Noninvasive evaluation of neutrophil extracellular traps signature predicts clinical outcomes and immunotherapy response in hepatocellular carcinoma.

Background: Neutrophil extracellular traps (NETs) have been shown to play a pivotal role in promoting metastasis and immune escape in hepatocellular carcinoma (HCC). Therefore, noninvasive tests to detect the formation of NETs in tumors can have significant implications for the treatment and prognoses of patients. Here, we sought to develop and validate a computed tomography (CT)-based radiomics model to predict the gene expression profiles that regulate the formation of NETs in HCC. Methods: This study included 1133 HCC patients from five retrospective cohorts. Based on the mRNA expression levels of 69 biomarkers correlated with NET formation, a 6-gene score (NETs score, NETS) was constructed in cohort 1 from TCIA database (n=52) and validated in cohort 2 (n=232) from ICGC database and cohort 3 (n=365) from TCGA database. And then based on the radiomics features of CT images, a radiomics signature (RNETS) was developed in cohort 1 to predict NETS status (high- or low-NETS). We further employed two cohorts from Nanfang Hospital (Guangzhou, China) to evaluate the predictive power of RNETS in predicting prognosis in cohort 4 (n=347) and the responses to PD-1 inhibitor of HCC patients in cohort 5 (n=137). Results: For NETS, in cohort 1, the area under the curve (AUC) values predicting 1, 2, and 3-year overall survival (OS) were 0.836, 0.879, and 0.902, respectively. The low-NETS was associated with better survival and higher levels of immune cell infiltration. The RNETS yielded an AUC value of 0.853 in distinguishing between high-NETS or low-NETS and patients with low-RNETS were associated with significantly longer survival time in cohort 1 (P<0.001). Notably, the RNETS was competent in predicting disease-free survival (DFS) and OS in cohort 4 (P<0.001). In cohort 5, the RNETS was found to be an independent risk factor for progression-free survival (PFS) (P<0.001). In addition, the objective response rate of HCC patients treated with PD-1 inhibitor was significantly higher in the low-RNETS group (27.8%) than in the high-RNETS group (10.8%). Conclusions: This study revealed that RNETS as a radiomics biomarker could effectively predict prognosis and immunotherapy response in HCC patients.

Heat Stress Suppresses Brassica napus Seed Oil Accumulation by Inhibition of Photosynthesis and BnWRI1 Pathway.

Heat stress during Brassica napus seed filling severely impairs yield and oil content. However, the mechanisms underlying heat-stress effects on B. napus seed photosynthesis and oil accumulation remain elusive. In this study, we showed that heat stress resulted in reduction of seed oil accumulation, whereas the seed sugar content was enhanced, which indicated that incorporation of carbohydrates into triacylglycerols was impaired. Photosynthesis and respiration rates, and the maximum quantum yield of photosystem II in developing seeds were inhibited by heat stress. Transcriptome analysis revealed that heat stress led to up-regulation of genes associated with high light response, providing evidence that photoinhibition was induced by heat stress. BnWRI1 and its downstream genes, including genes involved in de novo fatty acid biosynthesis pathway, were down-regulated by heat stress. Overexpression of BnWRI1 with a seed-specific promoter stabilized both oil accumulation and photosynthesis under the heat-stress condition, which suggested BnWRI1 plays an important role in mediating the effect of heat stress on fatty acid biosynthesis. A number of sugar transporter genes were inhibited by heat stress, resulting in defective integration of carbohydrates into triacylglycerols units. The results collectively demonstrated that disturbances of the seed photosynthesis machinery, impairment of carbohydrates incorporation into triacylglycerols and transcriptional deregulation of the BnWRI1 pathway by heat stress might be the major cause of decreased oil accumulation in the seed.

The water extract of Liuwei dihuang possesses multi-protective properties on neurons and muscle tissue against deficiency of survival motor neuron protein.

BACKGROUND: Deficiency of survival motor neuron (SMN) protein, which is encoded by the SMN1 and SMN2 genes, induces widespread splicing defects mainly in spinal motor neurons, and leads to spinal muscular atrophy (SMA). Currently, there is no effective treatment for SMA. Liuwei dihuang (LWDH), a traditional Chinese herbal formula, possesses multiple therapeutic benefits against various diseases via modulation of the nervous, immune and endocrine systems. Previously, we demonstrated water extract of LWDH (LWDH-WE) protects dopaminergic neurons and improves motor activity in models of Parkinson's disease. PURPOSE: This study aimed to investigate the potential protection of LWDH-WE on SMN deficiency-induced neurodegeneration and muscle weakness. STUDY DESIGN: The effects of LWDH-WE on SMN deficiency-induced neurotoxicity and muscle atrophy were examined by using SMN-deficient NSC34 motor neuron-like cells and SMA-like mice, respectively. METHODS: Inducible SMN-knockdown NSC34 motor neuron-like cells were used to mimic SMN-deficient condition. Doxycycline (1 µg/ml) was used to induce SMN deficiency in stable NSC34 cell line carrying SMN-specific shRNA. SMAΔ7 mice were used as a severe type of SMA mouse model. Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays. Apoptotic cells and neurite length were observed by inverted microscope. Protein expressions were examined by western blots. Muscle strength of animals was evaluated by hind-limb suspension test. RESULTS: LWDH-WE significantly increased SMN protein level, mitochondrial membrane potential and cell viability of SMN-deficient NSC34 cells. LWDH-WE attenuated SMN deficiency-induced down-regulation of B-cell lymphoma-2 (Bcl-2) and up-regulation of cytosolic cytochrome c and cleaved caspase-3. Moreover, LWDH-WE prevented SMN deficiency-induced inhibition of neurite outgrowth and activation of Ras homolog gene family, member A (RhoA)/ Rho-associated protein kinase (ROCK2)/ phospho-LIM kinase (p-LIMK)/ phospho-cofilin (p-cofilin) pathway. Furthermore, in SMA-like mice, LWDH-WE improved muscle strength and body weight accompanied with up-regulation of SMN protein in spinal cord, brain, and gastrocnemius muscle tissues. CONCLUSION: The present study demonstrated that LWDH-WE protects motor neurons against SMN deficiency-induced neurodegeneration, and it also improves the muscle strength of SMA-like mice, suggesting the potential benefits of LWDH-WE as a complementary prescription for SMN deficiency-related diseases.

[Recombinant adenovirus-mediated expression of 1.2-copy hepatitis B virus DNA in three hepatocytes].

OBJECTIVE: To study hepatitis B virus (HBV) expression in 3 hepatocytes infected with recombinant adenovirus containing 1.2-copy HBV DNA.a METHODS: A chicken hepatoma cell line and two human hepatocytes were infected by the recombinant adenovirus containing 1.2-copy HBV DNA at 25 pfu/cell. HBV-specific mRNA was detected by RT-PCR 3 days after the infection, and HBsAg and HBeAg were detected by ELISA and HBV DNA by real-time PCR daily after the infection. RESULTS: HBV mRNA expression was detected in all the 3 cells after recombinant adenovirus infection, and the quantities of HBV DNA and HBV antigens in the culture supernatant increased with the passage of time.a CONCLUSION: Infection with the recombinant adenovirus containing 1.2-copy HBV DNA can mediate HBV infection in the 3 cells in vitro.

[Dynamic analysis of HBV cccDNA in HepG2 cells infected with Ad-1.2 HBV].

OBJECTIVE: o study the replication of hepatitis B virus (HBV) in HepG2 cells infected with Ad-1.2 HBV. METHODS: HepG2 cells were transfected with adenovirus containing 1.2 copies of HBV DNA. The expression of HBV antigens were detected in the culture medium by means of enzyme-linked immunosorbent assay (ELISA), and the covalently closed circular DNA (cccDNA) in the cells was extracted with plasmid extraction kit and detected by real-time PCR with selective primer after treatment with mung bean nuclease. RESULTS: HBsAg, HBeAg and HBV cccDNA were all detected in HepG2 cells after tranfection with Ad-1.2 HBV. HBV cccDNA was detected 1 day after the infection, reaching the peak level 4 days after infection. CONCLUSION: Ad-1.2 HBV-infected cells can serve as the model for screening and evaluation of antiviral agents.

Tissue tropism of the TTV in experimentally infected rhesus monkeys.

OBJECTIVE: To determine whether the transfusion-transmitted virus (TTV) is hepatotropic. METHODS: Total DNA was extracted from various tissues of 5 experimentally infected Rhesus monkeys during the viremic period. A dot hybridization was done with viral double stranded DNA probes or single antisense probes. RESULTS: The double-stranded probe was hybridized with DNA from the liver, bone marrow, spleen,stomach, small intestine and colon. The single-stranded antisense probe was hybridized with DNA from the liver, small intestine and bone marrow of all 5 monkeys, but not with that from other tissues. CONCLUSIONS: As the viral genome was of negative polarity, the plus-stranded fragment identified in our study might be a replicative intermediate, and was only demonstrated in the liver, small intestine, and bone marrow by dot blot hybridization with single-stranded antisense probes. It is suggested that the TTV replicates in the liver, bone marrow and small intestine, and TTV might be hepatotropic.