing TNFα variants. In our previous works, we produced a pericyte-targeting Z-TNFα at high levels using the Escherichia coli (E. coli) M15-pQE30 system. To further compare Z-TNFα and NGR-TNFα, we attempted to express NGR-TNFα using the same system. Surprisingly, native NGR-TNFα was expressed at a low (~ 0.2 mg/L) level in E. coli M15 containing the pQE30 plasmid. However, a single nucleotide mutation of C to G, resulting in a substitution of leucine (L) with valine (V) at the start of TNFα, increased the expression of NGR-TNFα by ~ 100 times through improving transcription. In addition, the amino acid substitution showed a little impact on the receptor binding, in vitro cytotoxicity, and in vivo antitumor effect of NGR-TNFα. As fusing NGR to the N-terminus of TNFα with a valine substitution did not reduce the protein yield, the TNFα gene with a C > G mutation might be used to prepare novel tumor-homing TNFα when the native TNFα-based variant is expressed at an extremely low level in E. coli. Notably, in addition to the mutated valine, the impact of N-terminal additional amino acids provided by pQE30 vector on the function of TNFα variant must be carefully evaluated. KEY POINTS : • A single nucleotide mutation increased the expression of NGR-TNFα by two orders. • Nucleotide mutation-induced amino acid substitution did not reduce NGR-TNFα activity.

Molecular design of energetic tetrazine-triazole derivatives.

Nitrogen-rich compounds are promising candidates for preparing high energetic density materials (HEDMs) and show the potential in the application of propellants, explosives, and pyrotechnics. Two kinds of typical nitrogen-rich compounds, such as tetrazine and triazole, have attracted the attentions in recent years owing to their high densities, good thermal stabilities, and excellent energetic performances. In this work, four series of innovative energetic compounds based on the conjugates of tetrazine and triazole bearing various substituents (-NH2, -NO2, and -NHNO2) were designed. The optimized structures, crystal densities, heats of formation (HOFs) in gas phase and in condensed phase, detonation properties, bond dissociation energies (BDEs), and impact sensitivity (h50) of these compounds were studied systematically via density functional theory (DFT) method. The detonation velocities of four series of compounds are in the range between 7.03 and 8.59 km s-1 and their detonation pressures are in the range between 20.6 and 33.1 GPa. Results indicated that the linkage of -N=N- bond contributed significantly to HOFs and energy density of the energetic molecules, and 1,2,3-triazole showed better performances than 1,2,4-triazole slightly. As for the same series compounds with different substituents, the compounds with -NHNO2 possessed the highest HOFs (such as A6, B6, C6, D6). In terms of the energetic properties (D and P), four compounds (A7, B7, C7, and D7) exhibited the comparable performance with the widely used hexa-hydro-1,3,5-trinitro-1,3,5-triazine (RDX) and in the meanwhile displayed superior thermal stability and sensitivity to RDX, which indicated their potential application in the insensitive energetic materials.

Creating rat hepatocyte organoid as an in vitro model for drug testing.

BACKGROUND: Liver organoids have recently been applied as models for liver disease and drug screening, especially when combined with liver-on-a-chip technologies. Compared to hepatocyte-like cells, primary hepatocytes have high functionality but cannot maintain their function when cultured in vitro. Mesenchymal stem cells (MSCs) enhance hepatocyte function and maintain hepatocyte metabolism when co-cultured with hepatocytes. MSCs can help induced pluripotent stem cells to generate an organoid structure via the MSC-based traction force triggered by extracellular matrix (ECM) proteins. In this study, primary hepatocytes were co-cultured with MSCs on a liver-derived ECM to generate liver organoids within a short duration. AIM: To create hepatocyte organoids by co-culturing primary hepatocytes with MSCs on a porcine liver extracellular matrix (PLECM) gel. METHODS: Perfusion and enzymatic hydrolysis were used to form the PLECM gel. Rat hepatocytes and human MSCs were mixed and plated on pre-solidified PLECM gel in a 48-well plate for 48 h to generate organoids. Generated organoids were evaluated through hematoxylin and eosin, periodic acid-Schiff, immuno-histological, and immunofluorescence staining, and quantitative PCR for alb, CYP450 gene markers, and urea cycle genes. Culture medium was collected to detect albumin (ALB) and urea production on days 2, 4, 6, 8, 14, and 20. RESULTS: The whole porcine liver was perfused and enzymatically hydrolyzed to form a PLECM gel. The structural components and basement membrane composition of the ECM, such as collagen type I, collagen type IV, fibronectin, and laminin, were demonstrated to be retained. Through interaction of human MSCs with the liver-derived ECM, primary hepatocytes and human MSCs assembled together into a 3D construction and generated primary hepatocyte organoids for 48 h. The mRNAs of the gene alb, the CYP450 gene markers cyp1a1, cyp1a2, and cyp3a2 as well as urea cycle genes arg-1, asl, ass-1, cps-1, nags were highly expressed in hepatocyte organoids. Long-term survival of the primary hepatocyte organoids, as well as stable functionality, was demonstrated via ALB and urea production in vitro. CONCLUSION: Our new method of creating primary hepatocyte organoids by co-culturing hepatocytes with MSCs on liver-derived ECM hydrogels could be used to develop models for liver disease and for drug screening.

Customizing a Tridomain TRAIL Variant to Achieve Active Tumor Homing and Endogenous Albumin-Controlled Release of the Molecular Machine In Vivo.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is an attractive antitumor drug candidate for precision cancer therapy due to its superior selective cytotoxicity in a variety of tumor cells. However, the clinical application of TRAIL in cancer therapy has been limited by its poor tumor-homing capacities and short half-life. Herein, we designed a tridomain TRAIL variant, Z-ABD-TRAIL, by sequentially fusing the platelet-derived growth factor receptor beta (PDGFRß)-specific affibody ZPDGFRß and an albumin-binding domain (ABD) to the N-terminus of TRAIL. The fusion protein Z-ABD-TRAIL was produced as a soluble protein with high yield in Escherichia coli (E. coli). The ZPDGFRß domain provided Z-ABD-TRAIL with PDGFRß-binding properties and thus promoted its tumor homing via the engagement of PDGFRß-expressing pericytes on tumor microvessels. ABD-mediated binding of Z-ABD-TRAIL to albumin in the blood endowed TRAIL with long-lasting (>72 h for Z-ABD-TRAIL vs <0.5 h for TRAIL) abilities to kill tumor cells. Although the in vitro cytotoxicity of Z-ABD-TRAIL in tumor cells was similar to that of the parent TRAIL, the in vivo tumor uptake, apoptosis-inducing ability, and antitumor effect of Z-ABD-TRAIL were much greater than those of TRAIL, indicating that ZPDGFRß-mediated tumor homing and ABD-introduced albumin binding significantly improved the pharmacodynamics of TRAIL. In addition, repeated injection of high-dose Z-ABD-TRAIL showed no obvious acute toxicity in mice. These results demonstrate that the newly designed tridomain Z-ABD-TRAIL is a promising agent for precision cancer therapy.

Improved TDM scheme and data extracting algorithm for polymerization evaluation.

To quickly evaluate holographic photopolymers with different formulations, the most effective method is to record a volume holographic grating in the samples and detect the grating's diffraction in real time. Since the volume grating is highly sensitive to incident angle, existing schemes need to precisely control many space-related parameters. This study proposes an improved scheme, in which two different sized spots are used to reduce the requirements for the overlap of the two spots and the installation precision of the samples. Transmittances, diffractive efficiencies and diffractive asymmetries are obtained at a high sampling rate, through a specifically designed algorithm with the data from uncalibrated high-speed photodiodes. The experimental results show that the proposed scheme performance well in evaluating holographic photopolymer.

[Comparison Between Different Mitochondrial Staining Methods in Cell and Tissue Samples].

OBJECTIVE: To compare the effects of mitochondria staining between specific mitochondrial fluorescent probes and anti-mitochondrial protein antibody in cell and tissue samples. METHODS: The HepG2 cells fixed by 4% paraformaldehyde were stained with MitoTracker Deep Red (100 nmol/L) or anti-Grp75 antibody (75 nmol/L or 100 nmol/L). The human healthy liver tissue samples fixed by 4% paraformaldehyde were stained with 150 nmol/L MitoTracker Deep Red or anti-Grp75 antibody. The above stained cell and tissue samples were observed using confocal microscopy. RESULTS: We found non-specific staining in HeLa cells and obscure mitochondrial image using MitoTracker Deep Red probes, while clear tubular and punctate distribution using anti-Grp75 antibody. In contrast, we observed more specific and better effects of MitoTracker Deep Red probes-stained liver tissue samples as compared to the antibody. CONCLUSION: To visualize mitochondria, the anti-Grp75 antibody staining worked better on cells and the MitoTracker Deep Red probes are more suitable for tissue samples.