Trained immunity induced by high-salt diet impedes stroke recovery.

A high-salt diet (HSD) elicits sustained sterile inflammation and worsens tissue injury. However, how this occurs after stroke, a leading cause of morbidity and mortality, remains unknown. Here, we report that HSD impairs long-term brain recovery after intracerebral hemorrhage, a severe form of stroke, despite salt withdrawal prior to the injury. Mechanistically, HSD induces innate immune priming and training in hematopoietic stem and progenitor cells (HSPCs) by downregulation of NR4a family and mitochondrial oxidative phosphorylation. This training compromises alternative activation of monocyte-derived macrophages (MDMs) without altering the initial inflammatory responses of the stroke brain. Healthy mice transplanted with bone marrow from HSD-fed mice retain signatures of reduced MDM reparative functions, further confirming a persistent form of innate immune memory that originates in the bone marrow. Loss of NR4a1 in macrophages recapitulates HSD-induced negative impacts on stroke outcomes while gain of NR4a1 enables stroke recovery in HSD animals. Together, we provide the first evidence that links HSD-induced innate immune memory to the acquisition of persistent dysregulated inflammatory responses and unveils NR4a1 as a potential therapeutic target.

Twnbiome: a public database of the healthy Taiwanese gut microbiome.

With new advances in next generation sequencing (NGS) technology at reduced costs, research on bacterial genomes in the environment has become affordable. Compared to traditional methods, NGS provides high-throughput sequencing reads and the ability to identify many species in the microbiome that were previously unknown. Numerous bioinformatics tools and algorithms have been developed to conduct such analyses. However, in order to obtain biologically meaningful results, the researcher must select the proper tools and combine them to construct an efficient pipeline. This complex procedure may include tens of tools, each of which require correct parameter settings. Furthermore, an NGS data analysis involves multiple series of command-line tools and requires extensive computational resources, which imposes a high barrier for biologists and clinicians to conduct NGS analysis and even interpret their own data. Therefore, we established a public gut microbiome database, which we call Twnbiome, created using healthy subjects from Taiwan, with the goal of enabling microbiota research for the Taiwanese population. Twnbiome provides users with a baseline gut microbiome panel from a healthy Taiwanese cohort, which can be utilized as a reference for conducting case-control studies for a variety of diseases. It is an interactive, informative, and user-friendly database. Twnbiome additionally offers an analysis pipeline, where users can upload their data and download analyzed results. Twnbiome offers an online database which non-bioinformatics users such as clinicians and doctors can not only utilize to access a control set of data, but also analyze raw data with a few easy clicks. All results are customizable with ready-made plots and easily downloadable tables. Database URL: http://twnbiome.cgm.ntu.edu.tw/ .

High-performance deep learning pipeline predicts individuals in mixtures of DNA using sequencing data.

In this study, we proposed a deep learning (DL) model for classifying individuals from mixtures of DNA samples using 27 short tandem repeats and 94 single nucleotide polymorphisms obtained through massively parallel sequencing protocol. The model was trained/tested/validated with sequenced data from 6 individuals and then evaluated using mixtures from forensic DNA samples. The model successfully identified both the major and the minor contributors with 100% accuracy for 90 DNA mixtures, that were manually prepared by mixing sequence reads of 3 individuals at different ratios. Furthermore, the model identified 100% of the major contributors and 50-80% of the minor contributors in 20 two-sample external-mixed-samples at ratios of 1:39 and 1:9, respectively. To further demonstrate the versatility and applicability of the pipeline, we tested it on whole exome sequence data to classify subtypes of 20 breast cancer patients and achieved an area under curve of 0.85. Overall, we present, for the first time, a complete pipeline, including sequencing data processing steps and DL steps, that is applicable across different NGS platforms. We also introduced a sliding window approach, to overcome the sequence length variation problem of sequencing data, and demonstrate that it improves the model performance dramatically.

IL-1ß-driven neutrophilia preserves antibacterial defense in the absence of the kinase IKKß.

Transcription factor NF-κB and its activating kinase IKKß are associated with inflammation and are believed to be critical for innate immunity. Despite the likelihood of immune suppression, pharmacological blockade of IKKß-NF-κB has been considered as a therapeutic strategy. However, we found neutrophilia in mice with inducible deletion of IKKß (Ikkß(Δ) mice). These mice had hyperproliferative granulocyte-macrophage progenitors and pregranulocytes and a prolonged lifespan of mature neutrophils that correlated with the induction of genes encoding prosurvival molecules. Deletion of interleukin 1 receptor 1 (IL-1R1) in Ikkß(Δ) mice normalized blood cellularity and prevented neutrophil-driven inflammation. However, Ikkß(Δ)Il1r1(-/-) mice, unlike Ikkß(Δ) mice, were highly susceptible to bacterial infection, which indicated that signaling via IKKß-NF-κB or IL-1R1 can maintain antimicrobial defenses in each other's absence, whereas inactivation of both pathways severely compromises innate immunity.

Hypoxia-responsive circular RNA circAAGAB reduces breast cancer malignancy by activating p38 MAPK and sponging miR-378 h.

BACKGROUND: Breast cancer is a prevalent disease in women, with high prevalence worldwide. The hypoxic microenvironment of solid tumors develops during the progress of carcinogenesis and leads to greater malignancy and treatment resistance. Recently, accumulating evidence indicates that non-coding RNAs, such as circular RNAs (circRNAs), play a pivotal role in altering cellular functions. However, the underlying mechanisms of circRNAs in breast cancer are still unclear. Therefore, the purpose of this study was to investigate the role of a tumor-suppressive circRNA, circAAGAB, in breast cancer by assuming down-regulation of circAAGAB under hypoxia and the properties of a tumor suppressor. METHODS: Firstly, circAAGAB was identified from expression profiling by next generation sequencing. Next, the stability of circAAGAB increased by interacting with the RNA binding protein FUS. Moreover, cellular and nuclear fractionation showed that most circAAGAB resided in the cytoplasm and that it up-regulated KIAA1522, NKX3-1, and JADE3 by sponging miR-378 h. Lastly, the functions of circAAGAB were explored by identifying its down-stream genes using Affymetrix microarrays and validated by in vitro assays. RESULTS: The results showed that circAAGAB reduced cell colony formation, cell migration, and signaling through p38 MAPK pathway, as well as increased radiosensitivity. CONCLUSION: These findings suggest that the oxygen-responsive circAAGAB acts as a tumor suppressor in breast cancer, and may contribute to the development of a more specific therapeutic regimen for breast cancer.

Multi-ethnic Imputation System (MI-System): A genotype imputation server for high-dimensional data.

OBJECTIVE: Genotype imputation is a commonly used technique that infers un-typed variants into a study's genotype data, allowing better identification of causal variants in disease studies. However, due to overrepresentation of Caucasian studies, there's a lack of understanding of genetic basis of health-outcomes in other ethnic populations. Therefore, facilitating imputation of missing key-predictor-variants that can potentially improve a risk health-outcome prediction model, specifically for Asian ancestry, is of utmost relevance. METHODS: We aimed to construct an imputation and analysis web-platform, that primarily facilitates, but is not limited to genotype imputation on East-Asians. The goal is to provide a collaborative imputation platform for researchers in the public domain towards rapidly and efficiently conducting accurate genotype imputation. RESULTS: We present an online genotype imputation platform, Multi-ethnic Imputation System (MI-System) (https://misystem.cgm.ntu.edu.tw/), that offers users 3 established pipelines, SHAPEIT2-IMPUTE2, SHAPEIT4-IMPUTE5, and Beagle5.1 for conducting imputation analyses. In addition to 1000 Genomes and Hapmap3, a new customized Taiwan Biobank (TWB) reference panel, specifically created for Taiwanese-Chinese ancestry is provided. MI-System further offers functions to create customized reference panels to be used for imputation, conduct quality control, split whole genome data into chromosomes, and convert genome builds. CONCLUSION: Users can upload their genotype data and perform imputation with minimum effort and resources. The utility functions further can be utilized to preprocess user uploaded data with easy clicks. MI-System potentially contributes to Asian-population genetics research, while eliminating the requirement for high performing computational resources and bioinformatics expertise. It will enable an increased pace of research and provide a knowledge-base for genetic carriers of complex diseases, therefore greatly enhancing patient-driven research. STATEMENT OF SIGNIFICANCE: Multi-ethnic Imputation System (MI-System), primarily facilitates, but is not limited to, imputation on East-Asians, through 3 established prephasing-imputation pipelines, SHAPEIT2-IMPUTE2, SHAPEIT4-IMPUTE5, and Beagle5.1, where users can upload their genotype data and perform imputation and other utility functions with minimum effort and resources. A new customized Taiwan Biobank (TWB) reference panel, specifically created for Taiwanese-Chinese ancestry is provided. Utility functions include (a) create customized reference panels, (b) conduct quality control, (c) split whole genome data into chromosomes, and (d) convert genome builds. Users can also combine 2 reference panels using the system and use combined panels as reference to conduct imputation using MI-System.

First Report of Fusarium asiaticum Causing Sheath Rot of Zizania latifolia in China.

Zizania latifolia is perennial plant, belonging to the rice tribe (Oryzeae) of the grass family Poaceae (Xu et al. 2020), which is also called jiaobai in China and commonly consumed as a vegetable crop. In 2022, a sheath rot occurred on Z. latifolia plants in Lishui, the Zhejiang Province of China. Symptoms occurred on the leaf sheath and initially showed as water-soaked chlorotic spots, later enlarging to irregular, elliptic, and elongated dark brown necrotic lesions. Later, lesions fused and extended to most of the leaf sheath leading to wilting. Almost 60% of the surveyed Z. latifolia plants in 100 hectare were affected. Diseased samples were collected for pathogen isolation. Symptomatic tissues were taken from the edge of lesions, sterilized for 10 s in 70% ethanol, then 2 min in 1% NaClO, washed three times with sterile distilled water, and placed on potato dextrose agar (PDA) at 26 °C in the dark. Fungal colonies displaying similar morphology were picked and purified by single spore isolation. In total, 8 isolates were obtained from 8 plant samples. When cultured on PDA, fungal colonies were white, gradually turning pale yellow with time. Macroconidia only were produced on Carnation leaf agar (CLA) and were hyaline, slender, falcate with single foot cells, 3 to 5 septate, and measured 29 to 50 µm × 3.75 to 5.0 µm. Chlamydospores were globose to subglobose and measured 6.8 to 16.5 µm. These morphological features were consistent with the description of Fusarium asiaticum (Leslie and Summerell 2006). For molecular identification, the partial translation elongation factor 1 alpha (TEF1-α) gene and RNA polymerase II second largest subunit (RPB2) gene of three representative isolates were amplified and sequenced (O'Donnell et al. 1998). These sequences were identical to each other, and one representative, Z-3-1, was deposited in GenBank (Accession No. OQ129437 and OQ858619, respectively). Analysis of the TEF1-α and RPB2 sequences of Z-3-1 showed that they were 99.85% (688/689) and 100% (945/945) identical to F. asiaticum strain Daya350-3 (KT380124) and MRC 1976 (MH582121), respectively, in NCBI, and had 99.38% and 100% identity to F. asiaticum strain CBS 110257 (AF212451 and JX171573) in Fusarium-ID. A combined phylogenetic tree based on the TEF1-α and RPB2 sequences showed that Z-3-1 was clustered with F. asiaticum using the neighbor-joining algorithm. Pathogenicity testing was conducted by inoculating potted Z. latifolia plants with a 1×105 conidial suspension of isolate Z-3-1, which was prepared by culturing the fungal strain in PDB at 26°C for 4 days in a shaker incubator. Conidial suspensions (1 mL) were dropped onto sheaths of potted Z. latifolia plants with sterile water serving as controls. All inoculated plants were covered with plastic bags and maintained in a humid growth chamber at 26°C with a photoperiod of 16 h. The inoculation experiment was repeated twice with 5 replicates per test. Four days later, the sheaths of potted inoculated plants displayed symptoms similar to those observed in the field. No symptoms were observed on control plants. Fusarium asiaticum was re-isolated specifically from the symptomatic inoculated Z. latifolia plants and confirmed by morphological and molecular methods, thus fulfilling Koch's postulates. Fusarium asiaticum has been reported to be a pathogen of other plants in China, such as Ligusticum (Zhu et al. 2022) and Setaria italica (Kong et al. 2022). To our knowledge, this is the first report of F. asiaticum causing sheath rot of Z. latifolia in China. The identification of the pathogen is the first step in developing appropriate field management strategies for this new disease.

The association between retinal vascular fractal dimension and cognitive function in the community-dwelling older adults cohort TIGER.

BACKGROUND/PURPOSE: The small retinal vessels reflect cerebral microcirculation and its fractal dimension (Df), representing the complexity of the retinal microcirculation. However, the connection between retinal circulation and cognitive function lacked consistent and longitudinal evidence. This study aimed to explore the association between retinal vascular complexity and cognitive impairment over time in non-demented community-dwelling older adults. METHODS: This four-year prospective cohort study (2015-2019) is part of the ongoing Taiwan Initiative for Geriatric Epidemiological Research (TIGER, 2011 to present). Of the 434 older adults (age >65) recruited, 207 participants were included for analysis. The retinal vascular Df was assessed by baseline images from fundus photography (2015-2017). Global (Montreal Cognitive Assessment-Taiwanese version, MoCA-T) and domain-specific cognition were assessed at the baseline and 2-year follow-up (2017-2019). The multivariable linear regression models and generalized linear mixed models were used to evaluate the association of Df with cognitive decline/impairment over time. RESULTS: Decreased left retinal vascular complexity was associated with poor attention performance (ß = -0.40). As follow-up time increased, decreased vascular complexity was associated with poor memory performance (right: ß = -0.25; left: ß = -0.19), and decreased right vascular complexity was associated with poor attention performance (ß = -0.18). CONCLUSION: Low retinal vascular complexity of the right or left eye may be differentially associated with cognitive domains in community-dwelling older adults over two years. The retinal vascular Df of either eye may be served as a screening tool for detecting cognitive impairment in the preclinical phase of dementia.

Long-range charge transport in homogeneous and alternating-rigidity chains.

We study the interplay of intrinsic-electronic and environmental factors in long-range charge transport across molecular chains with up to N ∼ 80 monomers. We describe the molecular electronic structure of the chain with a tight-binding Hamiltonian. Thermal effects in the form of electron decoherence and inelastic scattering are incorporated with the Landauer-Büttiker probe method. In short chains of up to ten units, we observe the crossover between coherent (tunneling, ballistic) motion and thermally-assisted conduction, with thermal effects enhancing the current beyond the quantum coherent limit. We further show that unconventional (nonmonotonic with size) transport behavior emerges when monomer-to-monomer electronic coupling is made large. In long chains, we identify a different behavior, with thermal effects suppressing the conductance below the coherent-ballistic limit. With the goal to identify a minimal model for molecular chains displaying unconventional and effective long-range transport, we simulate a modular polymer with alternating regions of high and low rigidity. Simulations show that, surprisingly, while charge correlations are significantly affected by structuring environmental conditions, reflecting charge delocalization, the electrical resistance displays an averaging effect, and it is not sensitive to this patterning. We conclude by arguing that efficient long-range charge transport requires engineering both internal electronic parameters and environmental conditions.

Perilipin2 inhibits diabetic nephropathy-induced podocyte apoptosis by activating the PPARγ signaling pathway.

Podocyte apoptosis plays a pivotal role in the pathogenesis of diabetic nephropathy (DN). The main purpose of this study was to investigate the effects of perilipin2 on high glucose (HG)-induced podocyte apoptosis and associated mechanisms. Differentially expressed genes (DEGs) in BTBR ob/ob mice vs. nondiabetic mice kidneys were obtained from GSE106841 dataset and picked out using the 'limma' package. The protein-protein interaction (PPI) network was constructed using the Search Tool for the Retrieval of Interacting Genes (STRING) and was visualized by Cytoscape. Perilipin2 was a hub gene using the cytoHubba plug-in from Cytoscape. Gene ontology (GO) analysis revealed that the 126 overlapping DEGs were mainly enriched in 'oxidation reduction' [biological process, (BP)], metal ion binding' [molecular function, (MF)] and 'extracellular region' [cellular component, (CC)]. KEGG pathway analysis revealed that perilipin2 was mainly involved in 'PPAR signaling pathway'. DN inhibited perilipin2 expression and PPARγ expression, as by both in vitro and in vivo studies. In vitro experiments demonstrated that perilipin2 inhibition could not only reduced PPARγ expression in podocytes, it could also promote the apoptosis, and inhibit the viability in HG treated podocytes using western blot, CCK8 and flow cytometry assays. Perilipin2 overexpression reversed the effects of HG on inhibiting podocalyxin, nephrin, precursor (pro)-caspase-3/-9 and PPARγ protein expression and increasing cleaved caspase-3/-9 protein expression. Furthermore, the functions of perilipin2 overexpression reversing HG-induced podocyte apoptosis were inhibited by PPARγ inhibitor. In conclusion, the functions of DN-induced podocyte apoptosis were inhibited by activation of the PPARγ signaling pathway caused by perilipin2 overexpression.

anamiR: integrated analysis of MicroRNA and gene expression profiling.

BACKGROUND: With advancements in high-throughput technologies, the cost of obtaining expression profiles of both mRNA and microRNA in the same individual has substantially decreased. Integrated analysis of these profiles can help to elucidate the functional effects of RNA expression in complex diseases, such as cancer. However, fundamental discrepancies are observed in the results from microRNA-mRNA target gene prediction algorithms, and few packages can be used to analyze microRNA and mRNA expression levels simultaneously. RESULTS: To address these issues, an R package, anamiR, was developed. A total of 10 experimental/prediction databases were integrated. Two analytical functions are provided in anamiR, including the single marker test and functional gene set enrichment analysis, and several parameters can be changed by users. Here we demonstrate the potential application of the anamiR package to 2 publicly available microarray datasets. CONCLUSION: The anamiR package is effective for an integrated analysis of both RNA and microRNA profiles. By characterizing biological functions and signaling pathways, this package helps identify dysregulated genes/miRNAs from biological and medical experiments. The source code and manual of the anamiR package are freely available at https://bioconductor.org/packages/release/bioc/html/anamiR.html .

miR-338-5p inhibits cell proliferation, colony formation, migration and cisplatin resistance in esophageal squamous cancer cells by targeting FERMT2.

Esophageal cancer is one of the leading causes of cancer death in the male population of Eastern Asia. In addition, esophageal squamous cell carcinoma (ESCC) is the major type of esophageal cancer among the world. Owing to the poor overall 5-year survival rate, novel effective treatment strategies are needed. MicroRNAs are important gene regulators that are dysregulated in many cancer types. In our previous study, we applied next-generation sequencing to demonstrate that miR-338-5p was downregulated in the tumor tissue of patients with versus without recurrence. In this study, we further studied the roles of miR-338-5p in ESCC. The expression of endogenous miR-338-5p was at lower levels in ESCC cells compared with normal cells. Functional assays showed that miR-338-5p reduced cell proliferation, colony formation, migration and cisplatin resistance in an ESCC cell line, CE-81T. Potential target genes of miR-338-5p were identified by microarray and prediction tools, and 31 genes were selected. Among these, Fermitin family homolog 2 (FERMT2) plays an oncogenic role in ESCC, so it was chosen for further study. Luciferase assays showed the direct binding between miR-338-5p and the 3' untranslated region of FERMT2. Silencing of FERMT2 inhibited cell proliferation, colony formation, migration and cisplatin resistance. Pathway analysis revealed that the integrin-linked protein kinase signaling pathway, in which FERMT2 participates, was significantly affected by a miR-338-5p mimic. Our results suggest that miR-338-5p may play an antioncogenic role in ESCC via repressing FERMT2.

Using proteomic profiling to characterize protein signatures of different thymoma subtypes.

BACKGROUND: Histology is a traditional way to classify subtypes of thymoma, because of low cost and convenience. Yet, due to the diverse morphology of thymoma, this method increases the complexity of histopathologic classification, and requires experienced experts to perform correct diagnosis. Therefore, in this study, we developed an alternative method by identifying protein biomarkers in order to assist clinical practitioners to make right classification of thymoma subtypes. METHODS: In total, 204 differentially expressed proteins in three subtypes of thymoma, AB, B2, and B3, were identified using mass spectrometry. Pathway analysis showed that the differentially expressed proteins in the three subtypes were involved in activation-related, signaling transduction-related and complement system-related pathways. To predict the subtypes of thymoma using the identified protein signatures, a support vector machine algorithm was used. Leave-one-out cross validation methods and receiver operating characteristic analysis were used to evaluate the predictive performance. RESULTS: The mean accuracy rates were > 80% and areas under the curve were ≧0.93 across these three subtypes. Especially, subtype B3 had the highest accuracy rate (96%) and subtype AB had the greatest area under the curve (0.99). One of the differentially expressed proteins COL17A2 was further validated using immunohistochemistry. CONCLUSIONS: In summary, we identified specific protein signatures for accurately classifying subtypes of thymoma, which could facilitate accurate diagnosis of thymoma patients.

[Chemical constituents from fruiting bodies of Tremella sanguinea].

The chemical constituents from the fruiting bodies of Tremella sanguinea were separated and purified by column chromatography on silica gel,ODS,Sephadex LH-20,and RP-HPLC. The structures of the isolated compounds were identified on the basis of physicochemical properties and spectroscopic data analysis,as well as comparisons with the data reported in the literature. Sixteen compounds were isolated from the 90% ethanol extract of the fruiting bodies of T. sanguinea,which were identified as( 22 E)-5α,8α-epidioxy-24-methyl-cholesta-6,9( 11),22-trien-3ß-ol( 1),( 22 E)-5α,8α-epidioxyergosta-6,22-dien-3ß-ol( 2),cerevisterol( 3),ergosta-7-ene-3ß,5α,6ß-triol( 4),( 22 E)-6ß-methoxyergosta-7,22-diene-3ß,5α-diol( 5),ergosta-7-en-3ß-ol( 6),4-hydroxy-methylincisterol( 7),2-pyrrolidone( 8),nicotinamide( 9),1-( 3-indolyl)-3-dihydroxypropan-1-one( 10),yangambin( 11),linoleic acid( 12),( 9 Z,12 Z,15 Z)-2,3-dihydroxypropyl octadeca-trienoate( 13),( 9 Z,12 Z)-2,3-dihydroxypropyl-octadeca-dienoate( 14),crypticin B( 15)and 3-phenyllactic acid( 16). All compounds were isolated from T. sanguinea for the first time. Except for compounds 6,9 and 12,the remained compounds were isolated from the genus Tremella for the first time.

An automated microfluidic DNA microarray platform for genetic variant detection in inherited arrhythmic diseases.

In this study, we developed an automated microfluidic DNA microarray (AMDM) platform for point mutation detection of genetic variants in inherited arrhythmic diseases. The platform allows for automated and programmable reagent sequencing under precise conditions of hybridization flow and temperature control. It is composed of a commercial microfluidic control system, a microfluidic microarray device, and a temperature control unit. The automated and rapid hybridization process can be performed in the AMDM platform using Cy3 labeled oligonucleotide exons of SCN5A genetic DNA, which produces proteins associated with sodium channels abundant in the heart (cardiac) muscle cells. We then introduce a graphene oxide (GO)-assisted DNA microarray hybridization protocol to enable point mutation detection. In this protocol, a GO solution is added after the staining step to quench dyes bound to single-stranded DNA or non-perfectly matched DNA, which can improve point mutation specificity. As proof-of-concept we extracted the wild-type and mutant of exon 12 and exon 17 of SCN5A genetic DNA from patients with long QT syndrome or Brugada syndrome by touchdown PCR and performed a successful point mutation discrimination in the AMDM platform. Overall, the AMDM platform can greatly reduce laborious and time-consuming hybridization steps and prevent potential contamination. Furthermore, by introducing the reciprocating flow into the microchannel during the hybridization process, the total assay time can be reduced to 3 hours, which is 6 times faster than the conventional DNA microarray. Given the automatic assay operation, shorter assay time, and high point mutation discrimination, we believe that the AMDM platform has potential for low-cost, rapid and sensitive genetic testing in a simple and user-friendly manner, which may benefit gene screening in medical practice.

Different effects of long noncoding RNA NDRG1-OT1 fragments on NDRG1 transcription in breast cancer cells under hypoxia.

Hypoxia plays a crucial role in the aggressiveness of solid tumors by driving multiple signaling pathways. Recently, long non-coding RNA (lncRNA) has been reported to promote or inhibit tumor aggressiveness by regulating gene expression. Previous studies in our laboratory found that the lncRNA NDRG1-OT1 is significantly up-regulated under hypoxia and inhibits its target gene NDRG1 at both the mRNA and protein levels. At the protein level, NDRG1-OT1 increases NDRG1 degradation via ubiquitin-mediated proteolysis. However, the repressive mechanism of NDRG1 at the RNA level is still unknown. Therefore, the purpose of this study was to study how NDRG1-OT1 transcriptionally regulates its target gene NDRG1. Luciferase reporter assays showed that NDRG1-OT1 decreased NDRG1 promoter activities. Mass spectrometry, bioinformatics tools, genetic manipulation, and immunoblotting were used to identify the interacting proteins. Surprisingly, different fragments of NDRG1-OT1 had opposite effects on NDRG1. The first quarter fragment (1-149 nt) of NDRG1-OT1 had no effect on the NDRG1 promoter; the second quarter fragment (150-263 nt) repressed NDRG1 by increasing the binding affinity of HNRNPA1; the third quarter fragment (264-392 nt) improved NDRG1 promoter activity by recruiting HIF-1α; the fourth quarter fragment (393-508 nt) down-regulated NDRG1 promoter activity via down-regulation of KHSRP under hypoxia. In summary, we have found a novel mechanism by which different fragments of the same lncRNA can cause opposite effects within the same target gene.

Bioactive polyoxygenated seco-cyclohexenes from Artabotrys hongkongensis.

Six new polyoxygenated seco-cyclohexenes, artahongkongenes A-F (1-6), together with six known analogues (7-12) were isolated from the stems and leaves of Artabotrys hongkongensis. Their structures were elucidated by extensive spectroscopic methods. All new compounds were evaluated for their cytotoxicities against five human cancer cell lines: HL-60, SMMC-7721, A-549, MCF-7 and SW480 in vitro. New seco-cyclohexenes 1-6 showed significant inhibitory effects against various human cancer cell lines with IC50 values ranging from 0.26 to 16.58 µM.

iGC-an integrated analysis package of gene expression and copy number alteration.

BACKGROUND: With the advancement in high-throughput technologies, researchers can simultaneously investigate gene expression and copy number alteration (CNA) data from individual patients at a lower cost. Traditional analysis methods analyze each type of data individually and integrate their results using Venn diagrams. Challenges arise, however, when the results are irreproducible and inconsistent across multiple platforms. To address these issues, one possible approach is to concurrently analyze both gene expression profiling and CNAs in the same individual. RESULTS: We have developed an open-source R/Bioconductor package (iGC). Multiple input formats are supported and users can define their own criteria for identifying differentially expressed genes driven by CNAs. The analysis of two real microarray datasets demonstrated that the CNA-driven genes identified by the iGC package showed significantly higher Pearson correlation coefficients with their gene expression levels and copy numbers than those genes located in a genomic region with CNA. Compared with the Venn diagram approach, the iGC package showed better performance. CONCLUSION: The iGC package is effective and useful for identifying CNA-driven genes. By simultaneously considering both comparative genomic and transcriptomic data, it can provide better understanding of biological and medical questions. The iGC package's source code and manual are freely available at https://www.bioconductor.org/packages/release/bioc/html/iGC.html .

Deregulated microRNAs in triple-negative breast cancer revealed by deep sequencing.

BACKGROUND: MicroRNAs (miRNAs) are short, non-coding RNA molecules that play critical roles in human malignancy. However, the regulatory characteristics of miRNAs in triple-negative breast cancer, a phenotype of breast cancer that does not express the genes for estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2, are still poorly understood. METHODS: In this study, miRNA expression profiles of 24 triple-negative breast cancers and 14 adjacent normal tissues were analyzed using deep sequencing technology. Expression levels of miRNA reads were normalized with the quantile-quantile scaling method. Deregulated miRNAs in triple-negative breast cancer were identified from the sequencing data using the Student's t-test. Quantitative reverse transcription PCR validations were carried out to examine miRNA expression levels. Potential target candidates of a miRNA were predicted using published target prediction algorithms. Luciferase reporter assay experiments were performed to verify a putative miRNA-target relationship. Validated molecular targets of the deregulated miRNAs were retrieved from curated databases and their associations with cancer progression were discussed. RESULTS: A novel 25-miRNA expression signature was found to effectively distinguish triple-negative breast cancers from surrounding normal tissues in a hierarchical clustering analysis. We documented the evidence of seven polycistronic miRNA clusters preferentially harboring deregulated miRNAs in triple-negative breast cancer. Two of these miRNA clusters (miR-143-145 at 5q32 and miR-497-195 at 17p13.1) were markedly down-regulated in triple-negative breast cancer, while the other five miRNA clusters (miR-17-92 at 13q31.3, miR-183-182 at 7q32.2, miR-200-429 at 1p36.33, miR-301b-130b at 22q11.21, and miR-532-502 at Xp11.23) were up-regulated in triple-negative breast cancer. Moreover, miR-130b-5p from the miR-301b-130b cluster was shown to directly repress the cyclin G2 (CCNG2) gene, a crucial cell cycle regulator, in triple-negative breast cancer cells. Luciferase reporter assays showed that miR-130b-5p-mediated repression of CCNG2 was dependent on the sequence of the 3'-untranslated region. The findings described in this study implicate a miR-130b-5p-CCNG2 axis that may be involved in the malignant progression of triple-negative breast cancer. CONCLUSIONS: Our work delivers a clear picture of the global miRNA regulatory characteristics in triple-negative breast cancer and extends the current knowledge of microRNA regulatory network.

Transgenic expression of human cytoxic T-lymphocyte associated antigen4-immunoglobulin (hCTLA4Ig) by porcine skin for xenogeneic skin grafting.

Porcine skin is frequently used as a substitute of human skin to cover large wounds in clinic practice of wound care. In our previous work, we found that transgenic expression of human cytoxicT-lymphocyte associated antigen4-immunoglobulin (hCTLA4Ig) in murine skin graft remarkably prolonged its survival in xenogeneic wounds without extensive immunosuppression in recipients, suggesting that transgenic hCTLA4Ig expression in skin graft may be an effective and safe method to prolong xenogeneic skin graft survival. In this work, using a transgene construct containing hCTLA4Ig coding sequence under the drive of human Keratine 14 (k14) promoter, hCTLA4Ig transgenic pigs were generated by somatic nuclear transfer. The derived transgenic pigs were healthy and exhibited no signs of susceptibility to infection. The hCTLA4Ig transgene was stably transmitted through germline over generations, and thereby a transgenic pig colony was established. In the derived transgenic pigs, hCTLA4Ig expression in skin was shown to be genetically stable over generations, and detected in heart, kidney and corneal as well as in skin. Transgenic hCTLA4Ig protein in pigs exhibited expected biological activity as it suppressed human lymphocyte proliferation in human mixed lymphocyte culture to extents comparable to those of commercially purchased purified hCTLA4Ig protein. In skin grafting from pigs to rats, transgenic porcine skin grafts exhibited remarkably prolonged survival compared to the wild-type skin grafts derived from the same pig strain (13.33 ± 3.64 vs. 6.25 ± 2.49 days, P < 0.01), further indicating that the transgenic hCTLA4Ig protein was biologically active and capable of extending porcine skin graft survival in xenogeneic wounds. The transgenic pigs generated in this work can be used as a reproducible resource to provide porcine skin grafts with extended survival for wound coverage, and also as donors to investigate the impacts of hCTLA4Ig on xenotransplantation of other organs (heart, kidney and corneal) due to the ectopic transgenic hCTLA4Ig expression.