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Abstract The freezing and thawing cycle is one of the primary causes of damage and instability to buildings in seasonal frost regions. During this process, the mechanical properties of soil are affected, leading to settlement, cracking, or deformation of infrastructure. Mitigating or reducing the occurrence of building frost damage in seasonal frost regions has become an important subject of study. Freeze–thaw (F–T) action will influence the distribution of moisture inside the reinforced soil and change the strength of thawing soil, which is closely related to the main influencing factors, such as initial moisture content, compaction degree, reinforced spacing, number of freeze–thaw cycles (FTC), freezing temperature, and effective vertical stress. Cohesion is an important index to determine the shear strength of clay, which is important to analyze the change in cohesion after F–T. Meanwhile, cohesion is closely related to soil moisture content. This study conducted orthogonal experiments on these primary influencing factors (6 factors at 5 levels) through FTC tests, triaxial tests, and moisture content tests to determine the undrained cohesion and moisture content of the clay after FTC, thereby establishing the influence of reinforcement on soil strength under freeze–thaw conditions. Based on the experimental results, SPSS software was used to fit the regression equations of undrained cohesion and moisture content expressed by the main influencing factors at different heights of the clay. Optimization options for the main influencing factors were obtained with Matlab software when the highest undrained cohesion values 6.8, 10.6, 8.9 kPa and lowest moisture content values 24.0%, 24.3%, 26.2% appeared in upper, middle and lower parts of the testing clay structure respectively, in conditions of − 15 °C freezing temperature and 5 times FTC. And determined the optimal combinations of moisture content, reinforcement spacing, compaction density, and vertical load at different heights. Decreasing reinforced spacing in silty clay was beneficial for liquid underwater seepage after F–T. The redistribution of internal moisture in the soil sample strengthened its undrained cohesion, thereby increasing the soil's shear strength. Comparing reinforcement conditions at different locations, it was found that when there were 3 layers of reinforcement with a spacing of 150 mm between them, this spacing was optimal. It played a significant role in improving the soil's shear strength and enhancing its bearing capacity. For reinforced clay itself, the order of the main factors influencing the undrained cohesion of soil after F–T, from high to low, was initial moisture content, reinforced spacing, and compaction degree.

BackgroundHighly selective type Ib mesenchymal–epithelial transition gene (MET) tyrosine kinase inhibitors (TKIs) are the standard-of-care (SOC) therapy for previously untreated non-small cell lung cancer (NSCLC) harboring MET exon 14 (METex14) skipping mutations. However, there are rare reports describing effective regimens for patients who fail SOC without identifying resistant mutations or tissue transformation.Case reportWe report the first case of a 74-year-old woman with lung adenocarcinoma (cT1cNxM0) harboring METex14 splice region mutation, which was identified by a next-generation sequencing (NGS)-based assay. The patient was administered two treatments, including first-line tepotinib and second-line vebreltinib. The patient achieved progression-free survival (PFS) of 7.6 months, and then disease progression of tepotinib was observed. A re-biopsy was performed for NGS, which revealed the same mutations as before, with no new gene mutations detected. The woman received subsequent vebreltinib therapy and experienced durable clinical benefits. In the first 6.8 months, chest computed tomography demonstrated stable disease. Then, she achieved partial response (PR). The durable PR lasted for more than 13 months, and the PFS is currently over 20 months, exceeding the prior treatment.ConclusionThis case highlights the importance of considering re-biopsy and reanalysis of genetic profiles in NSCLC patients harboring METex14 skipping mutations after progressive disease in MET TKI treatment. This raises the possibility that vebreltinib may have long-term survival benefits for patients without mutations conferring resistance (funded by Beijing Pearl Biotechnology Co., Ltd; ClinicalTrials.gov number, NCT04258033).

Abstract N7 methylguanosine (m7G) has a crucial role the development of hepatocellular carcinoma (HCC). This study aimed to investigate the impact of the m7G methylation core genes (METTL1 and WDR4) and associated RNA risk signatures on HCC. we found m7G methylation core genes (METTL1 and WDR4) were upregulated in four HCC cell lines, and downregulation of METTL1 and WDR4 attenuated HCC cell proliferation, migration, and invasion. Moreover, METTL1 and WDR4 are upregulated in HCC tissues, and that there is a significant positive correlation between them. METTL1 and WDR4 were identified as independent prognostic markers for HCC by employing overall survival (OS), disease-specific survival (DSS), Progression Free Interval survival (PFI), and univariate/multivariate Cox analyses. We identified 1479 coding RNAs (mRNAs) and 232 long non-coding RNAs (lncRNAs) associated with METTL1 / WDR4 by using weighted coexpression network analysis (WGCNA) and co-clustering analysis. The least absolute shrinkage and selection operator (lasso) were used to constructing mRNA and lncRNA risk signatures associated with the METTL1 / WDR4. These risk were independent poor prognostic factors in HCC. Furthermore, we found that METTL1 / WDR4 expression and mRNA / lncRNA risk scores were closely associated with TP53 mutations. Clinicopathological features correlation results showed that METTL1 / WDR4 expression and mRNA / lncRNA risk score were associated with the stage and invasion depth (T) of HCC. To predict the overall survival of HCC individuals, we constructed a nomogram with METTL1/WDR4 expression, mRNA/lncRNA risk score, and clinicopathological features. In addition, we combined single-cell sequencing datasets and immune escape-related checkpoints to construct an immune escape-related protein–protein interaction(PPI) network. In conclusion, M7G methylated core genes (METTL1 and WDR4) and associated RNA risk signatures are associated with prognosis and immune escape in HCC.

Background: Rising evidence indicates the development of pyroptosis in the initiation and pathogenesis of spinal cord injury (SCI). However, the associated effects of pyroptosis-related genes (PRGs) in SCI are unclear. Methods: We obtained the gene expression profiles of SCI and normal samples in the GEO. Database: The R package limma screened for differentially expressed (DE) PRGs and performed functional enrichment analysis. Mechanical learning and PPI analysis helped filter essential PRGs to diagnose SCI. Peripheral blood was collected for validation from ten SCI patients and eight healthy individuals. The association of essential PRGs with immune infiltration was evaluated, and pyroptosis subtypes were recognized in SCI patients by unsupervised cluster analysis. Besides, a SCI model was built for in vivo validation of essential PRGs. Result: We identified 25 DE-PRGs between SCI and normal controls. Functional enrichment analysis revealed the principal involvement of DE-PRGs in pyroptosis, inflammasome complex, interleukin-1 beta production, etc. Subsequently, three essential PRGs were identified and validated, showing excellent diagnostic efficacy and significant correlation with immune cell infiltration. Additionally, we developed diagnostic nomograms to predict the occurrence of SCI. Two pyroptosis subtypes exhibited distinct biological functions and immune landscapes among SCI patients. Finally, the expression of these essential PRGswas verified in vivo. Conclusion: The current study described the vital effects of pyroptosis-related genes in SCI, providing a novel direction for effective assessment and management of SCI.

Abstract Ferroptosis, a nonapoptotic form of programmed cell death characterized by significant iron-dependent peroxidation of phospholipids, is regulated by cellular metabolism, redox homeostasis, and various cancer-related signaling pathways. Recently, considerable progress has been made in demonstrating the critical role of lipid metabolism in regulating ferroptosis, indicating the potential of combinational strategies for treating cancer in the future. In this study, we explored the combinational effects of lipid metabolism compounds and ferroptosis inducers on renal cell carcinoma (RCC) cells. We found potent synergy of the fatty acid amide hydrolase (FAAH) inhibitor URB597 with ferroptosis inducer (1S, 3R)-RSL3 (RSL3) in inhibiting the growth and metastasis of RCC cells both in vitro and in vivo via induction of G1 cell cycle arrest and promotion of the production of lipid peroxides, malondialdehyde (MDA), 4-hydroxynonenal (4-HNE), and cytosolic reactive oxygen species (ROS). In addition, inhibition of FAAH increased the sensitivity of RCC cells to ferroptosis. Genome-wide RNA sequencing indicated that the combination of URB597 and RSL3 has more significant effects on regulation of the expression of genes related to cell proliferation, the cell cycle, cell migration and invasion, and ferroptosis than either single agent alone. Moreover, we found that combinational treatment modulated the sensitivity of RCC cells to ferroptosis via the phosphatidylinositol 3 kinase (PI3K)-AKT signaling pathway. These data demonstrate that dual targeting of FAAH and ferroptosis could be a promising strategy for treating RCC.

In this paper, Cu thin films were deposited on Si (100) substrates by the high−power impulse magnetron sputtering (HiIPMS) technique, and the effects of different duty cycles (from 2.25% to 5.25%) on the plasma discharge characteristics, microstructure, and electrical properties of Cu thin films were investigated. The results of the target current test show that the peak target current remains stable under 2.25% and 3% duty cycle conditions. Under the conditions of a 4.5% and 5.25% duty cycle, the target peak current shows a decreasing trend. The average power of the target shows a rising trend with the increase in the duty cycle, while the peak power of the target shows a decreasing trend with the increase in the duty cycle. The results of OES show that with the increase in the duty cycle, the total peak intensity of copper and argon emissions shows an overall increasing trend. The duty cycle from 3% to 4.5% change in copper and argon emission peak total intensity change is not obvious. The deposition rate and surface morphology of the copper film were investigated by scanning electron microscopy, and the deposition rate of the copper film increased with the increase in the duty cycle, which was mainly due to the increase in the average power. The surface roughness of the copper film was evaluated by atomic force microscopy. X−ray diffraction (XRD) was used to analyze the grain size and texture of the Cu film, and the results showed that the average grain size of the Cu film increased from 38 nm to 59 nm on the (111) and (200) crystal planes. Four−probe square resistance test copper film resistivity in 2.25%, 3% low duty cycle conditions of the copper film resistivity is generally higher than 4.5%, 5.25% high duty cycle conditions, the copper film resistivity shows the trend of change is mainly affected by the copper film grain size and the (111) face of the double effect of the optimal orientation. The lowest resistivity of the copper film measured under the 4.5% duty cycle condition is 1.7005 μΩ·cm, which is close to the intrinsic resistivity of the copper film of 1.67 μΩ·cm.

High-power pulse magnetron sputtering is a new type of magnetron sputtering technology that has advantages such as high peak power density and a high ionization rate compared to DC magnetron sputtering. In this paper, we report the effects of different pulse widths on the current waveform and plasma spectrum of target material sputtering, as well as the structure and properties of Cu films prepared under the same sputtering voltage and duty cycle. Extending the pulse width can make the sputtering enter the self-sputtering (SS) stage and improve the ion quantity of sputtered particles. The Cu film prepared by HiPIMS with long pulse width has higher bond strength and lower electrical resistivity compared to the Cu film prepared by short pulse width. In terms of microstructure, the Cu film prepared by HiPIMS with the long pulse width has a larger grain size and lower micro-surface roughness. When the pulse width is bigger than 200 μs, the microstructure of the Cu film changes from granular to branched. This transformation reduces the interface on the Cu film, further reducing the resistivity of the Cu film. Compared to short pulses, long pulse width HiPIMS can obtain higher quality Cu films. This result provides a new process approach for preparing high-quality Cu films using HiPIMS technology.

High temperatures significantly injure the flowering, pollination, fruit growth, and quality of plants. Photosynthesis, the fundamental process supporting plant life, is crucial. Nevertheless, the quantitative evaluation of the physiological activity of the photosynthetic system of Nanfeng tangerine (NT) plants under high-temperature conditions remains a challenge. This research utilized NT plants, a distinctive citrus variety in Jiangxi Province, as the experimental subject. The study investigated the effects of varying degrees of high-temperature stress and duration on 16 photosynthetic physiological parameters of NT plants. The study examined the impact of four varying high-temperature treatment levels (32/22 °C, 35/25 °C, 38/28 °C, and 41/31 °C) for durations of 2, 4, 6, and 8 days, respectively. Principal component analysis was utilized to identify the key indicators of photosynthetic physiological activity in NT plants, with Fv/Fm, Pmax, LCP, H2O2, MDA, and POD being selected as key parameters. The high-temperature stress index model previously constructed was used to calculate the high-temperature stress index value of the NT plants exposed to varying degrees and durations of high temperature, in order to provide a comprehensive assessment of the photosynthetic system of NT plants under high-temperature stress. Subsequently, the high-temperature stress levels were categorized into five levels based on the calculated values: Level 0 for 0 < HSI ≤ 2, Level 1 for 2 < HSI ≤ 4, Level 2 for 4 < HSI ≤ 6, Level 3 for 6 < HSI ≤ 8, and Level 4 for HSI > 8. The research results provide valuable data for agricultural meteorological departments to carry out disaster risk zoning and risk assessment in the future.

Polygonatum cyrtonema Hua (family Asparagaceae) is an endemic plant in China that is valuable for its edible and medicinal uses. Plant growth regulators (PGRs) are natural or synthetic compounds that can regulate plant development and metabolism effectively. To explore potential applications of PGRs for improving the yield and bioactivity of this plant, four PGRs, including gibberellic acid (GA3), 6-benzylaminopurine (6-BA), naphthaleneacetic acid (NAA) and 24-epibrassinolide (EBL), were used in this study and sprayed on the growing seedlings of P. cyrtonema. All of these PGRs did not significantly affect the growth rate of P. cyrtonema, but they had varying effects on the polysaccharide and saponin content in the rhizome. NAA and 6-BA positively affected the polysaccharide content, while most PGR treatments negatively affected the saponin content. Widely targeted metabolomic analysis based on UPLC-MS/MS was conducted and revealed 101 differential metabolites in response to 6-BA, most of which were flavonoids, steroids and lipids. Most of the significantly changed flavonoids decreased under the 6-BA treatment. The study provides insights into the potential use of PGRs for improving the quality of P. cyrtonema, particularly in regulating the content of bioactive compounds.

The first laser–plasma interaction experiment using lasers of eight beams grouped into one octad has been conducted on the Shenguang Octopus facility. Although each beam intensity is below its individual threshold for stimulated Brillouin backscattering (SBS), collective behaviors are excited to enhance the octad SBS. In particular, when two-color/cone lasers with wavelength separation 0.3 nm are used, the backward SBS reflectivities show novel behavior in which beams of longer wavelength achieve higher SBS gain. This property of SBS can be attributed to the rotation of the wave vectors of common ion acoustic waves due to the competition of detunings between geometrical angle and wavelength separation. This mechanism is confirmed using massively parallel supercomputer simulations with the three-dimensional laser–plasma interaction code LAP3D.

Abstract Differentiation therapy using small molecules is a promising strategy for improving the prognosis of glioblastoma (GBM). Histone acetylation plays an important role in cell fate determination. Nevertheless, whether histone acetylation in specific sites determines GBM cells fate remains to be explored. Through screening from a 349 small molecule-library, we identified that histone deacetylase inhibitor (HDACi) MS-275 synergized with 8-CPT-cAMP was able to transdifferentiate U87MG GBM cells into neuron-like cells, which were characterized by cell cycle arrest, rich neuron biomarkers, and typical neuron electrophysiology. Intriguingly, acetylation tags of histone 3 at lysine 9 (H3K9ac) were decreased in the promoter of multiple oncogenes and cell cycle genes, while ones of H3K9ac and histone 3 at lysine 14 (H3K14ac) were increased in the promoter of neuron-specific genes. We then compiled a list of genes controlled by H3K9ac and H3K14ac, and proved that it is a good predictive power for pathologic grading and survival prediction. Moreover, cAMP agonist combined with HDACi also induced glioma stem cells (GSCs) to differentiate into neuron-like cells through the regulation of H3K9ac/K14ac, indicating that combined induction has the potential for recurrence-preventive application. Furthermore, the combination of cAMP activator plus HDACi significantly repressed the tumor growth in a subcutaneous GSC-derived tumor model, and temozolomide cooperated with the differentiation-inducing combination to prolong the survival in an orthotopic GSC-derived tumor model. These findings highlight epigenetic reprogramming through H3K9ac and H3K14ac as a novel approach for driving neuron-fate-induction of GBM cells.

Abstract Accumulating evidence supports the existence of glioma stem cells (GSCs) and their critical role in the resistance to conventional treatments for glioblastoma multiforme (GBM). Differentiation therapy represents a promising alternative strategy against GBM by forcing GSCs to exit the cell cycle and reach terminal differentiation. In this study, we demonstrated that cAMP triggered neuronal differentiation and compromised the self-renewal capacity in GSCs. In addition, cAMP induced negative feedback to antagonize the differentiation process by activating β-catenin pathway. Suppression of β-catenin signaling synergized with cAMP activators to eliminate GSCs in vitro and extended the survival of animals in vivo. The cAMP/PKA pathway stabilized β-catenin through direct phosphorylation of the molecule and inhibition of GSK-3β. The activated β-catenin translocated into the nucleus and promoted the transcription of APELA and CARD16, which were found to be responsible for the repression of cAMP-induced differentiation in GSCs. Overall, our findings identified a negative feedback mechanism for cAMP-induced differentiation in GSCs and provided potential targets for the reinforcement of differentiation therapy for GBM.

Abstract 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.

Ulcerative colitis (UC) is a subtype of inflammatory bowel disease (IBD) and occurs mainly in the colon. The etiology of UC is rather complex and involves various pathological factors, including genetic susceptibility, dietary intakes, environment, and microbiota. In China, the Chang-Yan-Ning (CYN) formula has been utilized in the clinic to treat gastrointestinal disorders, but its pharmacological evidence remains elusive. The investigation was designed to explore the molecular and cellular mechanisms of CYN. Liquid Chromatography with tandem mass spectrometry (LC/MS) was performed to identify the key components in the formula; Network pharmacology analysis was executed to predict the potential targets of CYN; An experimental murine colitis model was established by utilizing 2% dextran sodium sulfate (DSS), and CYN was administered for 14 days. The pharmacological mechanism of the CYN formula was corroborated by in-vivo and in-vitro experiments, and high throughput techniques including metabolomics and 16S rRNA sequencing. Results: LC/MS identified the active components in the formula, and network pharmacology analysis predicted 37 hub genes that were involved in tumor necrosis factor (TNF), interleukin (IL)-17, hypoxia-inducible factor (HIF) signaling pathways. As evidenced by in-vivo experiments, DSS administration shortened the length of the colon and led to weight loss, with a compromised structure of epithelium, and the CYN formula reversed these pathological symptoms. Moreover, CYN suppressed the levels of pro-inflammatory cytokines, including IL-4, IL-1b, and TNFαin the serum, inhibited the protein abundance of IL17 and HIF-1αand increased PPARγ and CCL2 in the colon, and facilitated the alternative activation of peritoneal macrophages. While peritoneal macrophages of colitis mice enhanced reactive oxygen species (ROS) production in murine intestinal organoids, the ROS level remained stable co-cultured with the macrophages of CYN-treated mice. Furthermore, the decreased microbiota richness and diversity and the prevalence of pathogenic taxa in colitis mice were rescued after the CYN treatment. The altered metabolic profile during colitis was also restored after the therapy. We posit that the CYN therapy attenuates the development and progression of colitis by maintaining the homeostasis of immune responses and microbiota.

Inflammatory bowel disease (IBD) is pathologically characterized by an immune response accommodative insufficiency and dysbiosis accompanied by persistent epithelial barrier dysfunction. The Cao-Xiang-Wei-Kang (CW) formula has been utilized to treat gastrointestinal disorders in the clinic. The present study was designed to delineate the pharmacological mechanisms of this formula from different aspects of the etiology of ulcerative colitis (UC), a major subtype of IBD. Dextran sodium sulfate (DSS) was given to mice for a week at a concentration of 2%, and the CW solution was administered for 3 weeks. 16S rRNA gene sequencing and untargeted metabolomics were conducted to examine the changes in the microbiome profile, and biochemical experiments were performed to confirm the therapeutic functions predicted by system pharmacology analysis. The CW treatment hampered DSS-induced experimental colitis progression, and the targets were enriched in inflammation, infection, and tumorigenesis, which was corroborated by suppressed caspase 3 (Casp3) and interleukin-1b (IL-1b) and increased cleaved caspase 3 expression and casp-3 activity in the colon samples from colitis mice subjected to the CW therapy. Moreover, the CW therapy rescued the decreased richness and diversity, suppressed the potentially pathogenic phenotype of the gut microorganisms, and reversed the altered linoleic acid metabolism and cytochrome P450 activity in murine colitis models. In our in vitro experiments, the CW administration increased the alternative activation of macrophages (Mφs) and inhibited the tumor necrosis factor-α (TNFα)-induced reactive oxygen species (ROS) level and subsequent death in intestinal organoids (IOs). We propose that the CW formula alleviates the progression of murine colitis by suppressing inflammation, promoting mucosal healing, and re-establishing a microbiome profile that favors re-epithelization.

Background: Osteosarcoma is a highly malignant bone tumor commonly occurring in adolescents with a poor 5-year survival rate. The unfolded protein response (UPR) can alleviate the accumulation of misfolded proteins to maintain homeostasis under endoplasmic reticulum stress. The UPR is linked to the occurrence, progression, and drug resistance of tumors. However, the function of UPR-related genes (UPRRGs) in disease progression and prognosis of osteosarcoma remains unclear.Methods: The mRNA expression profiling and corresponding clinical features of osteosarcoma were acquired from TARGET and GEO databases. Consensus clustering was conducted to confirm different UPRRG subtypes. Subsequently, we evaluated the prognosis and immune status of the different subtypes. Functional analysis of GO, GSEA, and GSVA was used to reveal the molecular mechanism between the subtypes. Finally, four genes (STC2, PREB, TSPYL2, and ATP6V0D1) were screened to construct and validate a risk signature to predict the prognosis of patients with osteosarcoma.Result: We identified two subtypes according to the UPRRG expression patterns. The subgroup with higher immune scores, lower tumor purity, and active immune status was linked to a better prognosis. Meanwhile, functional enrichment revealed that immune-related signaling pathways varied markedly in the two subtypes, suggesting that the UPR might influence the prognosis of osteosarcoma via influencing the immune microenvironment. Moreover, prognostic signature and nomogram models were developed based on UPRRGs, and the results showed that our model has an excellent performance in predicting the prognosis of osteosarcoma. qPCR analysis was also conducted to verify the expression levels of the four genes.Conclusion: We revealed the crucial contribution of UPRRGs in the immune microenvironment and prognostic prediction of osteosarcoma patients and provided new insights for targeted therapy and prognostic assessment of the disease.

Human TOR1AIP1 encodes LAP1, a nuclear envelope protein expressed in most human tissues, which has been linked to various biological processes and human diseases. The clinical spectrum of diseases related to mutations in TOR1AIP1 is broad, including muscular dystrophy, congenital myasthenic syndrome, cardiomyopathy, and multisystemic disease with or without progeroid features. Although rare, these recessively inherited disorders often lead to early death or considerable functional impairment. Developing a better understanding of the roles of LAP1 and mutant TOR1AIP1-associated phenotypes is paramount to allow therapeutic development. To facilitate further studies, this review provides an overview of the known interactions of LAP1 and summarizes the evidence for the function of this protein in human health. We then review the mutations in the TOR1AIP1 gene and the clinical and pathological characteristics of subjects with these mutations. Lastly, we discuss challenges to be addressed in the future.

Abstract Reversing the highly immunosuppressive tumor microenvironment (TME) is essential to achieve long-term efficacy with cancer immunotherapy. Despite the impressive clinical response to checkpoint blockade in multiple types of cancer, only a minority of patients benefit from this approach. Here, we report that the oncolytic virus M1 induces immunogenic tumor cell death and subsequently restores the ability of dendritic cells to prime antitumor T cells. Intravenous injection of M1 disrupts immune tolerance in the privileged TME, reprogramming immune-silent (cold) tumors into immune-inflamed (hot) tumors. M1 elicits potent CD8+ T cell-dependent therapeutic effects and establishes long-term antitumor immune memory in poorly immunogenic tumor models. Pretreatment with M1 sensitizes refractory tumors to subsequent checkpoint blockade by boosting T-cell recruitment and upregulating the expression of PD-L1. These findings reveal the antitumor immunological mechanism of the M1 virus and indicated that oncolytic viruses are ideal cotreatments for checkpoint blockade immunotherapy.

Citric acid plays significant roles in numerous physiological processes in plants, including carbon metabolism, signal transduction, and tolerance to environmental stress. For fruits, it has a major effect on fruit organoleptic quality by directly influencing consumer taste. Citric acid in citrus is mainly regulated by the balance between synthesis, degradation, and vacuolar storage. The genetic and molecular regulations of citric acid synthesis and degradation have been comprehensively elucidated. However, the transporters for citric acid in fruits are less well understood. Here, an aluminum-activated malate transporter, CitALMT, was characterized. Transient overexpression and stable transformation of CitALMT significantly reduced citrate concentration in citrus fruits and transgenic callus. Correspondingly, transient RNA interference-induced silencing of CitALMT and increased citrate significantly, indicating that CitALMT plays an important role in regulating citrate concentration in citrus fruits. In addition, dual-luciferase assays indicated that CitMYB52 and CitbHLH2 could trans-activate the promoter of CitALMT. EMSA analysis showed that CitbHLH2 could physically interact with the E-box motif in the CitALMT promoter. Bimolecular fluorescence complementation assays, yeast two-hybrid, coimmunoprecipitation and transient overexpression, and RNAi assay indicated that the interaction between CitMYB52 and CitbHLH2 could synergistically trans-activate CitALMT to negatively regulate citrate accumulation.

Abstract Background Viruses are obligate parasites that depend on host cells to provide the energy and molecular precursors necessary for successful infection. The main component of virus-induced metabolic reprogramming is the activation of glycolysis, which provides biomolecular resources for viral replication. However, little is known about the crosstalk between oncolytic viruses and host glycolytic processes. Methods A MTT assay was used to detect M1 virus-induced cell killing. Flow cytometry was used to monitor infection of M1 virus expressing the GFP reporter gene. qPCR and western blotting were used to detect gene expression. RNA sequencing was performed to evaluate gene expression under different drug treatments. Scanning electron microscopy was performed to visualize the endoplasmic reticulum (ER). Caspase activity was detected. Last, a mouse xenograft model was established to evaluate the antitumor effect in vivo. Most data were analyzed with a two-tailed Student’s t test or one-way ANOVA with Dunnett’s test for pairwise comparisons. Tumor volumes were analyzed by repeated measures of ANOVA. The Wilcoxon signed-rank test was used to compare nonnormally distributed data. Results Here, we showed that the glucose analog 2-deoxy-d-glucose (2-DG) inhibited infection by M1 virus, which we identified as a novel type of oncolytic virus, and decreased its oncolytic effect, indicating the dependence of M1 replication on glycolysis. In contrast, lonidamine, a reported hexokinase 2 (HK2) inhibitor, enhanced the infection and oncolytic effect of M1 virus independent of HK2. Further transcriptomic analysis revealed that downregulation of the antiviral immune response contributes to the lonidamine-mediated potentiation of the infection and oncolytic effect of M1 virus, and that MYC is the key factor in the pool of antiviral immune response factors inhibited by lonidamine. Moreover, lonidamine potentiated the irreversible ER stress-mediated apoptosis induced by M1 virus. Enhancement of M1′s oncolytic effect by lonidamine was also identified in vivo. Conclusions This research demonstrated the dependence of M1 virus on glycolysis and identified a candidate synergist for M1 virotherapy.

Thyroid cancer is now the most common endocrine malignancy and the effect of miR-429 in the development of thyroid cancer still need to be further investigated. The expression level of miR-429 was quantified by qPCR in both clinical samples and cultured cell lines. MTT, flow cytometry, migration analyses and Matrigel invasion assays were conducted to test the proliferation, apoptosis, migration and invasion of MiR-429 transfection in thyroid cancer cell lines. Luciferase activity assay and western blot were conducted to detect the direct effect of miR-429 on Zinc finger E-box-binding homeobox 1 (ZEB1) expression. In this study, it was found that miR-429 was frequently decreased in thyroid cancer tissues and cell lines. Transfection of miR-429 in thyroid cancer cell lines substantially suppressed cell proliferation, migration and invasion. Besides, miR-429 up-regulation would induce apoptosis in different cell lines. ZEB1 was identified as a direct target of miR-429 and miR-429 transfection could inhibit ZEB1 by direct binding to its 3’-untranslated region (3’-UTR). In conclusion, these data indicated that miR-429 could act as a tumour suppressor miRNA and contribute to the development and progression and metastasis of thyroid cancer.

The mandarin is an important fruit crop worldwide, and ‘Xinyu mandarin’—a local Citrus reticulata variety—is widely cultivated in Jiangxi Province, in China. Autopolyploidy has frequently been used for the improvement of crop varieties. In a previous study, we identified a tetraploid (4X) material of ‘Xinyu mandarin’ generated from its diploid (2X) mother seedling via a spontaneous mutation. However, the physiological and metabolic variations after polyploidy are not clear enough, which is not conducive to the early screening. In the present study, we analyzed the morphological, physiological, and metabolic differences between the tetraploid and diploid plants. The tetraploids had larger and thicker leaves, and the activities of key enzymes in photosynthesis, the total chlorophyll, phenolic, and proline contents, were all higher in tetraploids. In the non-targeted metabolomic profile, a total of 522 metabolites were identified, of which 61 were significantly different between diploids and tetraploids. The differential metabolites included similar proportions of primary and secondary metabolites, and most of these were up-regulated in tetraploids, especially stress-related metabolites such as phenolic acids, flavonoids, alkaloids, and so on, indicating that tetraploids may possess better stress tolerance ability and vigor. Therefore, the tetraploids of ‘Xinyu mandarin’ may serve as an excellent female parent for the improvement of citrus varieties.

Activation of the cyclic adenosine monophosphate/protein kinase A (cAMP/PKA) pathway induces glial differentiation of glioblastoma (GBM) cells, but the mechanism by which microRNA (miRNA) regulate this process remains poorly understood. In this study, by performing miRNA genomics and loss‐ and gain‐of‐function assays in dibutyryl‐cAMP‐treated GBM cells, we identified a critical negative regulator, hsa‐miR‐1275, that modulates a set of genes involved in cancer progression, stem cell maintenance, and cell maturation and differentiation. Additionally, we confirmed that miR‐1275 directly and negatively regulates the protein expression of glial fibrillary acidic protein (GFAP), a marker of mature astrocytes. Of note, tri‐methyl‐histone H3 (Lys27) (H3K27me3), downstream of the PKA/polycomb repressive complex 2 (PRC2) pathway, accounts for the downregulation of miR‐1275. Furthermore, decreased miR‐1275 expression and induction of GFAP expression were also observed in dibutyryl‐cAMP‐treated primary cultured GBM cells. In a patient‐derived glioma stem cell tumor model, a cAMP elevator and an inhibitor of H3K27me3 methyltransferase inhibited tumor growth, induced differentiation, and reduced expression of miR‐1275. In summary, our study shows that epigenetic inhibition of miR‐1275 by the cAMP/PKA/PRC2/H3K27me3 pathway mediates glial induction of GBM cells, providing a new mechanism and novel targets for differentiation‐inducing therapy.

Background: The influence of amyloid protein-binding protein 2 (APPBP2) on lung cancer is unknown. Methods: The function and mechanisms of APPBP2 were investigated in the NSCLC cell lines A549 and H1299. The ectopic expression of APPBP2, PPM1D and SPOP in NSCLS were examined in samples collected from ten pairs of human lung adenocarcinoma cancer tissues and adjacent normal lung tissues. shRNA vector was used for APPBP2 knockdown. Quantitative PCR and western blot assays quantified the mRNA and protein level of APPBP2, PPM1D, and SPOP. Cell proliferation was measured with BrdU, MTT, colony formation assays, and xenograft tumour growth experiments. Cell migration and invasion were analysed with transwell and wound healing assays. Co-Immunoprecipitation assay detected protein–protein interactions. Findings: APPBP2 was upregulated in NSCLC tissues. Silencing APPBP2 in A549 and H1299 cells resulted in the inhibition of cell proliferation, migration, and invasion, enhancement of apoptosis, and a significant decrease in the expression of PPM1D and SPOP. Overexpression of PPM1D and SPOP attenuated the APPBP2-knockdown inhibition of NSCLC cells. Co-IP assay showed that PPM1D interacted with APPBP2. Interpretation: The expression level of APPBP2 positively correlates with NSCLC cell proliferation, migration, and invasiveness. APPBP2 contributes to NSCLC progression through regulating the PPM1D and SPOP signalling pathway. This novel molecular mechanism, underlying NSCLC oncogenesis, suggests APPBP2 is a potential target for diagnosis and therapeutic intervention in NSCLC. Fund: Key Program of Natural Science Research of Higher Education of Anhui Province (No. KJ2017A241), the National Natural Science Foundation of China (No. 81772493). Keywords: APPBP2, Lung cancer, Non-small cell lung cancer, PPM1D, SPOP

Abstract Background Long non-coding RNAs are involved in the pathology of various tumors, including hepatocellular carcinoma. The expression of metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is increased in numerous types of tumors and is involved in tumor cell proliferation, migration, invasion and apoptosis. MALAT1 level was reported to be upregulated in hepatocellular carcinoma tissues, but its roles and the specific molecular mechanisms are still unclear. Methods The expression of MALAT1 and miR-142-3p in hepatocellular carcinoma tissues, cell lines and adjacent non-tumor tissues was assessed by Q-PCR. The putative-binding sites between MALAT1 and miR-142-3p were predicted by bioinformatics analysis. The expression of MALAT1 in HepG2 and SMMC-7721 cells was knocked down by transfection with MALAT1 siRNAs. Cell viability was assessed by the Cell Counting Kit-8 (CCK-8) assay after the indicated transfection in HepG2 and SMMC-7721 cells. Cell proliferation was assessed by EdU assay, and cell apoptosis was explored by flow cytometry. The migration and invasion potency of HepG2 and SMMC-7721 cells was assessed by the cell migration assay and matrigel invasion assay. Protein level of vimentin, E-cadherin and SMAD5 were assessed by Western blot. Results Overexpressed MALAT1 acts as a competing endogenous RNA sponge for miR-142-3p in hepatocellular carcinoma. The knockdown of MALAT1 inhibited the proliferation, migration, invasion, and epithelial cell-to-mesenchymal transition (EMT), and promoted apoptosis of hepatocellular carcinoma cells via miR-142-3p. MiR-142-3p inhibited cell proliferation, migration, invasion and EMT, and promoted the cell apoptosis by targeting SMAD5 in hepatocellular carcinoma. MALAT1 promoted tumor growth by regulating the expression of miR-142-3p in vivo. Conclusion MALAT1 promoted cell proliferation, migration, and invasion of hepatocellular carcinoma cells by antagonizing miR-142-3p.

Abstract Mutations to KRAS are recurrent in lung adenocarcinomas (LUAD) and are daunting to treat due to the difficulties in KRAS oncoprotein inhibition. A possible resolution to this problem may lie with co‐mutations to other genes that also occur in KRAS‐driven LUAD that may provide alternative therapeutic vulnerabilities. Approximately 3% of KRAS‐mutant LUADs carry functional mutations in NF1 gene encoding neurofibromin‐1, a negative regulator of focal adhesion kinase 1 (FAK1). We evaluated the impact of Nf1 loss on LUAD development using a CRISPR/Cas9 platform in a murine model of Kras‐mutant LUAD. We discovered that Nf1 deactivation is associated with Fak1 hyperactivation and phosphoserine aminotransferase 1 (Psat1) upregulation in mice. Nf1 loss also accelerates murine Kras‐driven LUAD tumorigenesis. Analysis of the transcriptome and metabolome reveals that LUAD cells with mutation to Nf1 are addicted to glutamine metabolism. We also reveal that this metabolic vulnerability can be leveraged as a treatment option by pharmacologically inhibiting glutaminase and/or Psat1. Lastly, the findings advocate that tumor stratification by co‐mutations to KRAS/NF1 highlights the LAUD patient population expected to be susceptible to inhibiting PSAT1.

Enhancement of x-ray sources from laser-produced plasmas is significant in wide-ranging applications. Solid and foam TiO2 targets were both used to generate bright x-ray sources at the Shenguang-III prototype laser facility, with a total laser power of 3.2 TW. The new foam targets were with an ultra-low initial density of 8.1 mg/cm3 and a high Ti fraction of 33 sat. %. By absolute measurements, the multi-keV x rays of the 4 keV–7 keV band and 1.6 keV–4.4 keV band and the total x rays above 0.1 keV of the foam target have simultaneously shown conversion enhancements of 1.4, 3.1, and 2.3 times, respectively, compared with the solid target. A much larger emission volume and an average electron temperature of 3.2 keV by moving the foci of laser beams inner were obtained for the foam target, providing a good condition for multi-keV x-ray production.

Background/Aims: To investigate the role of methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) in the clinical prognosis and cell biology of renal cell carcinoma (RCC). Methods: A total of 137 RCC tissues were evaluated by immunohistochemistry. The relationship between MTHFD2 overexpression and clinical parameters and vimentin expression was assessed. Kaplan-Meier curves and the log-rank test were applied for survival analysis according to MTHFD2 and vimentin expression in RCC tissues. The expression of MTHFD2 mRNA and protein was examined by quantitative reverse transcription PCR and western blotting, respectively. To determine further the biological activity of MTHFD2 in RCC, 786-O cells were transfected with short hairpin RNA specifically targeting MTHFD2 (shMTHFD2) with or without tumor necrosis factor (TNF)-α stimulation. Cell proliferation, cell migration and invasion and drug sensitivity were subsequently assessed using Cell Counting Kit-8, wound healing, and Transwell assays. Results: Immunohistochemical analysis demonstrated that both MTHFD2 and vimentin overexpression was positively associated with clinical staging, pathological grade, and poor overall survival (all P < 0.05). MTHFD2 expression was closely correlated with vimentin overexpression in RCC (r = 0.402, P < 0.001). After knocking down MTHFD2 expression in 786-O cells, decreased cell proliferation, migration, and invasion were observed and accompanied by the reduced expression of vimentin. The effects of MTHFD2 down-regulation could be partially restrained by TNF-α treatment. Vimentin expression and cell migration and invasion, but not cell proliferation, were reversed by TNF-α stimulation. Furthermore, treatment of 786-O cells with shMTHFD2 increased their sensitivity to chemotherapy drugs. Conclusion: The current results demonstrated that MTHFD2 was overexpressed in RCC and associated with poor clinical characteristics, vimentin expression, and cellular features connected to malignant disease, thus, implicating MTHFD2 as a potential target for RCC therapy.

Oncolytic viruses selectively kill tumour cells and induce anti-tumour immunity. Here, the AUs demonstrate the anti-viral effect of the mevalonate pathway on oncolytic virus M1 in refractory cancer cells and provide evidence for a combination strategy of targeting the mevalonate pathway for potentiating oncolytic virus therapy.

Advanced glycation end products (AGEs) are glycosylated metabolic products generated in vivo and are associated with aging-related diseases. They are also formed during heat treatment in food processing. In this work, we investigated the contribution of linoleic acid (LA) to AGE formation using a protein/glucose model. An electronic tongue, denaturing polyacrylamide gel electrophoresis, electron spin resonance spectroscopy, circular dichroism, and ultraperformance liquid chromatography-tandem mass spectrometry were used to analyse reaction intermediates and reactive radical formation. The results show that LA is the key factor responsible for the change in flavour including the rapid triggering of glycation reactions. The amount of lipid-induced reactive radicals was significantly higher than in the non-fat system, radical generation in the non-fat system was gradually quenched after a robust radical-yielding reaction in the first 25 minutes. Subsequent unsaturated lipid oxidation, and AGE accumulation surpass Maillard reaction-only outcomes. Initial LA-induced changes in protein structure are followed by glycation and are enhanced by hydrophobic interactions and increased carbonyl levels resulting from lipid oxidation. These findings implicate lipids and lipid oxidation as the main factors responsible for AGE formation during the processing of fat-rich unsaturated fatty acid-containing foods.

Volatile compounds principally contribute to flavor of strawberry (Fragaria × ananassa) fruit. Besides to genetics, cultivation conditions play an important role in fruit volatile formation. Compared to soil culture as control, effects of substrate culture on volatile compounds of two strawberry cultivars (‘Amaou’ and ‘Yuexin’) were investigated. GC-MS analysis revealed significant difference in volatile contents of ‘Amaou’ strawberry caused by substrate culture. No significant effect was observed for cultivar ‘Yuexin’. For ‘Amaou’ strawberry from soil culture produced higher volatile contents compared with substrate culture. This difference is contributed by high contents of esters, lactones, ketones, aldehydes, terpenes, hydrocarbons, acids, furans and phenols in ‘Amaou’ strawberry fruit from soil culture. Furanones, beta-linalool, trans-Nerolidol and esters are major contributor to strawberry aroma, whose contents are higher in soil culture planted fruit when compared to substrate culture. Moreover, strawberry fruit from soil culture had higher transcripts related to volatile biosynthesis were observed, including FaQR, FaOMT, FaNES1, FaSAAT and FaAAT2.

Mesenchymal stem cells (MSCs) and fibroblasts are two major seed cells for ligament tissue engineering. To understand the effects of mechanical stimulation on these cells and to develop effective approaches for cell therapy, it is necessary to investigate the biological effects of various mechanical loading conditions on cells. In this study, fibroblasts and MSCs were tested and compared under a novel Uniflex/Bioflex culture system that might mimic mechanical strain in ligament tissue. The cells were uniaxially or radially stretched with different strains (5%, 10%, and 15%) at 0.1, 0.5, and 1.0 Hz. The cell proliferation and collagen production were compared to find the optimal parameters. The results indicated that uniaxial stretch (15% at 0.5 Hz; 10% at 1.0 Hz) showed positive effects on fibroblast. The uniaxial strains (5%, 10%, and 15%) at 0.5 Hz and 10% strain at 1.0 Hz were favorable for MSCs. Radial strain did not have significant effect on fibroblast. On the contrary, the radial strains (5%, 10%, and 15%) at 0.1 Hz had positive effects on MSCs. This study suggested that fibroblasts and MSCs had their own appropriate mechanical stimulatory parameters. These specific parameters potentially provide fundamental knowledge for future cell-based ligament regeneration.

Our previous study demonstrated that 45S ribosomal DNA (45S rDNA) clusters were chromosome fragile sites expressed spontaneously in Lolium. In this study, fragile phenotypes of 45S rDNA were observed under aphidicolin (APH) incubation in several plant species. Further actinomycin D (ActD) treatment showed that transcriptional stress might interfere with chromatin packaging, resulting in 45S rDNA fragile expression. These data identified 45S rDNA sites as replication-dependent as well as transcription-dependent fragile sites in plants. In the presence of ActD, a dramatic switch to an open chromatin conformation and accumulated incomplete 5' end of the external transcribed spacer (5'ETS) transcripts were observed, accompanied by decreased DNA methylation, decreased levels of histone H3, and increased histone acetylation and levels of H3K4me2, suggesting that these epigenetic alterations are associated with failure of 45S rDNA condensation. Furthermore, the finding that γ-H2AX was accumulated at 45S rDNA sites following ActD treatment suggested that the DNA damage signaling pathway was associated with the appearance of 45S rDNA fragile phenotypes. Our data provide a link between 45S rDNA transcription and chromatin-packaging defects and open the door for further identifying the molecular mechanism involved.