Screening anlotinib responders via blood-based proteomics in non-small cell lung cancer.

Anlotinib has been demonstrated to be effective in advanced non-small cell lung cancer (NSCLC) patients. The response stratification of anlotinib remains unclear. In this study, plasma samples from 28 anlotinib-treated NSCLC patients (discovery cohort: 14 responders and 14 non-responders) were subjected to proteomic analysis, and plasma samples from 35 anlotinib-treated NSCLC patients (validation cohort) were subjected to validation analysis. Liquid chromatography-tandem mass spectrometry analysis was performed on samples with different time points, namely baseline (BL), best response (BR), and progression disease (PD). Bioinformatics analysis was performed to screen for the underlying differential proteins. Enzyme-linked immunosorbent assay was performed to detect plasma ARHGDIB, FN1, CDH1, and KNG1 levels respectively. The Kaplan-Meier survival analysis was used for biomarker-based responsive stratification. Our results indicated that differential proteins between responders and non-responders showed that proteomic technology potentially contributes to biomarker screening in plasma samples at BL. Furthermore, our results suggested that the detection of plasma ARHGDIB, FN1, CDH1, and KNG1 levels have potential predictive value for anlotinib response both in the discovery cohort and validation cohort. Collectively, this study offers novel insights into the value of plasma biomarker screening via proteomic examination and suggests that plasma ARHGDIB, FN1, CDH1, and KNG1 levels could be used as biomarkers for anlotinib stratification in NSCLC patients.

Aluminum and Fluoride Stresses Altered Organic Acid and Secondary Metabolism in Tea (Camellia sinensis) Plants: Influences on Plant Tolerance, Tea Quality and Safety.

Tea plants have adapted to grow in tropical acidic soils containing high concentrations of aluminum (Al) and fluoride (F) (as Al/F hyperaccumulators) and use secret organic acids (OAs) to acidify the rhizosphere for acquiring phosphorous and element nutrients. The self-enhanced rhizosphere acidification under Al/F stress and acid rain also render tea plants prone to accumulate more heavy metals and F, which raises significant food safety and health concerns. However, the mechanism behind this is not fully understood. Here, we report that tea plants responded to Al and F stresses by synthesizing and secreting OAs and altering profiles of amino acids, catechins, and caffeine in their roots. These organic compounds could form tea-plant mechanisms to tolerate lower pH and higher Al and F concentrations. Furthermore, high concentrations of Al and F stresses negatively affected the accumulation of tea secondary metabolites in young leaves, and thereby tea nutrient value. The young leaves of tea seedlings under Al and F stresses also tended to increase Al and F accumulation in young leaves but lower essential tea secondary metabolites, which challenged tea quality and safety. Comparisons of transcriptome data combined with metabolite profiling revealed that the corresponding metabolic gene expression supported and explained the metabolism changes in tea roots and young leaves via stresses from high concentrations of Al and F. The study provides new insight into Al- and F-stressed tea plants with regard to responsive metabolism changes and tolerance strategy establishment in tea plants and the impacts of Al/F stresses on metabolite compositions in young leaves used for making teas, which could influence tea nutritional value and food safety.

Tea plant roots respond to aluminum-induced mineral nutrient imbalances by transcriptional regulation of multiple cation and anion transporters.

BACKGROUND: Tea is one of the most popular non-alcoholic beverages in the world for its flavors and numerous health benefits. The tea tree (Camellia sinensis L.) is a well-known aluminum (Al) hyperaccumulator. However, it is not fully understood how tea plants have adapted to tolerate high concentrations of Al, which causes an imbalance of mineral nutrition in the roots. RESULTS: Here, we combined ionomic and transcriptomic profiling alongside biochemical characterization, to probe the changes of metal nutrients and Al responsive genes in tea roots grown under increasing concentrations of Al. It was found that a low level of Al (~ 0.4 mM) maintains proper nutrient balance, whereas a higher Al concentration (2.5 mM) compromised tea plants by altering micro- and macro-nutrient accumulation into roots, including a decrease in calcium (Ca), manganese (Mn), and magnesium (Mg) and an increase in iron (Fe), which corresponded with oxidative stress, cellular damage, and retarded root growth. Transcriptome analysis revealed more than 1000 transporter genes that were significantly changed in expression upon Al exposure compared to control (no Al) treatments. These included transporters related to Ca and Fe uptake and translocation, while genes required for N, P, and S nutrition in roots did not significantly alter. Transporters related to organic acid secretion, together with other putative Al-tolerance genes also significantly changed in response to Al. Two of these transporters, CsALMT1 and CsALS8, were functionally tested by yeast heterologous expression and confirmed to provide Al tolerance. CONCLUSION: This study shows that tea plant roots respond to high Al-induced mineral nutrient imbalances by transcriptional regulation of both cation and anion transporters, and therefore provides new insights into Al tolerance mechanism of tea plants. The altered transporter gene expression profiles partly explain the imbalanced metal ion accumulation that occurred in the Al-stressed roots, while increases to organic acid and Al tolerance gene expression partly explains the ability of tea plants to be able to grow in high Al containing soils. The improved transcriptomic understanding of Al exposure gained here has highlighted potential gene targets for breeding or genetic engineering approaches to develop safer tea products.

Thidiazuron Promotes Leaf Abscission by Regulating the Crosstalk Complexities between Ethylene, Auxin, and Cytokinin in Cotton.

Thidiazuron (TDZ) is widely used as a defoliant to induce leaf abscission in cotton. However, the underlying molecular mechanism is still unclear. In this study, RNA-seq and enzyme-linked immunosorbent assays (ELISA) were performed to reveal the dynamic transcriptome profiling and the change of endogenous phytohormones upon TDZ treatment in leaf, petiole, and abscission zone (AZ). We found that TDZ induced the gene expression of ethylene biosynthesis and signal, and promoted ethylene accumulation earlier in leaf than that in AZ. While TDZ down-regulated indole-3-acetic acid (IAA) biosynthesis genes mainly in leaf and IAA signal and transport genes. Furthermore, the IAA content reduced more sharply in the leaf than that in AZ to change the auxin gradient for abscission. TDZ suppressed CTK biosynthesis genes and induced CTK metabolic genes to reduce the IPA accumulation for the reduction of ethylene sensitivity. Furthermore, TDZ regulated the gene expression of abscisic acid (ABA) biosynthesis and signal and induced ABA accumulation between 12-48 h, which could up-regulate ABA response factor genes and inhibit IAA transporter genes. Our data suggest that TDZ orchestrates metabolism and signal of ethylene, auxin, and cytokinin, and also the transport of auxin in leaf, petiole, and AZ, to control leaf abscission.

Identification of C3H2C3-type RING E3 ubiquitin ligase in grapevine and characterization of drought resistance function of VyRCHC114.

BACKGROUND: RING is one of the largest E3 ubiquitin ligase families and C3H2C3 type is the largest subfamily of RING, which plays an important role in plant growth and development, and growth and responses to biotic and abiotic stresses. RESULTS: A total of 143 RING C3H2C3-type genes (RCHCs) were discovered from the grapevine genome and separated into groups (I-XI) according to their phylogenetic analysis, and these genes named according to their positions on chromosomes. Gene replication analysis showed that tandem duplications play a predominant role in the expansion of VvRCHCs family together. Structural analysis showed that most VvRCHCs (67.13 %) had no more than 2 introns, while genes clustered together based on phylogenetic trees had similar motifs and evolutionarily conserved structures. Cis-acting element analysis showed the diversity of VvRCHCs regulation. The expression profiles of eight DEGs in RNA-Seq after drought stress were like the results of qRT-PCR analysis. In vitro ubiquitin experiment showed that VyRCHC114 had E3 ubiquitin ligase activity, overexpression of VyRCHC114 in Arabidopsis improved drought tolerance. Moreover, the transgenic plant survival rate increased by 30 %, accompanied by electrolyte leakage, chlorophyll content and the activities of SOD, POD, APX and CAT were changed. The quantitative expression of AtCOR15a, AtRD29A, AtERD15 and AtP5CS1 showed that they participated in the response to drought stress may be regulated by the expression of VyRCHC114. CONCLUSIONS: This study provides valuable new information for the evolution of grapevine RCHCs and its relevance for studying the functional characteristics of grapevine VyRCHC114 genes under drought stress.

Clinicopathologic features and genomic analysis of pulmonary blastomatoid carcinosarcoma.

BACKGROUND: This study was designed to investigate the clinicopathologic features of pulmonary blastomatoid carcinosarcoma and explore the genomic profiles of epithelial and mesenchymal components in this tumor. METHODS: Three cases of pulmonary blastomatoid carcinosarcoma were enrolled in this study. Clinicopathologic information and prognostic data were retrospectively reviewed. Diagnostic immunohistochemistry was performed. The epithelial and mesenchymal components were microdissected to investigate the genomic profiles by performing capture-based targeted next generation sequencing. RESULTS: The epithelial components in patient one consisted of low-grade and high-grade fetal lung adenocarcinoma. Low-grade epithelial cells showed nuclear expression of ß-catenin and missense mutation of CTNNB1. The epithelial components in another two patients consisted of high-grade fetal lung adenocarcinoma/enteric adenocarcinoma. The epithelial cells showed membrane staining of ß-catenin and harbored no mutation of CTNNB1. The mesenchymal components in all three tumors were composed of primitive round/spindle cells without definite differentiation and showed cytoplasmic dot positive of ß-catenin and no corresponding mutation. Within a tumor, both components exhibited relatively comparable molecular profile. In patient one, 4 mutations: RB1, FAT3, PTCH1 and LRP1B were shared by both epithelial and mesenchymal components. Epithelial component had additional mutations in BCOR, CTNNB1, CTCF, FAT1 and DICER1. In patient two, 12 mutations were shared. The epithelial component had BRCA2 mutation and the mesenchymal had mutations in CREBBP, ALK, DNMT3A, ASXL2, MYCN and RICTOR. Patient three had 6 shared mutations. The epithelial component had an additional mutation in KAT6A and the mesenchymal had an additional mutation in APC. Collectively, we observed heterogeneity between epithelial and mesenchymal components of the same tumor. CONCLUSIONS: Blastomatoid carcinosarcoma showed characteristic morphology and immunophenotype. Parallel detection of genetic abnormalities in epithelial and mesenchymal components could provide further evidence for tumor differentiation, molecular targeting and differential diagnosis.

Reduction-sensitive poly(ethylene glycol)-polypeptide conjugate micelles for highly efficient intracellular delivery and enhanced antitumor efficacy of hydroxycamptothecin.

The non-specific biodistribution of traditional chemotherapeutic drugs against tumors is the key factor that causes systemic toxicity and hinders their clinical application. In this study, a reduction-sensitive polymer conjugate micelle was manufactured to achieve tumor-specific targeting, reduce toxic side-effects and improve anti-tumor activity of a natural anti-cancer drug, hydroxycamptothecin (HCPT). Therefore, HCPT was conjugated with methoxy-poly(ethylene glycol)-poly(ß-benzyl-L-aspartate) (mPEG-PBLA) by a disulfide bond or succinate bond for the first time to obtain the mPEG-PBLA-SS-HCPT (PPSH) and mPEG-PBLA-CC-HCPT (PPCH) that would form micelles after high-speed agitation and dialysis. The PPSH micelles showed an average particle size of 126.3 nm, a low polydispersity index of 0.209, and a negative surface charge of -21.1 mV zeta potential. Transmission electron microscopy showed the PPSH micelles to have spherical morphology. PPSH had a low critical micelle concentration of 1.29 µg ml-1 with high dilution stability, storage stability and reproducibility. Moreover, the particle size of the PPSH micelles had no significant change after incubation with rat plasma for 72 h, probably resulting in high long circulation in the blood. The PPSH micelles showed significant reduction sensitivity to glutathione. Their sizes increased by 403.2 nm after 24 h post-incubation, and 87.6% drug release was achieved 48 h post-incubation with 40 mM glutathione solutions. The PPSH micelles showed stronger inhibition of HepG2 cells in vitro and growth of H-22 tumor in vivo than the PPCH and HCPT solutions after intravenous injection. The accumulation of PPSH micelles in the tumor tissue contributed to the high anti-tumor effect with little side-effect on the normal tissues. The reduction-sensitive PPSH micelles were a promising carrier of HCPT and other poorly soluble anti-cancer drugs.

Plastid Transformation: How Does it Work? Can it Be Applied to Crops? What Can it Offer?

In recent years, plant genetic engineering has advanced agriculture in terms of crop improvement, stress and disease resistance, and pharmaceutical biosynthesis. Cells from land plants and algae contain three organelles that harbor DNA: the nucleus, plastid, and mitochondria. Although the most common approach for many plant species is the introduction of foreign DNA into the nucleus (nuclear transformation) via Agrobacterium- or biolistics-mediated delivery of transgenes, plastid transformation offers an alternative means for plant transformation. Since there are many copies of the chloroplast genome in each cell, higher levels of protein accumulation can often be achieved from transgenes inserted in the chloroplast genome compared to the nuclear genome. Chloroplasts are therefore becoming attractive hosts for the introduction of new agronomic traits, as well as for the biosynthesis of high-value pharmaceuticals, biomaterials and industrial enzymes. This review provides a comprehensive historical and biological perspective on plastid transformation, with a focus on current and emerging approaches such as the use of single-walled carbon nanotubes (SWNTs) as DNA delivery vehicles, overexpressing morphogenic regulators to enhance regeneration ability, applying genome editing techniques to accelerate double-stranded break formation, and reconsidering protoplasts as a viable material for plastid genome engineering, even in transformation-recalcitrant species.

Molecular cloning and characterization of a grapevine (Vitis vinifera L.) serotonin N-acetyltransferase (VvSNAT2) gene involved in plant defense.

BACKGROUND: Melatonin is a ubiquitous molecule and exists across kingdoms. Studies on melatonin in plants have mainly focused on its physiological influence on growth and development, and on its biosynthesis. A number of studies have been conducted on the melatonin content and exogenous melatonin treatment of grapevine (Vitis vinifera L.). However, key genes or enzymes of the melatonin biosynthetic pathway remain unclear. RESULTS: In this study, we cloned and identified the gene encoding serotonin N-acetyltransferase (SNAT) in grapevine (VvSNAT2). The VvSNAT2 protein was identified from a collection of 30 members of the grapevine GCN5-related N-acetyltransferase (GNAT) superfamily. Phylogenetic and protein sublocalization analyses showed that the candidate gene VvGNAT16 is VvSNAT2. Characterization of VvSNAT2 showed that its enzymatic activity is highest at a pH of 8.8 and a temperature of 45 °C. Analysis of enzyme kinetics showed the values of Km and Vmax of VvSNAT2 using serotonin were 392.5 µM and 836 pmol/min/mg protein, respectively. The expression of VvSNAT2 was induced by melatonin treatment and pathogen inoculation. Overexpression of VvSNAT2 in Arabidopsis resulted in greater accumulation of melatonin and chlorophyll and enhanced resistance to powdery mildew in the transgenic plants compared with the wild type (WT). Additionally, our data showed that the marker genes in the salicylic acid (SA) signaling pathway were expressed to higher levels in the transgenic plants compared with the WT. CONCLUSIONS: The VvSNAT2 gene was cloned and identified in grapevine for the first time. Our results indicate that VvSNAT2 overexpression activates the SA and JA signaling pathways; however, the SA pathway plays a central role in VvSNAT2-mediated plant defense.

Malignant perivascular epithelioid cell tumor of the lung synchronous with a primary adenocarcinoma: one case report and review of the literature.

BACKGROUND: Perivascular Epithelioid Cell Tumors (PEComa) is an extraordinarily rare mesenchymal neoplasm especially the malignant type originating from the lung. To date, only 8 cases of malignant or malignant potential pulmonary PEComa had been documented. Firm diagnostic criteria for malignant pulmonary PEComa need urgently to be established. CASE PRESENTATION: We report a challenging case of malignant pulmonary PEComa combined with a primary adenocarcinoma in a 54-year-old man. The PEComa-like tumor showed strong Melan-A and weak transcription factor E3 (TFE3) protein expression but no TFE3 gene rearrangement. The carcinoma-like nodule was recognized as a poorly differentiated primary lung adenocarcinoma. DISCUSSION AND CONCLUSIONS: Our case report was the first case of malignant pulmonary PEComa synchronous with a primary adenocarcinoma and studied the dilemma of diagnosing benign versus malignant criteria for this uncommon tumor.

Functional Characterization of Resistance to Powdery Mildew of VvTIFY9 from Vitis vinifera.

Powdery mildew is a disease caused by fungal pathogens that harms grape leaves and fruits. The TIFY gene family is a plant-specific super-family involved in the process of plants' development and their biotic and abiotic stress responses. This study aimed to learn the function of the VvTIFY9 gene to investigate molecular mechanisms of grape resistance to powdery mildew. A VvTIFY9 protein encoding a conserved motif (TIF[F/Y]XG) was characterized in grape (Vitis vinifera). Sequence analysis confirmed that VvTIFY9 contained this conserved motif (TIF[F/Y]XG). Quantitative PCR analysis of VvTIFY9 in various grape tissues demonstrated that the expression of VvTIFY9 was higher in grape leaves. VvTIFY9 was induced by salicylic acid (SA) and methyl jasmonate (MeJA) and it also quickly responded to infection with Erysiphe necator in grape. Analysis of the subcellular localization and transcriptional activation activity of VvTIFY9 showed that VvTIFY9 located to the nucleus and had transcriptional activity. Arabidopsis that overexpressed VvTIFY9 were more resistant to Golovinomyces cichoracearum, and quantitative PCR revealed that two defense-related genes, AtPR1 and AtPDF1.2, were up-regulated in the overexpressing lines. These results indicate that VvTIFY9 is intimately involved in SA-mediated resistance to grape powdery mildew. This study provides the basis for exploring the molecular mechanism of grape resistance to disease resistance and candidate genes for transgenic disease resistance breeding of grape plants.

Pretreatment direct bilirubin and total cholesterol are significant predictors of overall survival in advanced non-small-cell lung cancer patients with EGFR mutations.

This study was designed to examine the prediction of pretreatment circulating bilirubin and cholesterol for overall survival in 459 advanced non-small-cell lung cancer (NSCLC) patients with epidermal growth factor receptor (EGFR) mutations. Circulating total bilirubin, direct bilirubin (DB), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C) levels were measured at baseline. The mean age (standard deviation) of all study patients was 58.7 (10.5) years, and 42.9% of them was males. Ever smokers accounted for 27.0% and lung adenocarcinoma for 90.4%. The median follow-up time and survival time were 29.5 and 34.9 months, respectively. Patients with higher DB had a 1.68-fold increased risk of death compared with patients with lower DB (hazard ratio [HR] = 1.68, 95% confidence interval [CI]: 1.22-2.30, p = 0.001), while patients with higher TC were at a 63% reduced risk of death compared with patients with lower TC (HR = 0.37, 95% CI: 0.20-0.67, p = 0.001). As for HDL-C, patients with higher levels had the risk of death reduced by 46% (HR = 0.54, 95% CI: 0.29-1.00, p = 0.049) compared with patients with lower levels. After the Bonferroni correction, only DB and TC were significantly associated with NSCLC survival. Our findings demonstrate for the first time that pretreatment DB was identified as a significant risk factor, yet TC as a protective factor, for overall survival in NSCLC patients with EGFR mutations.

Ex vivo treatment with inhaled N-acetylcysteine in porcine lung transplantation.

BACKGROUND: We have shown the beneficial effects of N-acetylcysteine (NAC) on posttransplant lung function, when both donor and recipient were pretreated intravenously. However, systemic treatment of multiorgan donors may not be clinically relevant. Thus, we hypothesized that ex vivo treatment of donors with nebulized NAC would be adequate to prevent from ischemia-reperfusion injury after lung transplantation. METHODS: Lungs were retrieved from domestic pigs and stored at 4°C for 24 h followed by 2 h of ex vivo lung perfusion (EVLP) to administer 50 mg/kg of NAC via nebulization in the NAC group (n = 6). The control group received nebulized saline (n = 5). Left lungs were transplanted and isolated at 1 h of reperfusion by occluding the right main bronchus and pulmonary artery, followed by 5 h of observation. Physiological data during EVLP and after reperfusion were recorded. Inflammatory response, markers of oxidative stress, and microscopic lung injury were analyzed. RESULTS: There was a trend toward better oxygenation throughout reperfusion period in the treatment group, which was accompanied by inhibited inflammatory response related to reduction in myeloperoxidase activity during EVLP and nuclear factor-κB activation at the end of reperfusion. CONCLUSIONS: Ex vivo treatment of donor lungs with inhaled NAC reduced inflammatory response via its antioxidant activity in experimental porcine lung transplantation.

Effect of multiple cycles of freeze-thawing on the RNA quality of lung cancer tissues.

RNA degradation is a major problem in tissue banking. We explored the effect of thawing flash-frozen biospecimens on the quality and integrity of RNA for genetic testing as well as for other cancer research studies. The histological quality of the frozen tumor sections was evaluated by using hematoxylin and eosin staining. RNA extraction from 60 lung cancer tissue samples subjected to various freeze/thaw cycles was performed using the RNeasy Plus isolation kit. RNA integrity was assessed by using an Agilent bioanalyzer to obtain RNA integrity numbers (RIN). Furthermore, RNA from different groups was used for fluorescence Reverse transcription-polymerase chain reaction (RT-PCR) analysis of the echinoderm microtubule-associated protein-like 4 and anaplastic lymphoma kinase (EML4-ALK) fusion gene mutation to verify whether it can be used for research or clinical testing. Highly variable RIN values were observed among the samples, which showed no correlation with the number of freeze/thaw cycles conducted. However, after 3 freeze/thaw cycles (each thaw event lasted for 10 min), an increasing number of changes in peak intensity in RINs were observed. After 5 freeze/thaw cycles, RNA integrity decreased to approximately 35%. After 3 freeze/thaw cycles, the RNA could still be used for RT-PCR analysis of EML4-ALK fusion gene mutations; whereas those subjected to 5 freeze/thaw cycles could not. Limited (<3) freeze/thaw cycles did not adversely affect the quality of RNA extracted from tumor tissues and subsequent RT-PCR analysis. Our data could be utilized in the establishment of a standardized procedure for tissue biospecimen collection and storage.

Activation of the NF-κB pathway as a mechanism of alcohol enhanced progression and metastasis of human hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC), the most common form of primary liver cancer, is the third leading cause of cancer-related death in human. Alcohol is a known risk factor for HCC. However it is still unclear whether and how alcohol enhances the progression and metastasis of existing HCC. METHODS AND RESULTS: We first retrospectively investigated 52 HCC patients (24 alcohol-drinkers and 28 non-drinkers), and found a positive correlation between alcohol consumption and advanced Tumor-Node-Metastasis (TNM) stages, higher vessel invasion and poorer prognosis. In vitro and in vivo experiments further indicated that alcohol promoted the progression and migration/invasion of HCC. Specifically, in a 3-D tumor/endothelial co-culture system, we found that alcohol enhanced the migration/invasion of HepG2 cells and increased tumor angiogenesis. Consistently, higher expression of VEGF, MCP-1 and NF-κB was observed in HCC tissues of alcohol-drinkers. Alcohol induced the accumulation of intracellular reactive oxygen species (ROS) and the activation of NF-κB signaling in HepG2 cells. Conversely, blockage of alcohol-mediated ROS accumulation and NF-κB signaling inhibited alcohol-induced expression of VEGF and MCP-1, the tumor growth, angiogenesis and metastasis. CONCLUSION: This study suggested that chronic moderate alcohol consumption may promote the progression and metastasis of HCC; the oncogenic effect may be at least partially mediated by the ROS accumulation and NF-ĸB-dependent VEGF and MCP-1 up-regulation.

Correlation between squamous cell carcinoma of the lung and human papillomavirus infection and the relationship to expression of p53 and p16.

The number of non-small-cell lung cancer (NSCLC) patients reported as infected with human papillomavirus (HPV) varies among countries and by race and geographical location. Furthermore, the relationship between HPV and the expressions of p53 and p16 remains unclear. A large cohort of NSCLC patients from Shanghai was studied. Paraffin sections from 128 cases of lung adenocarcinoma (ADC) and 134 cases of squamous cell carcinoma (SQC) were collected from the Shanghai Chest Hospital. Samples were analyzed by polymerase chain reaction (PCR) and reverse dot blot for detection of HPV DNA and by immunohistochemistry for detection of p53 and p16 expressions. The rate of HPV infection in SQC cases was significantly higher than in ADC cases (12.69 versus 3.91%). Females with SQC had a significantly higher rate of HPV infection compared to males with SQC (18.75 versus 7.14%, p = 0.044). HPV infection was correlated with gender and age in SQC but not with the degree of tumor differentiation, TNM stage, or smoking. Koilocytosis was significantly correlated to the tumor differentiation grade, regardless of age and TNM stage. The expressions of p53 and p16 were correlated with HPV infection and the tumor histological type but not with the degree of tumor differentiation, TNM stage, smoking, gender, or age. p53-positive expression was significantly higher in HPV-infected SQC cases than in those not infected with HPV. There was no statistically significant difference in the expression of p16 between the two groups. Data showed that HPV infection may be an important virulence factor in SQC, particularly in female patients. HPV infection appears to be involved in cancer progression in SQC by promoting the expression of p53; however, p16 cannot be used as a surrogate marker for HPV infection in SQC.

Ethanol supports macrophage recruitment and reinforces invasion and migration of Lewis lung carcinoma.

BACKGROUND: Inflammation plays a critical role in cancer progression, and our data suggested that ethanol (EtOH) could promote the progression of breast cancer via increased monocyte chemo-attractant protein-1 (MCP-1). Thus, we investigated the effects of EtOH on lung cancer growth and metastasis to explore whether immunosuppression had a role. METHODS: C57BL/6 mice (n = 10) implanted with Lewis lung cancer (LLC) cells were used to model physiologically relevant EtOH intake on tumor growth and inflammation after macrophage polarization. Tumors were isolated and measured, and MCP-1 expression was measured via immunohistochemistry and Western blot. Recruitment of macrophages using CD11b and F4/80 antibodies was detected with immunohistochemistry and flow cytometry. Changes in arginase I and inducible nitric oxide synthase (iNOS) expression were measured with immunofluorescent microscopy. EtOH's effect on in vitro tumor angiogenesis was evaluated in culture, and the tumor microvessel density was assessed with CD31 immunohistochemistry. Matrix metalloproteinase 9 and interleukin 10 expressions were measured by Western blot, ELISA, and immunohistochemistry. Finally, we treated a macrophage cell line RAW264.7 with EtOH and measured changes in arginase I and iNOS expression. RESULTS: With EtOH exposure, macrophage density was positively correlated with MCP-1 expression. Macrophages infiltrated the tumor site, becoming tumor-associated macrophages that polarized to M2 phenotypes (ArgI(high) /iNOS(low) ) after EtOH treatment. Cancerous cells interacted with immune cells, especially M2 macrophages, and promoted tumor angiogenesis, progression, and invasiveness. RAW264.7 cells stimulated with EtOH expressed more arginase I and less iNOS than controls. These results agreed with the features of M2 phenotype macrophages (ArgI(high) /iNOS(low) ). CONCLUSIONS: Data show that EtOH induced M2 phenotype macrophages, suggesting that progression and metastasis of LLC may be mediated by recruitment of M2 macrophages, especially under the influence of EtOH.

Language Distance Moderates the Effect of a Mixed-Language Environment on New-Word Learning for 4-Year-Old Children.

As bilingual families increase, the phenomenon of language mixing among children in mixed-language environments has gradually attracted academic attention. This study aims to explore the impact of language mixing on vocabulary acquisition in bilingual children and whether language distance moderates this impact. We recruited two groups of bilingual children, Chinese-English bilinguals and Chinese-Japanese bilinguals, to learn two first-language new words in a monolingual environment and a mixed-language environment, respectively. The results showed that the participants could successfully recognize the novel words in the code-switching sentences. However, when we compared the performance of the two groups of bilingual children, we found that the gaze time proportion of the Chinese-English bilingual children under the code-switching condition was significantly higher than that of the Chinese-Japanese bilingual children, while there was no significant difference under the monolingual condition. This suggests that language mixing has an inhibitory effect on vocabulary acquisition in bilingual children and that this inhibitory effect is influenced by language distance, that is, the greater the language distance, the stronger the inhibitory effect. This study reveals the negative impact of language mixing on vocabulary acquisition in bilingual children and also implies that there may be some other influencing factors, so more research is needed on different types of bilingual children.

2,4-Dihydroxybenzoic Acid, a Novel SA Derivative, Controls Plant Immunity via UGT95B17-Mediated Glucosylation: A Case Study in Camellia Sinensis.

The plant hormone salicylic acid (SA) plays critical roles in plant innate immunity. Several SA derivatives and associated modification are identified, whereas the range and modes of action of SA-related metabolites remain elusive. Here, the study discovered 2,4-dihydroxybenzoic acid (2,4-DHBA) and its glycosylated form as native SA derivatives in plants whose accumulation is largely induced by SA application and Ps. camelliae-sinensis (Pcs) infection. CsSH1, a 4/5-hydroxylase, catalyzes the hydroxylation of SA to 2,4-DHBA, and UDP-glucosyltransferase UGT95B17 catalyzes the formation of 2,4-DHBA glucoside. Down-regulation reduced the accumulation of 2,4-DHBA glucosides and enhanced the sensitivity of tea plants to Pcs. Conversely, overexpression of UGT95B17 increased plant disease resistance. The exogenous application of 2,4-DHBA and 2,5-DHBA, as well as the accumulation of DHBA and plant resistance comparison, indicate that 2,4-DHBA functions as a potentially bioactive molecule and is stored mainly as a glucose conjugate in tea plants, differs from the mechanism described in Arabidopsis. When 2,4-DHBA is applied exogenously, UGT95B17-silenced tea plants accumulated more 2,4-DHBA than SA and showed induced resistance to Pcs infection. These results indicate that 2,4-DHBA glucosylation positively regulates disease resistance and highlight the role of 2,4-DHBA as potentially bioactive molecule in the establishment of basal resistance in tea plants.