Activation of farnesoid X receptor enhances the efficacy of normothermic machine perfusion in ameliorating liver ischemia-reperfusion injury.

The shortage of transplant organs remains a severe global issue. Normothermic machine perfusion (NMP) has the potential to increase organ availability, yet its efficacy is hampered by the inflammatory response during machine perfusion. Mouse liver ischemia-reperfusion injury (IRI) models, discarded human liver models, and porcine marginal liver transplantation models were utilized to investigate whether farnesoid X receptor (FXR) activation could mitigate inflammation-induced liver damage. FXR expression levels before and after reperfusion were measured. Gene editing and coimmunoprecipitation techniques were employed to explore the regulatory mechanism of FXR in inflammation inhibition. The expression of FXR correlates with the extent of liver damage after reperfusion. Activation of FXR significantly suppressed the inflammatory response triggered by IRI, diminished the release of proinflammatory cytokines, and improved liver function recovery during NMP, assisting discarded human livers to reach transplant standards. Mechanistically, FXR disrupts the interaction between p65 and p300, thus inhibiting modulating the nuclear factor kappa-B signaling pathway, a key instigator of inflammation. Our research across multiple species confirms that activating FXR can optimize NMP by attenuating IRI-related liver damage, thereby improving the utilization of marginal livers for transplantation.

The establishment of diseased human whole organ model by normothermic machine perfusion technique: Principle of concept.

BACKGROUND: The global incidence of liver diseases is rising, yet there remains a dearth of precise research models to mimic these diseases. The use of normothermic machine perfusion (NMP) to study diseased livers recovered from liver transplantation (LT) recipients presents a promising avenue. Accordingly, we have developed a machine perfusion system tailored specifically for the human whole diseased livers and present our experience from the NMP of diseased livers. METHODS: Six diseased livers recovered from LT recipients with different diagnoses were collected. The diseased livers were connected to the machine perfusion system that circulated tailored perfusate, providing oxygen and nutrients. The pressure and flow of the system were recorded, and blood gas analysis and laboratory tests of perfusate and bile were examined to analyze the function of the diseased livers. Liver tissues before and after NMP were collected for histological analysis. RESULTS: Experiments showed that the system maintained the diseased livers in a physiological state, ensuring stable hemodynamics and a suitable partial pressure of oxygen and carbon dioxide. The results of blood gas analysis and laboratory tests demonstrated a restoration and sustenance of metabolism with minimal damage. Notably, a majority of the diseased livers exhibited bile production continuously, signifying their vivid functional integrity. The pathological characteristics remained stable before and after NMP. CONCLUSION: We successfully established the machine perfusion system tailored specifically for diseased human whole livers. Through the application of this system, we have developed a novel in vitro model that faithfully recapitulates the main features of human liver disease. This model holds immense promise as an advanced disease modeling platform, offering profound insights into liver diseases and potential implications for research and therapeutic development.

Pharmacokinetic enhancement of oncolytic virus M1 by inhibiting JAK‒STAT pathway.

Oncolytic viruses (OVs), a group of replication-competent viruses that can selectively infect and kill cancer cells while leaving healthy cells intact, are emerging as promising living anticancer agents. Unlike traditional drugs composed of non-replicating compounds or biomolecules, the replicative nature of viruses confer unique pharmacokinetic properties that require further studies. Despite some pharmacokinetics studies of OVs, mechanistic insights into the connection between OV pharmacokinetics and antitumor efficacy remain vague. Here, we characterized the pharmacokinetic profile of oncolytic virus M1 (OVM) in immunocompetent mouse tumor models and identified the JAK‒STAT pathway as a key modulator of OVM pharmacokinetics. By suppressing the JAK‒STAT pathway, early OVM pharmacokinetics are ameliorated, leading to enhanced tumor-specific viral accumulation, increased AUC and Cmax, and improved antitumor efficacy. Rather than compromising antitumor immunity after JAK‒STAT inhibition, the improved pharmacokinetics of OVM promotes T cell recruitment and activation in the tumor microenvironment, providing an optimal opportunity for the therapeutic outcome of immune checkpoint blockade, such as anti-PD-L1. Taken together, this study advances our understanding of the pharmacokinetic-pharmacodynamic relationship in OV therapy.

TUBB2B facilitates progression of hepatocellular carcinoma by regulating cholesterol metabolism through targeting HNF4A/CYP27A1.

Cholesterol metabolism plays a critical role in the progression of hepatocellular carcinoma (HCC), but it is not clear how cholesterol metabolism is regulated. The tubulin beta class I genes (TUBBs) are associated with the prognosis of many different cancers. To confirm the function of TUBBs in HCC, the Kaplan-Meier method and Cox analyses were performed using TCGA and GSE14520 datasets. A higher expression of TUBB2B is an independent prognostic factor for shorter over survival in HCC patients. Deletion of TUBB2B in hepatocytes inhibits proliferation and promotes tumor cell apoptosis, while over-expression of TUBB2B has the opposite function. This result was confirmed in a mouse xenograft tumor model. Mechanistically, TUBB2B induces the expression of CYP27A1, an enzyme responsible for the conversion of cholesterol to 27-hydroxycholesterol, which leads to the up-regulation of cholesterol and the progression of HCC. In addition, TUBB2B regulates CYP27A1 via human hepatocyte nuclear factor 4alpha (HNF4A). These findings indicated that TUBB2B functions as an oncogene in HCC, and plays a role in promoting cell proliferation and anti-apoptosis through targeting HNF4A/CYP27A1/cholesterol.

STT3A-mediated viral N-glycosylation underlies the tumor selectivity of oncolytic virus M1.

Oncolytic viruses are emerging as promising anticancer agents. Although the essential biological function of N-glycosylation on viruses are widely accepted, roles of N-glycan and glycan-processing enzyme in oncolytic viral therapy are remain elusive. Here, via cryo-EM analysis, we identified three distinct N-glycans on the envelope of oncolytic virus M1 (OVM) as being necessary for efficient receptor binding. E1-N141-glycan has immediate impact on the binding of MXRA8 receptor, E2-N200-glycan mediates the maturation of E2 from its precursor PE2 which is unable to bind with MXRA8, and E2-N262-glycan slightly promotes receptor binding. The necessity of OVM N-glycans in receptor binding make them indispensable for oncolysis in vitro and in vivo. Further investigations identified STT3A, a key catalytic subunit of oligosaccharyltransferase (OST), as the determinant of OVM N-glycosylation, and STT3A expression in tumor cells is positively correlated with OVM-induced oncolysis. Increased STT3A expression was observed in various solid tumors, pointing to a broad-spectrum anticancer potential of OVM. Collectively, our research supports the importance of STT3A-mediated N-glycosylation in receptor binding and oncolysis of OVM, thus providing a novel predictive biomarker for OVM.

Oncolytic virus M1 functions as a bifunctional checkpoint inhibitor to enhance the antitumor activity of DC vaccine.

Although promising, dendritic cell (DC) vaccines still provide limited clinical benefits, mainly due to the immunosuppressive tumor microenvironment (TME) and the lack of tumor-associated antigens (TAAs). Oncolytic virus therapy is an ideal strategy to overcome immunosuppression and expose TAAs; therefore, they may work synergistically with DC vaccines. In this study, we demonstrate that oncolytic virus M1 (OVM) can enhance the antitumor effects of DC vaccines across diverse syngeneic mouse tumor models by increasing the infiltration of CD8+ effector T cells in the TME. Mechanically, we show that tumor cells counteract DC vaccines through the SIRPα-CD47 immune checkpoint, while OVM can downregulate SIRPα in DCs and CD47 in tumor cells. Since OVM upregulates PD-L1 in DCs, combining PD-L1 blockade with DC vaccines and OVM further enhances antitumor activity. Overall, OVM strengthens the antitumor efficacy of DC vaccines by targeting the SIRPα-CD47 axis, which exerts dominant immunosuppressive effects on DC vaccines.

Identification of the receptor of oncolytic virus M1 as a therapeutic predictor for multiple solid tumors.

Over the last decade, oncolytic virus (OV) therapy has shown its promising potential in tumor treatment. The fact that not every patient can benefit from it highlights the importance for defining biomarkers that help predict patients' responses. As particular self-amplifying biotherapeutics, the anti-tumor effects of OVs are highly dependent on the host factors for viral infection and replication. By using weighted gene co-expression network analysis (WGCNA), we found matrix remodeling associated 8 (MXRA8) is positively correlated with the oncolysis induced by oncolytic virus M1 (OVM). Consistently, MXRA8 promotes the oncolytic efficacy of OVM in vitro and in vivo. Moreover, the interaction of MXRA8 and OVM studied by single-particle cryo-electron microscopy (cryo-EM) showed that MXRA8 directly binds to this virus. Therefore, MXRA8 acts as the entry receptor of OVM. Pan-cancer analysis showed that MXRA8 is abundant in most solid tumors and is highly expressed in tumor tissues compared with adjacent normal ones. Further study in cancer cell lines and patient-derived tumor tissues revealed that the tumor selectivity of OVM is predominantly determined by a combinational effect of the cell membrane receptor MXRA8 and the intracellular factor, zinc-finger antiviral protein (ZAP). Taken together, our study may provide a novel dual-biomarker for precision medicine in OVM therapy.

Real-Time Visualization and Quantification of Oncolytic M1 Virus In Vitro and In Vivo.

Alphavirus M1 is a promising oncolytic virus for cancer therapy. Here, we constructed a fluorescent reporter virus for real-time visualization and quantification of M1 virus both in vitro and in vivo. The reporter-encoding M1 virus maintained the characteristics of parental virus in the aspects of structure, replication capacity, the feature to induce cytopathic cell death, and the property of tumor targeting. The fluorescence is positively correlated with virus replication both in vitro and in vivo. More importantly, the reporter can be stably expressed for at least 10 generations in a serial passage assay. In summary, we successfully constructed stable and authentic reporter viruses for studying M1 virus and provided a feasible technical route for gene modification of oncolytic virus M1.

Visualization of the Oncolytic Alphavirus M1 Life Cycle in Cancer Cells.

Oncolytic alphavirus M1 has been shown to selectively target and kill cancer cells, but cytopathic morphologies induced by M1 virus and the life cycle of the M1 strain in cancer cells remain unclear. Here, we study the key stages of M1 virus infection and replication in the M1 virus-sensitive HepG2 liver cancer cell line by transmission electron microscopy, specifically examining viral entry, assembly, maturation and release. We found that M1 virus induces vacuolization of cancer cells during infection and ultimately nuclear marginalization, a typical indicator of apoptosis. Specifically, our results suggest that the endoplasmic reticulum participates in the assembly of nucleocapsids. In the early and late stage of infection, three kinds of special cytopathic vacuoles are formed and appear to be involved in the replication, maturation and release of the virus. Taken together, our data displayed the process of M1 virus infection of tumor cells and provide the structural basis for the study of M1 virus-host interactions.

Necroptotic virotherapy of oncolytic alphavirus M1 cooperated with Doxorubicin displays promising therapeutic efficacy in TNBC.

Triple-negative breast cancer (TNBC) is the most aggressive molecular subtype among breast tumors and remains a challenge even for the most current therapeutic regimes. Here, we demonstrate that oncolytic alphavirus M1 effectively kills both TNBC and non-TNBC. ER-stress and apoptosis pathways are responsible for the cell death in non-TNBC as reported in other cancer types, yet the cell death in TNBC does not depend on these pathways. Transcriptomic analysis reveals that the M1 virus activates necroptosis in TNBC, which can be pharmacologically blocked by necroptosis inhibitors. By screening a library of clinically available compounds commonly used for breast cancer treatment, we find that Doxorubicin enhances the oncolytic effect of the M1 virus by up to 100-fold specifically in TNBC in vitro, and significantly stalls the tumor growth of TNBC in vivo, through promoting intratumoral virus replication and further triggering apoptosis in addition to necroptosis. These findings reveal a novel antitumor mechanism and a new combination regimen of the M1 oncolytic virus in TNBC, and highlight a need to bridge molecular diagnosis with virotherapy.

Zinc-finger antiviral protein acts as a tumor suppressor in colorectal cancer.

Avoiding immune destruction is essential for tumorigenesis. Current research into the interaction between tumor and immunological niches complement tumor pathology beyond cancer genetics. Intrinsic host defense immunity is a specialized innate immunity component to restrict viral infection. However, whether intrinsic immunity participates in tumor pathology is unclear. Previously, we identified a zinc-finger antiviral protein ZAP that is commonly downregulated in a panel of clinical cancer specimens. However, whether ZAP has an impact on tumor development was unknown. Here we report ZAP as a genuine tumor suppressor. Pan-caner analysis with TCGA data from 712 patients and large-scale immunohistochemistry in tissue microarrays from 1552 patients reveal that ZAP is prevalently downregulated, and associated with poor survival in liver, colon, and bladder cancer patients. Ectopic over-expression of ZAP inhibits the malignant phenotypes of colorectal tumor by cell cycle arrest. Using RNA immunoprecipitation and RNA decay assays, we demonstrate that ZAP directly and specifically binds to and degrades the transcript of TRAILR4, which in turn represses TRAILR4 expression and inhibits the aggressiveness of colorectal cancer cells. Furthermore, our CRISPR-engineered mice models show that loss-of-function of ZAP synergizes with APC-deficiency to drive malignant colorectal cancer in vivo. Overall, we identify a previously unknown function of the antiviral factor ZAP in colorectal tumorigenesis, linking intrinsic immunity to tumor pathogenetics.

Monte Carlo simulation of an anharmonic Debye-Waller factor to the T4 order.

In an increasing number of cases the harmonic approximation is incommensurate with the quality of Bragg diffraction data, while results of the anharmonic Debye-Waller factor are not typically available. This paper presents a Monte Carlo computation of a Taylor expansion of an anharmonic Debye-Waller factor with respect to temperature up to the fourth order, where the lattice was a face-centred cubic lattice and the atomic interaction was described by the Lennard-Jones potential. The anharmonic Debye-Waller factor was interpreted in terms of cumulants. The results revealed three significant points. Firstly, the leading term of anharmonicity had a negative contribution to the Debye-Waller factor, which was confirmed by Green's function method. Secondly, the fourth-order cumulants indicated a non-spherical probability density function. Thirdly, up to the melting point of two different densities, the cumulants up to the fourth order were well fitted by the Taylor expansion up to T4, which suggested that the Debye-Waller factor may be calculated by perturbation expansion up to the corresponding terms. In conclusion, Monte Carlo simulation is a useful approach for calculating the Debye-Waller factor.

Characterization of anti-leukemia components from Indigo naturalis using comprehensive two-dimensional K562/cell membrane chromatography and in silico target identification.

Traditional Chinese Medicine (TCM) has been developed for thousands of years and has formed an integrated theoretical system based on a large amount of clinical practice. However, essential ingredients in TCM herbs have not been fully identified, and their precise mechanisms and targets are not elucidated. In this study, a new strategy combining comprehensive two-dimensional K562/cell membrane chromatographic system and in silico target identification was established to characterize active components from Indigo naturalis, a famous TCM herb that has been widely used for the treatment of leukemia in China, and their targets. Three active components, indirubin, tryptanthrin and isorhamnetin, were successfully characterized and their anti-leukemia effects were validated by cell viability and cell apoptosis assays. Isorhamnetin, with undefined cancer related targets, was selected for in silico target identification. Proto-oncogene tyrosine-protein kinase (Src) was identified as its membrane target and the dissociation constant (Kd) between Src and isorhamnetin was 3.81 µM. Furthermore, anti-leukemia effects of isorhamnetin were mediated by Src through inducing G2/M cell cycle arrest. The results demonstrated that the integrated strategy could efficiently characterize active components in TCM and their targets, which may bring a new light for a better understanding of the complex mechanism of herbal medicines.

Diagnosing x-ray power and energy of tungsten wire array z-pinch with a flat spectral response x-ray diode.

Fast z-pinch is a very efficient way of converting electromagnetic energy to radiation. With an 8-10 MA current on primary test stand facility, about 1 MJ electromagnetic energy is delivered to vacuum chamber, which heats z-pinch plasma to radiate soft x-ray. To develop a pulsed high power x-ray source, we studied the applicability of diagnosing x-ray power from tungsten wire array z-pinch with a flat spectral response x-ray diode (FSR-XRD). The detector was originally developed to diagnose radiation of a hohlraum in SG-III prototype laser facility. It utilized a gold cathode XRD and a specially configured compound gold filter to yield a nearly flat spectral response in photon energy range of 0.1-4 keV. In practice, it was critical to avoid surface contamination of gold cathode. It is illustrated that an exposure of an XRD to multiple shots caused a significant change of response. Thus, in diagnosing x-ray power and energy, we used each XRD in only one shot after calibration. In a shot serial, output of FSR-XRD was compared with output of a nickel bolometer. In these shots, the outputs agreed with each other within their uncertainties which were about 12% for FSR-XRD and about 15% for bolometer. Moreover, the ratios between the FSR-XRD and the bolometer among different shots were explored. In 8 shots, the standard deviation of the ratio was 6%. It is comparable to XRD response change of 7%.

A nomogram for predicting the benefit of adjuvant cytokine-induced killer cell immunotherapy in patients with hepatocellular carcinoma.

The benefits of adjuvant cytokine-induced killer (CIK) cell immunotherapy for hepatocellular carcinoma (HCC) remain mixed among patients. Here, we constructed a prognostic nomogram to enable individualized predictions of survival benefit of adjuvant CIK cell treatment for HCC patients. Survival analysis showed that the median overall survival (OS) and progression-free survival (PFS) for patients in the hepatectomy/CIK combination group were 41 and 16 months, respectively, compared to 28 and 12 months for patients in the hepatectomy alone group (control). Based on multivariate analysis of the entire cohort, independent factors for OS were tumor size, tumor capsule, pathological grades, total bilirubin, albumin, prothrombin time, alpha-fetoprotein, and tumor number, which were incorporated into the nomogram. The survival prediction model performed well, as assessed by the c-index and calibration curve. Internal validation revealed a c-index of 0.698, which was significantly greater than the c-index value of the TNM (tumor-node-metastasis) staging systems of 0.634. The calibration curves fitted well. In conclusions, our developed nomogram resulted in more accurate individualized predictions of the survival benefit from adjuvant CIK cell treatment after hepatectomy. The model may provide valuable information to aid in the decision making regarding the application of adjuvant CIK cell immunotherapy.

[Diversity and stability of arthropod community in peach orchard under effects of ground cover vegetation].

A comparative study was conducted on the arthropod community in peach orchards with and without ground cover vegetation. In the orchard with ground cover vegetation, the individuals of beneficial, neutral, and phytophagous arthropods were 1.48, 1.84 and 0.64 times of those in the orchard without ground cover vegetation, respectively, but the total number of arthropods had no significant difference with that in the orchard without ground cover vegetation. The species richness, Shannon's diversity, and Pielou's evenness index of the arthropods in the orchard with ground cover vegetation were 83.733 +/- 4.932, 4.966 +/- 0.110, and 0.795 +/- 0.014, respectively, being significantly higher than those in the orchard without ground cover vegetation, whereas the Berger-Parker's dominance index was 0.135 +/- 0.012, being significantly lower than that (0.184 +/- 0.018) in the orchard without ground cover vegetation. There were no significant differences in the stability indices S/N and Sd/Sp between the two orchards, but the Nn/Np, Nd/Np, and Sn/Sp in the orchard with ground cover vegetation were 0.883 +/- 0.123. 1714 +/- 0.683, and 0.781 +/- 0.040, respectively, being significantly higher than those in the orchard without ground cover vegetation. Pearson's correlation analysis indicated that in the orchard with ground cover vegetation, the Shannon's diversity index was significantly negatively correlated with Nd/Np, Sd/Sp, and S/N but had no significant correlations with Nn/Np and Sn/Sp, whereas in the orchard without ground cover vegetation, the diversity index was significantly positively correlated with Nn/Np and Nd/Np and had no significant correlations with Sd/Sp, Sn/Sp, and S/N.