Therapeutic targeting at genome mutations of liver cancer by the insertion of HSV1 thymidine kinase through Cas9-mediated editing.

BACKGROUND: Liver cancer is one of the most lethal malignancies for humans. The treatment options for advanced-stage liver cancer remain limited. A new treatment is urgently needed to reduce the mortality of the disease. METHODS: In this report, we developed a technology for mutation site insertion of a suicide gene (herpes simplex virus type 1- thymidine kinase) based on type II CRISPR RNA-guided endonuclease Cas9-mediated genome editing to treat liver cancers. RESULTS: We applied the strategy to 3 different mutations: S45P mutation of catenin beta 1, chromosome breakpoint of solute carrier family 45 member 2-alpha-methylacyl-CoA racemase gene fusion, and V235G mutation of SAFB-like transcription modulator. The results showed that the herpes simplex virus type 1-thymidine kinase insertion rate at the S45P mutation site of catenin beta 1 reached 77.8%, while the insertion rates at the breakpoint of solute carrier family 45 member 2 - alpha-methylacyl-CoA racemase gene fusion were 95.1%-98.7%, and the insertion at V235G of SAFB-like transcription modulator was 51.4%. When these targeting reagents were applied to treat mouse spontaneous liver cancer induced by catenin beta 1S45P or solute carrier family 45 member 2-alpha-methylacyl-CoA racemase, the mice experienced reduced tumor burden and increased survival rate. Similar results were also obtained for the xenografted liver cancer model: Significant reduction of tumor volume, reduction of metastasis rate, and improved survival were found in mice treated with the targeting reagent, in comparison with the control-treated groups. CONCLUSIONS: Our studies suggested that mutation targeting may hold promise as a versatile and effective approach to treating liver cancers.

Long-read single-cell sequencing reveals expressions of hypermutation clusters of isoforms in human liver cancer cells.

The protein diversity of mammalian cells is determined by arrays of isoforms from genes. Genetic mutation is essential in species evolution and cancer development. Accurate long-read transcriptome sequencing at single-cell level is required to decipher the spectrum of protein expressions in mammalian organisms. In this report, we developed a synthetic long-read single-cell sequencing technology based on LOOPSeq technique. We applied this technology to analyze 447 transcriptomes of hepatocellular carcinoma (HCC) and benign liver from an individual. Through Uniform Manifold Approximation and Projection analysis, we identified a panel of mutation mRNA isoforms highly specific to HCC cells. The evolution pathways that led to the hyper-mutation clusters in single human leukocyte antigen molecules were identified. Novel fusion transcripts were detected. The combination of gene expressions, fusion gene transcripts, and mutation gene expressions significantly improved the classification of liver cancer cells versus benign hepatocytes. In conclusion, LOOPSeq single-cell technology may hold promise to provide a new level of precision analysis on the mammalian transcriptome.

Identification of prognostic risk score of disulfidptosis-related genes and molecular subtypes in glioma.

Background: Programmed cell death is closely related to glioma. As a novel kind of cell death, the mechanism of disulfidptosis in glioma remains unclear. Therefore, it is of great importance to study the role of disulfidptosis-related genes (DRGs) in glioma. Methods: We first investigated the genetic and transcriptional alterations of 15 DRGs. Two consensus cluster analyses were used to evaluate the association between DRGs and glioma subtypes. In addition, we constructed prognostic DRG risk scores to predict overall survival (OS) in glioma patients. Furthermore, we developed a nomogram to enhance the clinical utility of the DRG risk score. Finally, the expression levels of DRGs were verified by immunohistochemistry (IHC) staining. Results: Most DRGs (14/15) were dysregulated in gliomas. The 15 DRGs were rarely mutated in gliomas, and only 50 of 987 samples (5.07 %) showed gene mutations. However, most of them had copy number variation (CNV) deletions or amplifications. Two distinct molecular subtypes were identified by cluster analysis, and DRG alterations were found to be related to the clinical characteristics, prognosis, and tumor immune microenvironment (TIME). The DRG risk score model based on 12 genes was developed and showed good performance in predicting OS. The nomogram confirmed that the risk score had a particularly strong influence on the prognosis of glioma. Furthermore, we discovered that low DRG scores, low tumor mutation burden, and immunosuppression were features of patients with better prognoses. Conclusion: The DRG risk model can be used for the evaluation of clinical characteristics, prognosis prediction, and TIME estimation of glioma patients. These DRGs may be potential therapeutic targets in glioma.

Effect of different loads on facet joint motion during lumbar lateral bending in sitting position.

OBJECTIVE: To study the effect of weight-bearing on lumbar facet joint during lateral bending in sitting position. METHODS: Ten normal healthy people (5 males and 5 females) aged 25-39 years (mean 32 ± 4.29 years) were recruited. CT scanning was used to reconstruct the lumbar spine model, and then dual fluoroscopic image system (DFIS) was used to restore the lumbar facet joint movement in sitting position. Finally, the lumbar facet joint translation distance and rotation angle were measured. RESULTS: In L3-4 level, the displacement of right facet joint in Y-axis was the smallest at 0.05 ± 0.40 mm, the displacement of 0 kg left facet joint in X-axis was the largest at 1.68 ± 0.85 mm, and the rotation angle was - 0.57 ± 1.43° to 5.66 ± 2.70° at 10 kg; in L4-5 level, the displacement of right facet joint in Y-axis was the smallest at 10 kg, - 0.13 ± 0.91 mm, and the displacement of left facet joint in Z-axis was the largest at - 2.11 ± 0.88 mm, and the rotation angle was 0.21 ± 2.14° to 7.89 ± 2.59° at 10 kg; in L5-S1 level, the displacement of right facet joint in Y-axis was the smallest at 10 kg, - 0.17 ± 1.10 mm, and the displacement of 0 kg left facet joint in X-axis was the largest at 2.19 ± 2.28 mm, and the rotation angle was 0.03 ± 2.02° to 3.98 ± 0.37°. CONCLUSION: In sitting position, weight-bearing has certain influence on the displacement of facet joints during lumbar lateral bending movement, and this influence occurs simultaneously in translation and rotation; the left and right facet joints are not symmetrical during lumbar lateral bending movement.

HIF-1α contributes to metastasis in choriocarcinoma by regulating DEC1 expression

Purpose To elucidate the underlying mechanism of HIF-1α in migration and invasion of choriocarcinoma. Methods Cell proliferation was determined by CCK-8 assay when cell invasion was detected by transwell assay. The protein expression was detected by western blotting, immunohistochemistry, and qPCR assay. Result HIF-1α was shown to be strongly expressed in both clinical tumour tissues and cell lines in choriocarcinoma. When HIF-1α was efficiently knocked down in JEG3 cells, the proliferation rate was reduced by approximately 50% and the number of cells that migrated through the transwell insert was greatly decreased. The cell invasion rate was also significantly reduced. Moreover, typical markers of epithelial–mesenchymal transition such as E-cadherin, were increased, while vimentin and α–SMA were decreased after HIF-1α knockdown. In contrast, overexpression of DEC1 reversed the effects of HIF-1α knockdown. Cell proliferation, migration, and invasion were partially recovered. The level of E-cadherin was decreased, while the level of vimentin and α–SMA was increased. In addition, the level of β-catenin and LEF1 was downregulated after HIF-1α knockdown. The expression of MMP2 and MMP9 also declined. However, overexpression of DEC1 after HIF-1α knockdown partially reversed the expression pattern of these molecules. Conclusion HIF-1α contributed to EMT and metastasis through activation of canonical β-catenin signalling in choriocarcinoma and this process was dependent on DEC1. This study provides a new mechanism of HIF-1α in choriocarcinoma and suggests that intervention with DEC1 might be a promising therapeutic choice for choriocarcinoma (AU)

[Research progress in control strategies of biological clock disorder].

Circadian clock is an internal mechanism evolved to adapt to cyclic environmental changes, especially diurnal changes. Keeping the internal clock in synchronization with the external clock is essential for health. Mismatch of the clocks due to phase shift or disruption of molecular clocks may lead to circadian disorders, including abnormal sleep-wake cycles, as well as disrupted rhythms in hormone secretion, blood pressure, heart rate, body temperature, etc. Long-term circadian disorders are risk factors for various common critical diseases such as metabolic diseases, cardiovascular diseases, and tumor. To prevent or treat the circadian disorders, scientists have conducted extensive research on the function of circadian clocks and their roles in the development of diseases, and screened hundreds of thousands of compounds to find candidates to regulate circadian rhythms. In addition, melatonin, light therapy, exercise therapy, timing and composition of food also play a certain role in relieving associated symptoms. Here, we summarized the progress of both drug- and non-drug-based approaches to prevent and treat circadian clock disorders.

Long-read single-cell sequencing reveals expressions of hypermutation clusters of isoforms in human liver cancer cells.

The protein diversity of mammalian cells is determined by arrays of isoforms from genes. Genetic mutation is essential in species evolution and cancer development. Accurate Long-read transcriptome sequencing at single-cell level is required to decipher the spectrum of protein expressions in mammalian organisms. In this report, we developed a synthetic long-read single-cell sequencing technology based on LOOPseq technique. We applied this technology to analyze 447 transcriptomes of hepatocellular carcinoma (HCC) and benign liver from an individual. Through Uniform Manifold Approximation and Projection (UMAP) analysis, we identified a panel of mutation mRNA isoforms highly specific to HCC cells. The evolution pathways that led to the hyper-mutation clusters in single human leukocyte antigen (HLA) molecules were identified. Novel fusion transcripts were detected. The combination of gene expressions, fusion gene transcripts, and mutation gene expressions significantly improved the classification of liver cancer cells versus benign hepatocytes. In conclusion, LOOPseq single-cell technology may hold promise to provide a new level of precision analysis on the mammalian transcriptome.

Detection of Quantum Signals Free of Classical Noise via Quantum Correlation.

Extracting useful signals is key to both classical and quantum technologies. Conventional noise filtering methods rely on different patterns of signal and noise in frequency or time domains, thus limiting their scope of application, especially in quantum sensing. Here, we propose a signal-nature-based (not signal-pattern-based) approach which singles out a quantum signal from its classical noise background by employing the intrinsic quantum nature of the system. We design a novel protocol to extract the quantum correlation signal and use it to single out the signal of a remote nuclear spin from its overwhelming classical noise backgrounds, which is impossible to be accomplished by conventional filter methods. Our Letter demonstrates the quantum or classical nature as a new degree of freedom in quantum sensing. The further generalization of this quantum nature-based method opens a new direction in quantum research.

Fusion Gene Detection in Prostate Cancer Samples Enhances the Prediction of Prostate Cancer Clinical Outcomes from Radical Prostatectomy through Machine Learning in a Multi-Institutional Analysis.

Prostate cancer remains one of the most fatal malignancies in men in the United States. Predicting the course of prostate cancer is challenging given that only a fraction of prostate cancer patients experience cancer recurrence after radical prostatectomy or radiation therapy. This study examined the expressions of 14 fusion genes in 607 prostate cancer samples from the University of Pittsburgh, Stanford University, and the University of Wisconsin-Madison. The profiling of 14 fusion genes was integrated with Gleason score of the primary prostate cancer and serum prostate-specific antigen level to develop machine-learning models to predict the recurrence of prostate cancer after radical prostatectomy. Machine-learning algorithms were developed by analysis of the data from the University of Pittsburgh cohort as a training set using the leave-one-out cross-validation method. These algorithms were then applied to the data set from the combined Stanford/Wisconsin cohort (testing set). The results showed that the addition of fusion gene profiling consistently improved the prediction accuracy rate of prostate cancer recurrence by Gleason score, serum prostate-specific antigen level, or a combination of both. These improvements occurred in both the training and testing cohorts and were corroborated by multiple models.

Nanothermometry with Enhanced Sensitivity and Enlarged Working Range Using Diamond Sensors.

Nanothermometry is increasingly demanded in frontier research in physics, chemistry, materials science and engineering, and biomedicine. An ideal thermometer should have features of reliable temperature interpretation, high sensitivity, fast response, minimum disturbance of the target's temperature, applicability in a variety of environments, and a large working temperature range. For applications in nanosystems, high spatial resolution is also desirable. Such requirements impose great challenges in nanothermometry since the shrinking of the sensor volume usually leads to a reduction in sensitivity.Diamond with nitrogen-vacancy (NV) centers provides opportunities for nanothermometry. NV center spins have sharp resonances due to their superb coherence. NV centers are multimodal sensors. They can directly sense magnetic fields, electric fields, temperature, pressure, and nuclear spins and, through proper transduction, measure other quantities such as the pH and deformation. In particular, their spin resonance frequencies vary with temperature, making them a promising thermometer. The high thermal conductivity, high hardness, chemical stability, and biocompatibility of diamond enable reliable and fast temperature sensing in complex environments ranging from erosive liquids to live systems. Chemical processing of diamond surfaces allows various functionalities such as targeting. The small size and the targeting capability of nanodiamonds then enable site-specific temperature sensing with nanoscale spatial resolution. However, the sensitivity of NV-based nanothermometry is yet to meet the requirement of practical systems with a large gap of a few orders of magnitude. On the other hand, although NV-based quantum sensing works well from 0.3 to 600 K, extending the sensing scheme to high temperature remains challenging due to uncertainty in identifying the exact physical limits and possible solution at elevated temperatures.This Account focuses on our efforts to enhance the temperature sensitivity and widen the working temperature range of diamond-based nanothermometry. We start with explaining the working principle and features of NV-based thermometry with examples of applications. Then a transducer-based concept is introduced with practical schemes to improve the sensitivity of the nanodiamond thermometer. Specifically, we show that the temperature signal can be transduced and amplified by adopting hybrid structures of nanodiamond and magnetic nanoparticles, which results in a record temperature sensitivity of 76 µK/√Hz. We also demonstrate quantum sensing with NV at high temperatures of up to 1000 K by adopting a pulsed heating-cooling scheme to carry out the spin polarization and readout at room temperature and the spin manipulation (sensing) at high temperatures. Finally, unsolved problems and future endeavors of diamond nanothermometry are discussed.

Effect of dietary supplementation of Bacillus subtilis TLRI 211-1 on laying performance, egg quality and blood characteristics of Leghorn layers.

OBJECTIVE: TLRI 211-1 is a novel Bacillus subtilis strain. This experiment was to investigate dietary supplementation of TLRI 211-1 on laying performance, egg quality and blood characteristics of layers. METHODS: One hundred and twenty 65-wk-old Leghorn layers were divided into four treatment groups for 8 weeks experiment. Each treatment had three replicates. The basal diet was formulated as control group with crude protein 17% and metabolizable energy 2,850 kcal/kg and supplemented with TLRI 211-1 0.1%, 0.3%, and commercial Bacillus amyloliquefaciens 0.1% as treatment 2, 3 and 4 groups, respectively. Both TLRI 211-1 and commercial Bacillus amyloliquefaciens were adjusted to contain 1×109 colony-forming unit (CFU)/mL (g), hence the 0.1% supplemental level was 1×109 CFU/kg. RESULTS: The results showed that TLRI 211-1 0.3% and commercial B. amyloliquefaciens groups had higher weight gain than the other groups; TLRI 211-1 0.1% group had better feed to eggs conversion ratio than the control and commercial B. amyloliquefaciens groups (p<0.05). Bacillus subtilis supplementation increased yolk weight (p<0.05). In egg quality during storage, TLRI 211-1 0.1% had higher breaking strength than the control group at the second week of storage (p<0.05). At the third week of storage, TLRI 211-1 0.3% had higher Haugh unit (p<0.05). Hens fed diets supplemented with TLRI 211-1 0.3% significantly decreased blood triglyceride levels and increased blood calcium levels (p< 0.05). TLRI 211-1 0.3% group had lower H2S (p<0.05) and hence had less unpleasant odor in excreta of hens. CONCLUSION: In conclusion, supplementation with 0.1% TLRI 211-1 can significantly improve feed to eggs conversion ratio. TLRI 211-1 supplementation also can maintain eggs at their optimum quality level during storage. The study showed that B. subtilis TLRI 211-1 can be used as feed additives for improving egg production performance and egg quality.

Resveratrol Attenuates Ankylosing Spondylitis in Mice by Inhibiting the TLR4/NF-κB/NLRP3 Pathway and Regulating Gut Microbiota.

Ankylosing spondylitis (AS) is an autoimmune disease associated with disturbed gut microbiota. Currently, the treatments and outcomes of AS are not satisfactory. It is reported that resveratrol (RES) is a major phytoalexin with anti-inflammatory, antibacterial and some other pharmacological effects. However, there are no studies on the role of RES in AS. Therefore, this study aimed to explore the effect and mechanism of RES on AS. Proteoglycan and complete freund's adjuvant were used to conduct an AS mouse model, and then the AS mice were gavaged with RES (20 mg/kg and 50 mg/kg) daily for 4 weeks. Subsequently, the effect of RES on AS mice was assessed by detecting disease severity, inflammatory cytokines, NLRP3 inflammasome, TLR4/NF-κB pathway, intestinal mucosal barrier function, intestinal microbial barrier function. The assessment results indicated that RES could significantly relieve progression and severity of AS, inhibit the expression of pro-inflammatory cytokines (tumor necrosis factor-α, interleukin-6, interleukin-17A, interferon-γ), and promote the expression of anti-inflammatory cytokines (interleukin-4). RES intervention caused the inhibition of NLRP3 inflammasome and TLR4/NF-κB pathway. In terms of intestinal barrier function, experimental results found RES increased zonula occludens-1 and occludin expression, and additionally, changed the composition of the gut microbiota by increasing levels of Lactobacillus and Bifidobacterium and reducing levels of Enterococcus faecalis and Escherichia coli. Collectively, RES protects PG-induced AS mice by inhibiting inflammatory responses and TLR4/NF-κB/NLRP3 pathway, restoring intestinal mucosal barrier function, and regulating the composition of the gut microbiota. In other words, RES is a potential candidate for the treatment of AS.

HIF-1α contributes to metastasis in choriocarcinoma by regulating DEC1 expression.

PURPOSE: To elucidate the underlying mechanism of HIF-1α in migration and invasion of choriocarcinoma. METHODS: Cell proliferation was determined by CCK-8 assay when cell invasion was detected by transwell assay. The protein expression was detected by western blotting, immunohistochemistry, and qPCR assay. RESULT: HIF-1α was shown to be strongly expressed in both clinical tumour tissues and cell lines in choriocarcinoma. When HIF-1α was efficiently knocked down in JEG3 cells, the proliferation rate was reduced by approximately 50% and the number of cells that migrated through the transwell insert was greatly decreased. The cell invasion rate was also significantly reduced. Moreover, typical markers of epithelial-mesenchymal transition such as E-cadherin, were increased, while vimentin and α-SMA were decreased after HIF-1α knockdown. In contrast, overexpression of DEC1 reversed the effects of HIF-1α knockdown. Cell proliferation, migration, and invasion were partially recovered. The level of E-cadherin was decreased, while the level of vimentin and α-SMA was increased. In addition, the level of ß-catenin and LEF1 was downregulated after HIF-1α knockdown. The expression of MMP2 and MMP9 also declined. However, overexpression of DEC1 after HIF-1α knockdown partially reversed the expression pattern of these molecules. CONCLUSION: HIF-1α contributed to EMT and metastasis through activation of canonical ß-catenin signalling in choriocarcinoma and this process was dependent on DEC1. This study provides a new mechanism of HIF-1α in choriocarcinoma and suggests that intervention with DEC1 might be a promising therapeutic choice for choriocarcinoma.

Molecular Cloning and Characterization of a New Family VI Esterase from an Activated Sludge Metagenome.

A new esterase gene, est6, was discovered in an activated sludge metagenomic library. The 729-bp gene encodes a 242-amino acid protein (designated Est6) with a molecular mass of 26.1 kDa. Est6 shared only a moderate identity to a putative hydrolase with the highest BLASTP analysis score. Most of the closely related proteins are uncharacterized and are predicted from genome sequencing data of microorganisms or metagenomic DNA sequences. The phylogenetic analysis of Est6 showed that the protein was assigned to family VI esterases/lipases. The catalytic triad of Est6 was predicted to be Ser135, Asp188, and His219, with Ser135 in a typically conserved pentapeptide (GFSQG) of family VI members, which was further confirmed by site-directed mutagenesis. The est6 gene was overexpressed successfully in its soluble form in Escherichia coli and then purified to its tag-free form and homogeneity by affinity chromatography. The purified Est6 in pH 8.0 buffer was active as a monomer. The optimal conditions for Est6 activity were at a temperature of 45 °C and pH of 8.0 when using p-nitrophenyl acetate as a substrate. The enzyme was stable over wide temperature and pH ranges, and it exhibited activity in the presence of organic solvents, metal cations, or detergents. Furthermore, the enzyme showed significant regioselectivity in the spectrophotometric analysis. In conclusion, Est6 might have the potential for applications in biotechnological processes.

Near-Surface

Impurity spins in crystal matrices are promising components in quantum technologies, particularly if they can maintain their spin properties when close to surfaces and material interfaces. Here, we investigate an attractive candidate for microwave-domain applications, the spins of group-VI ^{125}Te^{+} donors implanted into natural Si at depths as shallow as 20 nm. We show that surface band bending can be used to ionize such near-surface Te to spin-active Te^{+} state, and that optical illumination can be used further to control the Te donor charge state. We examine spin activation yield, spin linewidth, and relaxation (T_{1}) and coherence times (T_{2}) and show how a zero-field 3.5 GHz "clock transition" extends spin coherence times to over 1 ms, which is about an order of magnitude longer than other near-surface spin systems.

Quantum nonlinear spectroscopy of single nuclear spins.

Conventional nonlinear spectroscopy, which use classical probes, can only access a limited set of correlations in a quantum system. Here we demonstrate that quantum nonlinear spectroscopy, in which a quantum sensor and a quantum object are first entangled and the sensor is measured along a chosen basis, can extract arbitrary types and orders of correlations in a quantum system. We measured fourth-order correlations of single nuclear spins that cannot be measured in conventional nonlinear spectroscopy, using sequential weak measurement via a nitrogen-vacancy center in diamond. The quantum nonlinear spectroscopy provides fingerprint features to identify different types of objects, such as Gaussian noises, random-phased AC fields, and quantum spins, which would be indistinguishable in second-order correlations. This work constitutes an initial step toward the application of higher-order correlations to quantum sensing, to examining the quantum foundation (by, e.g., higher-order Leggett-Garg inequality), and to studying quantum many-body physics.

Revealing Capillarity in AFM Indentation of Cells by Nanodiamond-Based Nonlocal Deformation Sensing.

Nanoindentation based on atomic force microscopy (AFM) can measure the elasticity of biomaterials and cells with high spatial resolution and sensitivity, but relating the data to quantitative mechanical properties depends on information on the local contact, which is unclear in most cases. Here, we demonstrate nonlocal deformation sensing on biorelevant soft matters upon AFM indentation by using nitrogen-vacancy centers in nanodiamonds, providing data for studying both the elasticity and capillarity without requiring detailed knowledge about the local contact. Using fixed HeLa cells for demonstration, we show that the apparent elastic moduli of the cells would have been overestimated if the capillarity was not considered. In addition, we observe that both the elastic moduli and the surface tensions are reduced after depolymerization of the actin cytoskeleton in cells. This work demonstrates that the nanodiamond sensing of nonlocal deformation with nanometer precision is particularly suitable for studying mechanics of soft biorelevant materials.

Transcriptome and Exome Analyses of Hepatocellular Carcinoma Reveal Patterns to Predict Cancer Recurrence in Liver Transplant Patients.

Hepatocellular carcinoma (HCC) is one of the most lethal human cancers. Liver transplantation has been an effective approach to treat liver cancer. However, significant numbers of patients with HCC experience cancer recurrence, and the selection of suitable candidates for liver transplant remains a challenge. We developed a model to predict the likelihood of HCC recurrence after liver transplantation based on transcriptome and whole-exome sequencing analyses. We used a training cohort and a subsequent testing cohort based on liver transplantation performed before or after the first half of 2012. We found that the combination of transcriptome and mutation pathway analyses using a random forest machine learning correctly predicted HCC recurrence in 86.8% of the training set. The same algorithm yielded a correct prediction of HCC recurrence of 76.9% in the testing set. When the cohorts were combined, the prediction rate reached 84.4% in the leave-one-out cross-validation analysis. When the transcriptome analysis was combined with Milan criteria using the k-top scoring pairs (k-TSP) method, the testing cohort prediction rate improved to 80.8%, whereas the training cohort and the combined cohort prediction rates were 79% and 84.4%, respectively. Application of the transcriptome/mutation pathways RF model on eight tumor nodules from 3 patients with HCC yielded 8/8 consistency, suggesting a robust prediction despite the heterogeneity of HCC. Conclusion: The genome prediction model may hold promise as an alternative in selecting patients with HCC for liver transplant.