Circ_0098823 binding with IGF2BP3 regulates DNM1L stability to promote metastasis of hepatocellular carcinoma via mitochondrial fission.

Hepatocellular carcinoma (HCC) is highly metastatic and invasive. CircRNA participates in gene regulation of multiple tumor metastases, but little is known whether it is a bystander or an actual player in HCC metastasis. We aim to explore the molecular mechanisms of novel circRNAs in HCC metastasis. RT-qPCR was used to detect the expression of 13 circRNAs derived by the ERBB3 gene. The function of circ_0098823 and DNM1L in HCC cells were estimated by CCK-8, transwell assays, flow cytometry, electron microscope, and in vivo experiments. RNA binding protein of circ_0098823 was confirmed by RNA pull-down, mass spectrometry, and RNA immunoprecipitation. The expression of DNM1L after IGF2BP3 knockdown was detected by RT-qPCR and western blot. Circ_0098823 was significantly up-regulated both in HCC tissues and HGF induced cell lines. Circ_0098823 overexpression significantly enhanced proliferation, migration, and invasion but decreased apoptosis of HCC cells, particularly promoted mitochondrial fission. Compared with the control group, the tumors in the circ_0098823 knockdown mice were significantly smaller and lighter. Circ_0098823 silencing suppressed DNM1L expression, a key molecule for fission, which enhanced proliferation, migration and invasion, and inhibited apoptosis of HCC cell. IGF2BP3 was a binding protein of circ_0098823. The expression and mRNA stability of DNM1L were down-regulated by IGF2BP3 knockdown. IGF2BP3 knockdown significantly alleviated the excessive migration, invasion and apoptosis of HCC cells caused by circ_0098823 overexpression. This study uncovered a novel circ_0098823 with tumor-promoting effect, and the mechanism by which circ_0098823 participates in HCC progression through IGF2BP3-guided DNM1L. Our study broadens molecular understanding of HCC progression.

Atrachinenins D-S, novel meroterpenoids with geranyl hydroquinone moiety from Atractylodes chinensis by the LC/MS-based molecular decoy and targeted isolation.

To mine fascinating molecules from the rhizomes of Atractylodes chinensis, the known molecular formula of atrachinenin A was used as a bait to search LC-HRMS data in different subfractions. Sixteen new meroterpenoids, atrachinenins D-S (1-16) including three unprecedented carbon skeletons (1-5) and eleven new oxygen-bridged hybrids (6-16) were obtained by the targeted isolation. Their structures and absolute configurations were elucidated by the spectroscopic data and electronic circular dichroism (ECD) calculations. The isolated compounds were evaluated for their inhibitory activity of NO production and compounds 1, 4, 8, and 13 showed moderate anti-inflammatory activity. The proposed biosynthetic pathways of 1-5 were also discussed.

SPP1 is a prognostic related biomarker and correlated with tumor-infiltrating immune cells in ovarian cancer.

BACKGROUND: Secreted phosphoprotein 1 (SPP1) plays a vital role in tumor progression of multiple cancer types However, it still awaits further exploration whether SPP1 is a bystander or an actual player in the modulation of immune infiltration in ovarian cancer. METHODS: In this study, the expression level of SPP1 was identified by Oncomine, GEPIA and TIMER databases, and the result of SPP1 immumohistochemical staining was acquired by The HPA database. The impact of SPP1 expression level on the clinical outcome of ovarian cancer patients were evaluated via Kaplan-Meier Plotter and PrognoScan dataset. Immune infiltration analyses were conducted using TIMER and TISIDB dataset. In addition, Functional enrichment analyses were performed with Metascape and GeneMANIA database. To verify these findings from the public database, the results were validated in a cohort of ovarian cancer patients. RESULTS: SPP1 was found to be overexpressed in ovarian tumor tissues and high SPP1 expression was correlated with shorter survivals. Notably, SPP1 expression was positively correlated with infiltrating levels of CD4 + T cells, CD8 + T cells, macrophages, neutrophils, and dendritic cells. Furthermore, SPP1 expression level showed strong correlation with diverse immune cells in ovarian cancer. Of note, functional enrichment analysis suggested that SPP1 was strongly correlated with immune response. CONCLUSIONS: These findings imply that SPP1 is correlated with prognosis and immune cell infiltrating, offering a new potential immunotherapeutic target in ovarian cancer. TRIAL REGISTRATION: Not applicable.

Effects of YM155 on the proliferation and apoptosis of pulmonary artery smooth muscle cells in a rat model of high pulmonary blood flow-induced pulmonary arterial hypertension.

INTRODUCTION: Proliferation and apoptosis of pulmonary artery smooth muscle cells (PASMCs) play an important role in the occurrence and development of pulmonary arterial hypertension (PAH). The purpose of this study was to investigate the effects of survivin inhibitor YM155 on the proliferation and apoptosis of PASMCs in rats with PAH induced by high pulmonary blood flow. METHODS: Thirty male Sprague-Dawley (SD) rats were randomly divided into control, model, and YM155 intervention groups. A rat model of PAH induced by high pulmonary blood flow was established, and it was confirmed by assessments of right-ventricular pressure (RVP) and right ventricular hypertrophy index (RVHI). Immunohistochemical staining and western blot analysis were used to detect the expression of survivin, and the proliferation and apoptosis of PASMCs. Lastly, the effects of in vivo treatment of YM155 were tested. RESULTS: The increased expression of survivin mRNA and protein were observed in the model group, accompanied by pulmonary arteriolar wall thickening, lumen stenosis, and perivascular inflammatory cell infiltration. Elevated expression of survivin and pulmonary vascular remodeling were significantly mitigated after YM155 treatment. Specifically, the YM155 intervention group had a significantly lower PASMC proliferation rate and a higher PASMC apoptotic rate. CONCLUSION: YM155 suppressed PASMC proliferation and promoted PASMC apoptosis by inhibiting survivin expression and thereby reducing pulmonary vascular remodeling in high pulmonary blood flow-induced PAH in vivo.

ROR1 and ROR2 expression in pancreatic cancer.

BACKGROUND: The Wnt receptors ROR1 and ROR2 are generating increased interest as cancer therapeutic targets but remain understudied in pancreatic ductal adenocarcinoma (PDAC). Compared to canonical Wnt/ ß-catenin signalling, the role of noncanonical Wnt signalling in PDAC remains largely unknown. Only one study has investigated the prognostic significance of the noncanonical Wnt signalling receptor, ROR2 in PDAC. No studies have investigated the prognostic role of ROR1 in PDAC. METHODS: Here, we performed analysis of ROR1 and ROR2 mRNA expression in three publicly available datasets ICGC-PACA-AU (n = 81), TCGA-PAAD (n = 150) and CPTAC-PDAC (n = 137). ROR1 and ROR2 protein expression from the CPTAC-PDAC discovery cohort were also analysed. Immunohistochemistry (IHC) using the validated anti ROR1 monoclonal antibody (4A5) was performed on the Australian Pancreatic Cancer Genome Initiative (APGI) cohort of PDAC samples (n = 152). Association between ROR1 cytoplasmic staining intensity and clinicopathological parameters including stage, grade and overall survival (OS) was investigated. RESULTS: High ROR1 mRNA expression levels correlated with a favourable OS outcome in all of the ICGC-PACA-AU, TCGA-PAAD and CPTAC-PDAC cohorts. ROR1 protein expression was not associated with stage, grade or OS in the APGI cohort. CONCLUSION: ROR1 and ROR2 have potential as prognostic markers when measured at the mRNA level in PDAC. Our IHC cohort did not support ROR1 protein expression in predicting OS, and highlighted the discrepancy of prognostic biomarkers when measured by MS, IHC and RNAseq.

Cell-free DNA is abundant in ascites and represents a liquid biopsy of ovarian cancer.

OBJECTIVE: Malignant ascites is a common clinical feature of ovarian cancer and represents a readily accessible sample of tumour cells and tumour DNA. This study aimed to characterise the cell-free DNA (cfDNA) in ascites in terms of its size profile, stability and cell-free tumour DNA (cftDNA) content. METHODS: Cell spheroids, loose cells and cell-free fluid was collected from ascites from 18 patients with ovarian cancer. cfDNA was isolated and assessed for size by electrophoresis, concentration by fluorometry,cftDNA content by methylation specific qPCR of HOXA9 and IFFO1 promoter regions and by targeted sequencing. Stability was assessed after ascites fluid was stored at 4 °C for 24 and 72 h before fractionating. RESULTS: The concentration of cfDNA in ascites ranged from 6.6 to 300 ng/mL. cfDNA size distribution resembled blood plasma-derived cfDNA, with major peaks corresponding to mono- and di-nucleosome DNA fragments. High molecular weight cfDNA was observed in 7 of 18 patients and appeared to be associated with extracellular vesicles. IFFO1 and HOXA9 methylation was proportionately higher in cfDNA than spheroid- and loose-cell fractions and was not observed in healthy primary cells. Variant allele frequency was highest in cfDNA compared to single cells and spheroids from ascites. Though cancer cell numbers in ascites declined to near zero in recurrent ascites from one patient undertaking chemotherapy, cftDNA could still be sampled. cfDNA size, concentration and tumour content was stable over 72 h. CONCLUSION: cfDNA in ovarian cancer ascites demonstrates inter-patient variability, yet is consistently a rich source of cftDNA, which is a stable substrate. This supports the wider clinical use of ascites in the molecular analysis of ovarian cancer.

Leaf spot disease of Orychophragmus violaceus caused by Alternaria tenuissima in China.

Orychophragmus violaceus (L.) O. E. Schulz, also called February orchid or Chinese violet cress, belongs to the Brassicaceae family and is widely cultivated as a green manure and garden plant in China. During the prolonged rainy period in August 2020, leaf spot disease of O. violaceus was observed in the garden of Northeast Agricultural University, Harbin, Heilongjiang province. One week after the rainy days, the disease became more serious and the disease incidence ultimately reached approximately 80%. The disease symptoms began as small brown spots on the leaves, and gradually expanded to irregular or circular spots. As the disease progressed, spots became withered with grayish-white centers and surrounded by dark brown margins. Later on, the centers collapsed into holes. For severely affected plants, the spots coalesced into large necrotic areas and resulted in premature defoliation. No conidiophores or hyphae were present, and disease symptoms were not observed on other tissues of O. violaceus. To isolate the pathogen, ten leaves with typical symptoms were collected from different individual plants. Small square leaf pieces (5×5 mm) were excised from the junction of diseased and healthy tissues, disinfected in 75% ethanol solution for 1 min, rinsed in sterile distilled water, and then transferred to Petri dishes (9 cm in diameter) containing potato dextrose agar (PDA). After 3 days of incubation at 25 oC in darkness, newly grown-out mycelia were transferred onto fresh PDA and purified by single-spore isolation. Nine fungal isolates (NEAU-1 ~ NEAU-9) showing similar morphological characteristics were obtained and no other fungi were isolated. The isolation frequency from the leaves was almost 90%. On PDA plates, all colonies were grey-white with loose and cottony aerial hyphae, and then turned olive-green and eventually brown with grey-white margins. The fungus formed pale brown conidiophores with sparsely branched chains on potato carrot agar (PCA) plates after incubation at 25 oC in darkness for 7 days. Conidia were ellipsoidal or ovoid, light brown, and ranged from 18.4 to 59.1 × 9.2 to 22.3 µm in size, with zero to two longitudinal septa and one to five transverse septa and with a cylindrical light brown beak (n = 50). Based on the cultural and morphological characteristics, the fungus was tentatively identified as Alternaria tenuissima (Simmons 2007). Genomic DNA was extracted from the mycelia of five selected isolates (NEAU-1 ~ NEAU-5). The internal transcribed spacer region (ITS) was amplified and sequenced using primers ITS1/ITS4 (White et al., 1990). Blast analysis demonstrated that these five isolates had the same ITS sequence, and the ITS sequence of representative strain NEAU-5 (GenBank accession No. MW139354) showed 100% identity with the type strains of Alternaria alternata CBS916.96 and Alternaria tenuissima CBS918.96. Furthermore, the translation elongation factor 1-α gene (TEF), RNA polymerase II second largest subunit (RPB2), and glyceraldehyde 3-phosphate dehydrogenase (GPD) of representative strain NEAU-5 were amplified and sequenced using primers EF1-728F/EF1-986R (Carbone and Kohn 1999), RPB2-5F2/RPB2-5R (Sung et al., 2007), and Gpd1/Gpd2 (Berbee et al., 1999), respectively. The sequences of RPB2, GPD, and TEF of strain NEAU-5 were submitted to GenBank with accession numbers of MW401634, MW165223, and MW165221, respectively. Phylogenetic trees based on ITS, RPB2, GPD, and TEF were constructed with the neighbour-joining and maximum-likelihood algorithms using MEGA software version 7.0. The results demonstrated that strain NEAU-5 formed a robust clade with A. tenuissima CBS918.96 (supported by 99% and 96% bootstrap values) in the neighbour-joining and maximum-likelihood trees. As mentioned above, strain NEAU-5 produced seldomly branched conidial chains on PCA plates. The pattern is consistent with that of A. tenuissima (Kunze) Wiltshire, but distinct from that of A. alternata which could produce abundant secondary ramification (Simmons 2007). Thus, strain NEAU-5 was identified as A. tenuissima based on its morphology and phylogeny. Pathogenicity tests were carried out by inoculating five unwounded leaves with a conidial suspension of strain NEAU-5 (approximately 106 conidia/ml) on five different healthy plants cultivated in garden, and an equal number of leaves on the same plants inoculated with sterilized ddH2O served as negative controls. Inoculated and control leaves were covered with clear plastic bags for 3 days. After 6 days, small brown and irregular or circular spots were observed on all leaves inoculated with conidial suspension, while no such symptoms were observed in the control. The tests were repeated three times. Furthermore, the pathogenicity tests were also performed using 2-month-old potted plants in a growth chamber (28 oC, 90% relative humidity, 12 h/12 h light/dark) with two repetitions. Five healthy plants were inoculated by spraying 20 ml of a conidial suspension of strain NEAU-5 (approximately 106 conidia/ml) onto unwounded leaves. Five other healthy plants were inoculated with sterilized ddH2O as controls. After 7 days, similar symptoms were observed on leaves inoculated with strain NEAU-5, whereas no symptoms were observed in the control. The pathogen was reisolated from the inoculated leaves and identified as A. tenuissima by morphological and molecular methods. In all pathogenicity tests, A. tenuissima could successfully infect unwounded leaves of O. violaceus, indicating a direct interaction between leaves and A. tenuissima. It is known that high humidity and fairly high temperatures can favor the epidemics of Alternaria leaf spot (Yang et al., 2018), and this may explain why severe leaf spot disease of O. violaceus was observed after prolonged rain. Previously, it has been reported that Alternaria brassicicola and Alternaria japonica could cause leaf blight and spot disease on O. violaceus in Hebei and Jiangsu Provinces, China, respectively (Guo et al., 2019; Sein et al., 2020). Although these pathogens could lead to similar disease symptoms on the leaves of O. violaceus, it is easy to distinguish them by the morphological characteristics of conidiophores and ITS gene sequences. To our knowledge, this is the first report of A. tenuissima causing leaf spot disease of O. violaceus in China.

Solvent-Free Nanofabrication Based on Ice-Assisted Electron-Beam Lithography.

Advances in electron-beam lithography (EBL) have fostered the prominent development of functional micro/nanodevices. Nonetheless, traditional EBL is predominantly applicable to large-area planar substrates and often suffers from chemical contamination and complex processes for handling resists. This paper reports a streamlined and ecofriendly approach to implement e-beam patterning on arbitrary shaped substrates, exemplified by solvent-free nanofabrication on optical fibers. The procedure starts with the vapor deposition of water ice as an electron resist and ends in the sublimation of the ice followed by a "blow-off" process. Without damage and contamination from chemical solvents, delicate nanostructures and quasi-3D structures are easily created. A refractive index sensor is further demonstrated by decorating plasmonic nanodisk arrays on the end face of a single-mode fiber. Our study provides a fresh perspective in EBL-based processing, and more exciting research exceeding the limits of traditional approaches is expected.

TMEM16A Regulates Pulmonary Arterial Smooth Muscle Cells Proliferation via p38MAPK/ERK Pathway in High Pulmonary Blood Flow-Induced Pulmonary Arterial Hypertension.

OBJECTIVE: Pulmonary arterial hypertension (PAH) is a complex disease of the small pulmonary arteries that is mainly characterized by vascular remodeling. It has been demonstrated that excessive proliferation of pulmonary arterial smooth muscle cells (PASMCs) plays a pivotal role in vascular remodeling during PAH. The present study was undertaken to explore the role of TMEM16A in regulating PASMCs proliferation in high pulmonary blood flow-induced PAH. METHODS: Aortocaval shunt surgery was undertaken to establish an animal model. Pulmonary artery pressure and pulmonary vascular structure remodeling (PVSR) were tested. Immunohistochemical staining and Western blot were performed to investigate the expression of TMEM16A. The proliferation of PASMCs was tested by the MTT assay. After treating PASMCs with TMEM16A-siRNA, the expression of proliferating cell nuclear antigen (PCNA), phosphorylated p38 mitogen-activated protein kinase (p-p38MAPK), and phosphorylated extracellular signal-regulated kinase (p-ERK) signaling in PASMCs were tested. RESULTS: PAH and PVSR developed 11 weeks postoperation. Elevated expression of TMEM16A accompanied by high expression of PCNA in pulmonary arteries of the shunt group was observed. The increased proliferation of PASMCs and increased expression of TMEM16A and PCNA, along with activated p-p38MAPK and p-ERK signaling in PASMCs of the shunt group, were all attenuated by siRNA-specific TMEM16A knockdown. CONCLUSION: TMEM16A regulates PASMCs proliferation in high pulmonary blood flow-induced PAH, and the p38MAPK/ERK signaling pathway is probably involved.

Microbiota Analysis of Eggshells in Different Areas and During Different Storage Time by Non-cultural Methods.

This study is to investigate and characterize the microbiota composition on eggshells from 3 different areas of Shaanxi province (Yulin, Hanzhong and Xi'an). The eggs were stored at 25 °C for 56 days and bacterial samples were collected from eggshells on day 0, 14, 28, 42 and 56. Denaturing gradient gel electrophoresis and high-throughput sequencing of 16S rRNA hypervariable region V3-V4 were performed. Alpha diversity was applied for analyzing the diversity of samples through 6 indices, including Observed-species, Chao1, Shannon, Simpson, ACE and Good's-coverage. Beta diversity was used to study the similarities or differences in the community composition of the samples. Totally, 36 phyla and 595 genera were classified by 16S rRNA gene sequencing. The composition of the microbial communities of different regions was quite different. Firmicutes (33-38% of total phyla) and Actinobacteria (36-61% of total phyla) were the most abundant phyla in all three regions. Proteobacteria were relatively more abundant (about 18% of total phyla) on eggs from Hanzhong. During storage time, the microbial communities mainly changed from Firmicutes to Actinobacteria on eggs from Yulin and Xi'an. Lactobacillus, Kocuria and Streptomyces were much higher at the genus level. Spoilage bacteria Staphylococcus, Streptococcus, Pseudomonas and Enterococcus were detected at the genus level. Campylobacter jejuni (< 1% of total bacteria), which might be related to human illness, was also detected. In conclusion, the structure, abundance, and composition of microbiota on eggshells differ among areas. The microbiota changed regularly during storage time. The current study may offer a new insight into bacterial species on eggshells.

Arthrobacter celericrescens sp. nov., isolated from forest soil.

A novel bacterial strain, designated NEAU-SA2T, was isolated from forest soil collected from the Zhangjiajie city, Hunan Province, PR China and characterised using a polyphasic approach. The cells were aerobic, Gram-stain-positive, non-flagellated and rod-coccus-shaped. The strain grew optimally at 28 °C, pH 7.0 and with 0 % NaCl (w/v). Phylogenetic analysis based on the 16S rRNA gene sequence indicated that the organism should be assigned to the genus Arthrobacter and was closely related to Arthrobacter cupressi DSM 24664T (98.89 %) and Arthrobacter silvisoli CCTCC AB 2017271T (98.41 %), which was further confirmed by multilocus sequence analysis. The major cellular fatty acids were anteiso-C15 : 0, anteiso-C17 : 0 and C16 : 0; MK-9(H2) was the predominant respiratory quinone. The polar lipids comprised diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol and an unidentified glycolipid. The peptidoglycan type was A3α, and the cell-wall sugars were glucose and galactose. The genomic G+C content of strain NEAU-SA2T was 67.04 mol%. The average nucleotide identity values between NEAU-SA2T and A. cupressi DSM 24664T and A. silvisoli CCTCC AB 2017271T were 88.57-90.94 %. The digital DNA-DNA hybridisation values between strain NEAU-SA2T and its most closely related species were 37.00 and 41.10 %, respectively, again indicating that they belong to different taxa. Therefore, strain NEAU-SA2T represents a novel species of the genus Arthrobacter, for which the name Arthrobacter celericrescens sp. nov. is proposed. The type strain is NEAU-SA2T (=DSM 106718T=CCTCC AB 2017272T).

Trinickia diaoshuihuensis sp. nov., a plant growth promoting bacterium isolated from soil.

A novel Gram-stain-negative, aerobic, rod-shaped plant growth promoting bacterium, NEAU-SY24T, was isolated from soil in Diaoshuihu, Heilongjiang, China. The isolate grew at temperatures 10-40 °C (optimum, 30 °C), pH 5-8 (optimum, pH 6) and in the presence of up to 1 % (w/v) NaCl, although NaCl was not required for growth. Phylogenetic analysis based on the 16S rRNA gene sequence indicated that strain NEAU-SY24T belonged to the genus Trinickia and was closely related to Trinickia dabaoshanensis NRRL B-59553T (99.16 % similarity) and Trinickia soli DSM 18235T (99.11 %). The average nucleotide identity values between NEAU-SY24T and its most closely related species were 79.30-87.09 %. The in silico DNA-DNA hybridization values between NEAU-SY24T and T. dabaoshanensis NRRL B-59553T and T. soli DSM 18235T were 29.30 and 24.00 %, respectively, again indicating they belong to different taxa. The genomic DNA G+C content was 63.3 mol%. The major cellular fatty acids were C17 : 0cyclo, C18 : 1ω7c, C16 : 0, summed feature 2 (comprising C14 : 0 3-OH and/or C16 : 1iso I) and C16 : 0 3-OH. The predominant respiratory quinone was Q-8 and the whole-cell sugars contained ribose, glucose and galactose. The polar lipids consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, hydroxyphosphatidylethanolamine and two unidentified aminolipids. On the basis of morphological, physiological, biochemical and chemotaxonomic analysis, strain NEAU-SY24T was classified as a novel species in the genus Trinickia, for which the name Trinickiadiaoshuihuensis sp. nov. is proposed. The type strain is NEAU-SY24T (=DSM 106065T=CCTCC AA 2018003T).

A low-fat diet up-regulates expression of fatty acid taste receptor gene FFAR4 in fungiform papillae in humans: a co-twin randomised controlled trial.

Fatty acid taste (FAT) perception is involved in the regulation of dietary fat intake, where impaired FAT is associated with increased fatty food intake. There are a number of FAT receptors identified on human taste cells that are potentially responsible for FAT perception. Manipulating dietary fat intake, and in turn FAT perception, would elucidate the receptors that are associated with long-term regulation of FAT perception. The present study aimed to assess associations between diet-mediated changes to FAT receptors and FAT perception in humans. A co-twin randomised controlled trial was conducted, where each matching twin within a pair were randomly allocated to either an 8-week low-fat (LF; <20 % energy fat) or an 8-week high-fat (HF; >35 % energy fat) diet. At baseline and week 8, fungiform papillae were biopsied in the fasted state and FAT receptor gene expressions (cluster of differentiation 36 (CD36), free fatty acid receptor 2 (FFAR2), FFAR4, G protein-coupled receptor 84 (GPR84) and a delayed rectifying K+ channel (K+ voltage-gated channel subfamily A member 2; KCNA2)) were measured using RT-PCR; and FAT threshold (FATT) was assessed using three-alternate forced choice methodology. Linear mixed models were fitted, adjusting for correlation between co-twins. Intake was compliant with the study design, with the LF and HF groups consuming 14·8 and 39·9 % energy from fat, respectively. Expression of FFAR4 increased by 38 % in the LF group (P = 0·023; time-diet interaction P = 0·063). ΔFFAR4 (Δ, week 8-baseline) was associated with Δfat intake (g) ( = -159·4; P < 0·001) and ΔFATT ( = -8·8; P = 0·016). In summary, FFAR4 is involved in long-term diet-mediated changes to FAT perception. Manipulating dietary fat intake, and therefore FFAR4 expression, might aid in reducing taste-mediated passive overconsumption of fatty foods.

Three-Dimensional in Situ Electron-Beam Lithography Using Water Ice.

Three-dimensional (3D) nanofabrication techniques are of paramount importance in nanoscience and nanotechnology because they are prerequisites to realizing complex, compact, and functional 3D nanodevices. Although several 3D nanofabrication methods have been proposed and developed in recent years, it is still a formidable challenge to achieve a balance among resolution, accuracy, simplicity, and adaptability. Here, we propose a 3D nanofabrication method based on electron-beam lithography using ice resists (iEBL) and fabricate 3D nanostructures by stacking layered structures and those with dose-modulated exposure, respectively. The entire process of 3D nanofabrication is realized in one vacuum system by skipping the spin-coating and developing steps required for commonly used resists. This needs far fewer processing steps and is contamination-free compared with conventional methods. With in situ alignment and correction in the iEBL process, a pattern resolution of 20 nm and an alignment error below 100 nm can be steadily achieved. This 3D nanofabrication technique using ice thus shows great potential in the fabrication of complicated 3D nanodevices.

Composition and Physicochemical Properties of Three Chinese Yam (Dioscorea opposita Thunb.) Starches: A Comparison Study.

The aim of this work was to compare the composition and physicochemical properties (SEM, XRD, solubility, swelling power, paste clarity, retrogradation, freeze-thaw stability, thermal property, and pasting property) of three Chinese yam (Dioscorea opposita Thunb.) starches (CYYS-1, CYYS-2, and CYYS-3) in Yunlong town, Haikou, Hainan Province, China. Our results show that all the CYYS gave a typical C-type X-ray diffraction pattern. The swelling power of CYYS varied from 10.79% to 30.34%, whereas solubility index was in the range of 7.84-4.55%. The freeze-thaw stability of each CYYS showed a contrary tendency with its amylose content. In addition, CYYS-3 showed the highest To (81.1 °C), Tp (84.8 °C), Tc (91.2 °C), and ΔH (14.1 J/g). The pasting temperature of CYYS-1 increased significantly with sucrose addition. NaCl could inhibit the swelling power of CYYS. There were significant decreases in pasting temperature and pasting time of CYYS when pH decreased.

Arthrobacter silvisoli sp. nov., isolated from forest soil.

A novel Gram-stain-positive, strictly aerobic strain, NEAU-SA1T, which showed a rod-coccus growth life cycle, was isolated from forest soil from Zhangjiajie, Hunan Province, China. The isolate grew at 10-40 °C (optimum 28 °C), at pH 5.0-10.0 (optimum pH 7.0) and in the presence of up to 5 % (w/v) NaCl, although NaCl was not required for growth. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain NEAU-SA1T belonged to the genus Arthrobacter and was closely related to Arthrobacter cupressi DSM 24664T (98.1 % similarity). Average nucleotide identity values between NEAU-SA1T and A. cupressi DSM 24664T were 88.91 and 87.41 % by ANIm and ANIb analysis, respectively. The in silico DNA-DNA hybridization value between strain NEAU-SA1T and A. cupressi DSM 24664T was 34.20 %, again indicating they belong to different taxa. The genomic DNA G+C content was 66.74 mol%. The major cellular fatty acids (>10 %) were anteiso-C15 : 0, anteiso-C17 : 0 and iso-C16 : 0. The polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol and two unidentified glycolipids. The predominant menaquinone was MK-9(H2). The peptidoglycan type was A3α with an interpeptide bridge comprising l-Lys and l-Ala. Glucose, ribose and galactose were the whole-cell sugars. On the basis of morphological, physiological, biochemical and chemotaxonomic analysis, strain NEAU-SA1T was classified as representing a novel species in the genus Arthrobacter, for which the name Arthrobacter silvisoli sp. nov. is proposed. The type strain is NEAU-SA1T (=DSM 106716T=CCTCC AB 2017271T).

Expression of the candidate fat taste receptors in human fungiform papillae and the association with fat taste function.

Significant experimental evidence supports fat as a taste modality; however, the associated peripheral mechanisms are not well established. Several candidate taste receptors have been identified, but their expression pattern and potential functions in human fungiform papillae remain unknown. The aim of this study is to identify the fat taste candidate receptors and ion channels that were expressed in human fungiform taste buds and their association with oral sensory of fatty acids. For the expression analysis, quantitative RT-PCR (qRT-PCR) from RNA extracted from human fungiform papillae samples was used to determine the expression of candidate fatty acid receptors and ion channels. Western blotting analysis was used to confirm the presence of the proteins in fungiform papillae. Immunohistochemistry analysis was used to localise the expressed receptors or ion channels in the taste buds of fungiform papillae. The correlation study was analysed between the expression level of the expressed fat taste receptors or ion channels indicated by qRT-PCR and fat taste threshold, liking of fatty food and fat intake. As a result, qRT-PCR and western blotting indicated that mRNA and protein of CD36, FFAR4, FFAR2, GPR84 and delayed rectifying K+ channels are expressed in human fungiform taste buds. The expression level of CD36 was associated with the liking difference score (R -0·567, ß=-0·04, P=0·04) between high-fat and low-fat food and FFAR2 was associated with total fat intake (ρ=-0·535, ß=-0·01, P=0·003) and saturated fat intake (ρ=-0·641, ß=-0·02, P=0·008).

A c-Myc/miR-17-5p feedback loop regulates metastasis and invasion of hepatocellular carcinoma.

The molecular mechanisms that control metastasis of hepatocellular cancer (HCC) are still poorly understood. It has been determined that microRNA (miRNA) expression has tissue and cell specific, and decreased expression of specific miRNA could induce tumor genesis or metastasis. In this study, we identified that miR-17-5p was expressed lower in high metastatic capability HCC cell lines HCCLM3 and MHCC97H than low metastatic HCC cell line HepG2 by real-time (RT)-PCR. Restoration of miR-17-5p could significantly repress the invasiveness and metastasis of MHCC97H cell line. Furthermore, we validated c-Myc as a downstream and functional target of miR-17-5p using luciferase reporter assay. Immunohistochemical assay revealed that the expression of c-Myc protein levels was significantly increased in cancerous tissues compared with para-tumor tissues. After clinical data analysis, we observed that the higher level of c-Myc was significantly associated with a reduced overall survival (p = 0.0209). Consistent with previous research, we also demonstrated that c-Myc could upregulate the expression of miR-17-5p. Taken together, our data indicated that there is a regulatory feedback loop between miR-17-5p and c-Myc, in which miR-17-5p could suppress some of the distinguishing features, invasion, and metastasis, of oncogenic c-Myc in HCC cells, and meanwhile, miR-17-5p is upregulated by c-Myc role as a transcription factor, although further studies are still needed.

Malic enzyme 1 induces epithelial-mesenchymal transition and indicates poor prognosis in hepatocellular carcinoma.

Malic enzyme 1 (ME1) links the glycolytic and citric acid cycles and is important for NADPH production, glutamine metabolism, and lipogenesis. Recently, its deregulation has been implicated in the progression of various cancers. However, the role of ME1 in the progression of hepatocellular carcinoma (HCC) remains unclear. In this study, we utilized short hairpin RNA-mediated gene silencing to investigate the biological effects of ME1 depletion in HCC and determined its prognostic significance in HCC. ME1 expression was examined by real-time (RT)-PCR and Western blot using five HCC cell lines and one normal liver cell line. We used polyethylenimine nanoparticles to deliver a short hairpin RNA to induce cessation of ME1 expression in HCC cells. Changes in NADPH production and reactive oxygen species (ROS) production were studied. Metastatic potentials of HCC cells were evaluated in vitro. Furthermore, we evaluated the protein level of ME1 in para-tumor and cancerous tissues of 65 HCC patients with detailed clinical, pathological, and clinical follow-up data. Patients' survivals were further assessed as well. Upregulated ME1 expression was observed in HCC cell lines. Downregulation of ME1 attenuated NADPH production and stimulated ROS production. Silencing ME1 was noted to inhibit migratory and invasive properties of HCC cells by inducing the E-cadherin expression and decreasing of N-cadherin and vimentin expression in a ROS-dependent pathway. Overexpression of ME1 was observed in a major fraction of HCC samples. Higher level of ME1 in tumors was significantly associated with reduced overall survival (Kaplan-Meier analysis, P = 0.024) and reduced progression-free survival (Kaplan-Meier analysis, P = 0.011). Inhibition of ME1 expression decreases HCC metastasis via suppression of epithelial-mesenchymal transition (EMT) processes in ROS-induced pathways. ME1 overexpression associates with unfavorable prognoses in patients with HCC, suggesting that ME1 is a poor prognostic predictor of hepatocellular carcinoma.

Radical surgery versus standard surgery for primary cytoreduction of bulky stage IIIC and IV ovarian cancer: an observational study.

BACKGROUND: The aim of this study was to evaluate the survival benefit of radical surgery with additional extensive upper abdominal procedures (EUAS) for the treatment of stage IIIC and IV ovarian cancer with bulky upper abdominal disease (UAD). METHODS: An observational study was conducted between 2009 and 2012 involving two different surgical teams. Team A was composed of the "believers" in EUAS and Team B the "non-believers" in EUAS. Patients were divided into a radical surgery group (EUAS group) or a standard surgery group (non-EUAS group) according to whether or not they had received EUAS. All patients underwent primary cytoreductive surgery with the goal of optimal debulking (≤ 1 cm); this was reviewed in the pelvis, middle abdomen, and upper abdomen. The baseline for the two groups was optimal cytoreduction in both the pelvis and middle abdomen. Progression-free survival (PFS) was evaluated. RESULTS: Radical surgery was performed in 70.7% (82/116) and 12.7% (30/237) of the patients by Teams A and B, respectively. The study groups had similar clinicopathologic characteristics. The median PFS and OS were significantly improved in the radical surgery group, compared with standard surgery groups (PFS: 19.5 vs. 13.3 months, HR: 0.61; 95% CI: 0.46-0.80, P < 0.001; OS: not reached vs. 39.3 months, HR: 0.47; 95% CI: 0.30-0.72, P < 0.001). Positive predictors of complete cytoreduction were treatment with neoadjuvant chemotherapy, improved American Society of Anesthesiologists performance status, and the absence of bowel mesenteric carcinomatosis. CONCLUSIONS: Radical surgery lengthens the PFS and overall survival times of ovarian cancer patients with bulky UAD. However, a well-designed randomized trial is needed to confirm the present results.