Comparing gene discovery from Affymetrix GeneChip microarrays and Clontech PCR-select cDNA subtraction: a case study

BACKGROUND: Several high throughput technologies have been employed to identify differentially regulated genes that may be molecular targets for drug discovery. Here we compared the sets of differentially regulated genes discovered using two experimental approaches: a subtracted suppressive hybridization (SSH) cDNA library methodology and Affymetrix GeneChip(® )technology. In this "case study" we explored the transcriptional pattern changes during the in vitro differentiation of human monocytes to myeloid dendritic cells (DC), and evaluated the potential for novel gene discovery using the SSH methodology. RESULTS: The same RNA samples isolated from peripheral blood monocyte precursors and immature DC (iDC) were used for GeneChip microarray probing and SSH cDNA library construction. 10,000 clones from each of the two-way SSH libraries (iDC-monocytes and monocytes-iDC) were picked for sequencing. About 2000 transcripts were identified for each library from 8000 successful sequences. Only 70% to 75% of these transcripts were represented on the U95 series GeneChip microarrays, implying that 25% to 30% of these transcripts might not have been identified in a study based only on GeneChip microarrays. In addition, about 10% of these transcripts appeared to be "novel", although these have not yet been closely examined. Among the transcripts that are also represented on the chips, about a third were concordantly discovered as differentially regulated between iDC and monocytes by GeneChip microarray transcript profiling. The remaining two thirds were either not inferred as differentially regulated from GeneChip microarray data, or were called differentially regulated but in the opposite direction. This underscores the importance both of generating reciprocal pairs of SSH libraries, and of real-time RT-PCR confirmation of the results. CONCLUSIONS: This study suggests that SSH could be used as an alternative and complementary transcript profiling tool to GeneChip microarrays, especially in identifying novel genes and transcripts of low abundance

Data Assimilation with Machine Learning for Dynamical Systems: Modelling Indoor Ventilation

Data assimilation is a method of combining physical observations with prior knowledge (for instance, a computational simulation) in order to produce an improved model; that is, improved over what thephysical observations or the computational simulation could offer in isolation. Recently, machine learning techniques have been deployed in order to address the significant computational burden that is associated with the procedures involved in data assimilation.In this paper we propose an approach that uses a non-intrusive reduced-order model (NIROM) as a surrogate for a high-resolution model thereby saving computational effort. The mismatch between observations and the surrogate model is propagated forwards and backwards in time in a manner similar to 4D-variational data assimilation methods. The observations and prior are reconciled in a new way which takes full advantage of the neural network used in the NIROM and also means that there is no need to form the sensitivities explicitly when propagating the mismatch. Instead, the observations are part of the input and output of the network.Modelling the air quality in a school classroom is the test case for our demonstration. Firstly, the data assimilation approach is shown to perform very well in a dual-twin type experiment, and secondly, theapproach is used to assimilate observations collected from a classroom in Houndsfield Primary School with predictions from the NIROM

Nodal s± pairing symmetry in an iron-based superconductor with only hole pockets

The origin of high-temperature superconductivity in iron-based superconductors is still not understood; determination of the pairing symmetry is essential for understanding the superconductivity mechanism. In the iron-based superconductors that have hole pockets around the Brillouin zone centre and electron pockets around the zone corners, the pairing symmetry is generally considered to be s±, which indicates a sign change in the superconducting gap between the hole and electron pockets. For the iron-based superconductors with only hole pockets, however, a couple of pairing scenarios have been proposed, but the exact symmetry is still controversial. Here we determine that the pairing symmetry in KFe2As2—which is a prototypical iron-based superconductor with hole pockets both around the zone centre and around the zone corners—is also of the s± type. Our laser-based angle-resolved photoemission measurements have determined the superconducting gap distribution and identified the locations of the gap nodes on all the Fermi surfaces around the zone centres and the zone corners. These results unify the pairing symmetry in hole-doped iron-based superconductors and point to spin fluctuation as the pairing glue in generating superconductivity

Myeloid DLL4 Does Not Contribute to the Pathogenesis of Non-Alcoholic Steatohepatitis in Ldlr-/- Mice.

Non-alcoholic steatohepatitis (NASH) is characterized by liver steatosis and inflammation. Currently, the underlying mechanisms leading to hepatic inflammation are not fully understood and consequently, therapeutic options are poor. Non-alcoholic steatohepatitis (NASH) and atherosclerosis share the same etiology whereby macrophages play a key role in disease progression. Macrophage function can be modulated via activation of receptor-ligand binding of Notch signaling. Relevantly, global inhibition of Notch ligand Delta-Like Ligand-4 (DLL4) attenuates atherosclerosis by altering the macrophage-mediated inflammatory response. However, the specific contribution of macrophage DLL4 to hepatic inflammation is currently unknown. We hypothesized that myeloid DLL4 deficiency in low-density lipoprotein receptor knock-out (Ldlr-/-) mice reduces hepatic inflammation. Irradiated Ldlr-/- mice were transplanted (tp) with bone marrow from wild type (Wt) or DLL4f/fLysMCre+/0 (DLL4del) mice and fed either chow or high fat, high cholesterol (HFC) diet for 11 weeks. Additionally, gene expression was assessed in bone marrow-derived macrophages (BMDM) of DLL4f/fLysMCreWT and DLL4f/fLysMCre+/0 mice. In contrast to our hypothesis, inflammation was not decreased in HFC-fed DLL4del-transplanted mice. In line, in vitro, there was no difference in the expression of inflammatory genes between DLL4-deficient and wildtype bone marrow-derived macrophages. These results suggest that myeloid DLL4 deficiency does not contribute to hepatic inflammation in vivo. Since, macrophage-DLL4 expression in our model was not completely suppressed, it can't be totally excluded that complete DLL4 deletion in macrophages might lead to different results. Nevertheless, the contribution of non-myeloid Kupffer cells to notch signaling with regard to the pathogenesis of steatohepatitis is unknown and as such it is possible that, DLL4 on Kupffer cells promote the pathogenesis of steatohepatitis

Co-Targeting IGF-1R and Autophagy Enhances the Effects of Cell Growth Suppression and Apoptosis Induced by the IGF-1R Inhibitor NVP-AEW541 in Triple-Negative Breast Cancer Cells.

BACKGROUND:Triple-negative breast cancer (TNBC) is the most intractable type of breast cancer, and there is a lack of effective targeted therapy. Insulin-like growth factor-1 receptor (IGF-1R) is reportedly a potential target for TNBC treatment. However, satisfying treatment outcomes in breast cancer patients have yet to be achieved with IGF-1R-targeted agents. METHODS:To confirm whether inhibiting IGF-1R could induce autophagy, we detected autophagy-related proteins by western blotting and immunofluorescence staining of LC3-II. The IGF-1R inhibitor NVP-AEW541, autophagy inhibitor 3-methyladenine (3-MA) and Atg7 small interfering RNA (siRNA) were used to further investigate the effects of autophagy induced by IGF-1R inhibition in TNBC cells. The CCK8 assay, EdU assay, apoptosis and cell cycle analyses were applied to test cell function after treatment. RESULTS:NVP-AEW541 markedly induced autophagy in TNBC cells by increasing the levels of the autophagy-related protein Beclin-1 and the LC3-II/LC-I ratio and reducing the selective autophagy substrate p62. Joint application of 3-MA or Atg7 siRNA enhanced the cell growth inhibition and apoptosis effects of NVP-AEW541 by arresting cells at G1/G0 phase and increasing Bax expression and decreasing that of Bcl-2. CONCLUSION:Targeting IGF-1R in TNBC induces cell-protective autophagy, thereby weakening the therapeutic effect of agents directed toward IGF-1R. Our findings reveal that combined use autophagy-disrupting agents can enhance the therapeutic efficacy of IGF-1R inhibitors in TNBC cells and may provide a valuable treatment strategy for IGF-1R inhibitor-based therapies for TNBC and other IGF-1 signaling-associated tumors

Gas chromatography-mass spectrometry-based untargeted metabolomics analysis reveals circulating biomarkers related to wooden breast myopathy in broilers: a preliminary study

ABSTRACT: Approaches for the diagnosis of wooden breast (WB) myopathy in live birds are urgently required before applying intervention strategies to reduce occurrence and severity for the poultry industry. The objective of this study was to characterize the serum metabolic profiles in male broilers affected by WB and to identify biomarkers related to this myopathy. Broilers were categorized into normal (CON) and WB groups based on gross scoring and histological evaluation. Gas chromatography-mass spectrometry-based metabolomics, multivariate analysis, and orthogonal partial least squares discriminant analysis revealed a clear separation between CON and WB. A total of 73 significantly different (P < 0.05) metabolites with 17 upregulated and 56 downregulated were identified, which were mainly involved in pathways of alanine, aspartate, and glutamate metabolism, carbohydrate metabolism, and taurine and hypotaurine metabolism. By using the nested cross-validation function of random forest analysis, 9 significantly altered (P < 0.05) metabolites (cerotinic acid, arabitol, phosphoenolpyruvate, terephthalic acid, cis-gondoic acid, N-acetyl-d-glucosamine, 4-hydroxymandelic acid, caffeine, and xanthurenic acid) were identified as biomarkers with an excellent discriminant performance for WB myopathy. Collectively, this study provides new insights for a deeper understanding of the pathogenesis and provides metabolites as biomarkers for diagnostic utilization of WB myopathy

METTL3-Mediated m6A Methylation Regulates Muscle Stem Cells and Muscle Regeneration by Notch Signaling Pathway

The Pax7+ muscle stem cells (MuSCs) are essential for skeletal muscle homeostasis and muscle regeneration upon injury, while the molecular mechanisms underlying muscle stem cell fate determination and muscle regeneration are still not fully understood. N6-methyladenosine (m6A) RNA modification is catalyzed by METTL3 and plays important functions in posttranscriptional gene expression regulation and various biological processes. Here, we generated muscle stem cell-specific METTL3 conditional knockout mouse model and revealed that METTL3 knockout in muscle stem cells significantly inhibits the proliferation of muscle stem cells and blocks the muscle regeneration after injury. Moreover, knockin of METTL3 in muscle stem cells promotes the muscle stem cell proliferation and muscle regeneration in vivo. Mechanistically, METTL3-m6A-YTHDF1 axis regulates the mRNA translation of Notch signaling pathway. Our data demonstrated the important in vivo physiological function of METTL3-mediated m6A modification in muscle stem cells and muscle regeneration, providing molecular basis for the therapy of stem cell-related muscle diseases

Revealing Spatial Patterns of Cultural Ecosystem Services in Four Agricultural Landscapes: A Case Study from Hangzhou, China

Monitoring and mapping agricultural cultural ecosystem services (CES) is essential, especially in areas with a sharp contradiction between agricultural land protection and urban development. Despite research assessing CES increasing exponentially in recent years, our knowledge of the CES of agricultural landscapes is still inadequate. This study used four types of agricultural landscapes in Hangzhou, China, as the study area, analyzed their CES spatial patterns, and explored their societal preferences by integrating the multi-sourced datasets, clustering algorithms, and Maxent model. The results indicated that hot spots of agricultural CES correspond to river valley plains, which were also easily vulnerable to urbanization. Moreover, we found that the CES level of paddy field and dry farmland were higher than tea garden and orchard. Based on the above spatial patterns of supply, demand, and flow of CES, we identified four groups of agricultural land by cluster analysis, distinguishing between significant, unimportant, little used, and potential CES. Further, our results showed that natural and human factors could explain societal preferences. This study can provide a valuable basis for stakeholders to develop balanced strategies by the aforementioned results

Effect of Exogenous Methyl Jasmonate on Resistance to Bacterial Leaf Stripe in Rice

【Objective】The effect of exogenous methyl jasmonate on resistance to bacterial leaf stripe (BLS) was studied, which would provide a theoretical basis for the prevention and control of BLS in rice.【Method】With rice BLS-resistant and susceptible near isogenic lines (K9 and G9) as materials, 0.1 mmol/L methyl jasmonate (MeJA) were sprayed on BLS-resistant and susceptible near isogenic lines (K9 and G9) plants respectively at tillering stage, and water spraying was used as control (CK). The inoculation with Xanthomonas oryzae pv. Oryzicola (Xoc) were conducted at 0, 12, 24, 48, 72 h after MeJA treatment, then the symptoms of BLS were investigated. In K9 and G9 plants sprayed with MeJA for 48 h then inoculated with Xoc, the expression changes of hormone-pathway related genes were further analyzed at 0, 6, 12, 24 and 48 h after inoculation.【Result】In K9 plants, the lesion lengths of leaves inoculated at 0, 24, 48, 72h after being sprayed with MeJA were significantly shorter than those in CK; in G9 plants, the lesion lengths of leaves inoculated at 0 h and 12 h after being sprayed with MeJA were significantly longer than those in CK, while inoculated at 72 h after being sprayed with MeJA were significantly shorter than those in CK. Compared with the CK, relative expression levels of jasmonate-pathway related genes LOX2 and AOS2, and pathogenesis-related gene PR10 were upregulated, while relative expression levels of alicylic acid-pathway related genes PAD4 and PAL, ethylene-pathway related genes EIN2 and ERF70, and pathogenesis-related gene PR1a were downregulated in K9 plants inoculated at 48h after being sprayed with MeJA; relative expression levels of jasmonate-pathway related genes LOX2 and AOS2, salicylic acid-pathway related genes PAD4 and PAL and pathogenesis-related genes PR1a and PR10 were upregulated, while relative expression level of ethylene-pathway related gene EIN2 was downregulated in G9 plants inoculated at 48h after being sprayed with MeJA.【Conclusion】Exogenous MeJA affects the BLS-resistance in rice resistant and susceptible near isogenic lines. The resistance responses of resistant and susceptible near isogenic lines to Xoc are different at different inoculation time points after MeJA treatment. Exogenous MeJA involves in the defense response to Xoc infection by inducing the expression changes of jasmonate, salicylic acid and ethylene pathway-related genes and pathogenesis-related genes

Effects of β-diketone antibiotics on F1-zebrafish (Danio rerio) based on high throughput miRNA sequencing under exposure to parents.

The toxicity of β-diketone antibiotics (DKAs), a class of ''pseudo-persistent'' environmental pollutants, to F0-zebrafish (Danio rerio) was investigated using 7-dpf F1-zebrafish miRNA sequencing and bioinformatics analyses. Based on relative expression, 47, 134 and 118 of 193 mature miRNAs were differentially expressed between control vs 6.25 mg/L, control vs 12.5 mg/L and 6.25 vs 12.5 mg/L treatments, respectively. Utilizing three databases, 2523 potential target genes were predicted, and they were assigned to 19 high-abundance KEGG pathways and 20 functional categories by COG analysis. Among 11 significantly differential expression and high-abundance miRNAs, the expression levels for 7 miRNAs (miR-144, -124, -499, -125b, -430b, -430c and -152) assessed by qRT-PCR were consistent with those determined by sRNA-seq. A potential network was plotted between 11 miRNAs and their target genes based on differential expression and binding effectiveness. The high degree of connectivity between miRNA-gene pairs suggests that these miRNAs play critical roles in zebrafish development. The expression of miR-124 and miR-499 in whole-mount in situ hybridization was in general agreement with those from qRT-PCR and miRNA-seq and were DKA concentration-dependent. DKA exposure induced severe histopathological changes and damage in F0-zebrafish ovary tissue, as reflected by an increased number of early developmental oocytes, irregular cell distribution, decreased yolk granules, cytoplasmic shrinkage, cell lysis in mature oocytes, and dissolution of internal corona radiata. Chronic DKA exposure affected reproduction of F0-zebrafish and development of F1-zebrafish. These observations demonstrate the toxic effect transfer relation across parent and their offspring, and enhance our understanding of drug-induced diseases