Transcriptomic inspection revealed a possible pathway regulating the formation of the high-quality brush hair in Chinese Haimen goat (
            Capra hircus
            )

The high-quality brush hair, or Type III brush hair, is coarse hair but with a tip and little medulla, which uniquely grows in the cervical carina of Chinese Haimen goat ( Capra hircus ). To unveil the mechanism of the formation of Type III brush hair in Haimen goats, transcriptomic RNAseq technology was used for screening of differentially expressed genes (DEGs) in the skin samples of the Type III and the non-Type III hair goats, and these DEGs were analysed by KEGG pathway analysis. The results showed that a total of 295 DEGs were obtained, mainly from three main functional types: cellular component, molecular function and biological process. These DEGs were mainly enriched in three KEGG pathways, such as protein processing in endoplasmic reticulum, MAPK, and complement and coagulation cascades. These DEGs gave hints to a possible mechanism, under which heat stress possibly initiated the formation. The study provided some useful biological information, which could give a new view about the roles of certain factors in hair growth and give hints on the mechanism of the formation of the Type III brush hair in Chinese Haimen goat.

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Multi-Level Analyses of Genome-Wide Association Study to Reveal Significant Risk Genes and Pathways in Neuromyelitis Optica Spectrum Disorder

Frontiers in Genetics ◽

10.3389/fgene.2021.690537 ◽

2021 ◽

Vol 12 ◽

Author(s):

Ting Li ◽

He Li ◽

Yue Li ◽

Shu-An Dong ◽

Ming Yi ◽

...

Keyword(s):

Genome Wide Association Study ◽

Biological Process ◽

Susceptibility Gene ◽

Susceptibility Genes ◽

Cellular Component ◽

Molecular Function ◽

Risk Genes ◽

Kegg Pathways ◽

Gene Sets ◽

Genome Wide

BackgroundNeuromyelitis optica spectrum disorder (NMOSD) is an inflammatory disease of the central nervous system and it is understandable that environmental and genetic factors underlie the etiology of NMOSD. However, the susceptibility genes and associated pathways of NMOSD patients who are AQP4-Ab positive and negative have not been elucidated.MethodsSecondary analysis from a NMOSD Genome-wide association study (GWAS) dataset originally published in 2018 (215 NMOSD cases and 1244 controls) was conducted to identify potential susceptibility genes and associated pathways in AQP4-positive and negative NMOSD patients, respectively (132 AQP4-positive and 83 AQP4-negative).ResultsIn AQP4-positive NMOSD cases, five shared risk genes were obtained at chromosome 6 in AQP4-positive NMOSD cases by using more stringent p-Values in both methods (p < 0.05/16,532), comprising CFB, EHMT2, HLA-DQA1, MSH5, and SLC44A4. Fifty potential susceptibility gene sets were determined and 12 significant KEGG pathways were identified. Sixty-seven biological process pathways, 32 cellular-component pathways, and 29 molecular-function pathways with a p-Value of <0.05 were obtained from the GO annotations of the 128 pathways identified. In the AQP4 negative NMOSD group, no significant genes were obtained by using more stringent p-Values in both methods (p < 0.05/16,485). The 22 potential susceptibility gene sets were determined. There were no shared potential susceptibility genes between the AQP4-positive and negative groups, furthermore, four significant KEGG pathways were also identified. Of the GO annotations of the 165 pathways identified, 99 biological process pathways, 37 cellular-component pathways, and 29 molecular-function pathways with a p-Value of <0.05 were obtained.ConclusionThe potential molecular mechanism underlying NMOSD may be related to proteins encoded by these novel genes in complements, antigen presentation, and immune regulation. The new results may represent an improved comprehension of the genetic and molecular mechanisms underlying NMOSD.

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Semantic Annotation for Biological Information Retrieval System

Advances in Bioinformatics ◽

10.1155/2015/597170 ◽

2015 ◽

Vol 2015 ◽

pp. 1-11 ◽

Cited By ~ 2

Author(s):

Mohamed Marouf Z. Oshaiba ◽

Enas M. F. El Houby ◽

Akram Salah

Keyword(s):

Query Expansion ◽

Retrieval System ◽

Biological Process ◽

Semantic Annotation ◽

Information Retrieval System ◽

Cellular Component ◽

Biological Information ◽

Molecular Function ◽

Problem Finding ◽

Biological Domain

Online literatures are increasing in a tremendous rate. Biological domain is one of the fast growing domains. Biological researchers face a problem finding what they are searching for effectively and efficiently. The aim of this research is to find documents that contain any combination of biological process and/or molecular function and/or cellular component. This research proposes a framework that helps researchers to retrieve meaningful documents related to their asserted terms based on gene ontology (GO). The system utilizes GO by semantically decomposing it into three subontologies (cellular component, biological process, and molecular function). Researcher has the flexibility to choose searching terms from any combination of the three subontologies. Document annotation is taking a place in this research to create an index of biological terms in documents to speed the searching process. Query expansion is used to infer semantically related terms to asserted terms. It increases the search meaningful results using the term synonyms and term relationships. The system uses a ranking method to order the retrieved documents based on the ranking weights. The proposed system achieves researchers’ needs to find documents that fit the asserted terms semantically.

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CP Poly(A) RNA Binding Immunity via Outside-in Glycosyltransfer with MT of BTRC-Activating L12535 and PIN1 Subnetworks for Cognition in PFC|CD14

10.21203/rs.3.rs-41614/v1 ◽

2020 ◽

Author(s):

Lin Wang ◽

Qingchun Chen ◽

Haitao Feng ◽

Minghu Jiang ◽

Juxiang Huang ◽

...

Keyword(s):

Immune Response ◽

Network Inference ◽

Rna Binding ◽

Biological Process ◽

Cellular Component ◽

Innate Immune ◽

Molecular Function ◽

Ubiquitin Protein Ligase ◽

The Common ◽

Cluster Of Differentiation

Abstract Background: Ras suppressor protein 1 (L12535) and peptidylprolyl cis/trans isomerase NIMA-interacting 1 (PIN1) common molecular and knowledge subnetworks containing microtubule associated protein 1B-MAP1B_1 (upstream) related to cognition by references were identified in human left hemisphere, based on our established significant high expression beta-transducin repeat containing E3 ubiquitin protein ligase (BTRC)-activating downstream Gene (protein) reconstruction network inference (GRNInfer) and Database for Annotation, Visualization and Integrated Discovery (DAVID).Results: Our results show the common molecules exostosin-like glycosyltransferase 2 (EXTL2) interaction with MAP1B_1 both activating TERF1_1 with HSP90AB1 from BTRC-activating downstream GRNInfer database; The common biological process and molecular function of MAP1B_1, TERF1_1 as microtubule (MT) binding; HSP90AB1 as poly(A) RNA binding; BTRC, HSP90AB1, PIN1 as innate immune response from BTRC-activating downstream DAVID database; The common cellular component of EXTL2 at integral component of membrane; MAP1B_1, HSP90AB1, TERF1_1 at cytoplasm (CP); The common tissue distributions of L12535 and PIN1 in Prefrontal Cortex (PFC), PB cluster of differentiation (CD)14+Monocytes.Conclusions: We propose and mutual positively verify CP poly(A) RNA binding immunity via outside-in glycosyltransfer with MT of BTRC-activating L12535 and PIN1 subnetworks for cognition in PFC|CD14.

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NUDT1-Knockdown Inhibits Proliferation, Invasion and Migration of CAL27

10.21203/rs.3.rs-56328/v1 ◽

2020 ◽

Author(s):

Qiusheng Shan

Keyword(s):

Squamous Cell Carcinoma ◽

Cell Carcinoma ◽

Squamous Cell ◽

Cell Apoptosis ◽

Biological Process ◽

Cellular Component ◽

Expression Level ◽

Molecular Function ◽

Invasion And Migration ◽

And Migration

Abstract Background: The high level of reactive oxygen species (ROS) in cancer could oxidize guanine into 8-oxoG resulting in A: T changed into G: C or G: C into A: T. Nudix hydrolase 1 (NUDT1) overexpressed in many kind of cancers could hydrolyze the 8-oxo-dGTP into 8-oxo-dGMP or pyrophosphate to prevent the DNA damage or gene mutation. However, the role and function of NUDT1 in oral squamous cell carcinoma (OSCC) has not been investigated.Methods: Firstly, the bioinformatics methods were used to predict the biological process, cellular component and molecular function of NUDT1 and to confirm the mRNA and protein expression level of NUDT1 in OSCC. Furthermore, the MTT assay, Flow Cytometry, Transwell (Invasion), Scratch Test and Transwell (Migration) were used to test the effect of NUDT1 inhibitor on the proliferation, cell apoptosis, invasion and migration of CAL27. In addition, western blot was used to exam the expression level of NUDT1 and cleaved caspase-3 in different groups. Finally, the laser scanning microscope (LSM) was used to test the invadopodia formation level in different groups.Results: The bioinformatics analysis suggested that both mRNA and protein of NUDT1 overexpressed in head and neck squamous cell carcinoma (HNSCC) and OSCC. Furthermore, the biological process of NUDT1 mainly enriched in response to oxidative stress, aging, response to cadmium ion and male gonad development in OSCC. the cellular component of NUDT1 mainly enriched in extracellular exosome, cytoplasm, plasma membrane and nucleus in OSCC. The molecular function of NUDT1 mainly enriched in protein binding in OSCC. Finally, the biological experiments confirmed that NUDT1-knockdown could inhibit the proliferation, invasion, migration and promote cell apoptosis of CAL27.Conclusion: The overexpressed NUDT1 is positively associated with the progression of OSCC which has much potential to be new target in targeted therapy of OSCC.

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Comparative transcriptome investigation of Nosema ceranae infecting eastern honeybee workers

10.1101/2022.01.12.476029 ◽

2022 ◽

Author(s):

Fan Yuanchan ◽

Dafu Chen ◽

Rui Guo

Keyword(s):

Virulence Factor ◽

Clustering Analysis ◽

Biological Process ◽

Kegg Pathway ◽

Apis Cerana ◽

Nosema Ceranae ◽

Kegg Pathway Analysis ◽

Genes Encoding ◽

Comparison Groups ◽

Shed Light

Apis cerana is the original host for Nosema ceranae, a widespread fungal parasite resulting in bee nosemosis, which leads to severe losses for apiculture industry throughout the world. However, knowledge of N. ceranae infecting eastern honeybees is extremely limited. Currently, the mechanism underlying N. ceranae infection is still largely unknown. Based on our previously gained high-quality transcriptome datasets, comparative transcriptomic investigation was conducted in this work, with a focus on virulence factor-associated differentially expressed genes (DEGs). Microscopic observation showed that A. c. cerana workers midguts were effectively infected after inoculation with clean spores of N. ceranae. Totally, 1411, 604, and 38 DEGs were identified from NcCK vs. NcT1, NcCK vs. NcT2 and NcT1 vs. NcT2 comparison groups. Venn analysis showed that ten up-regulated genes and nine down-regulated ones were shared by aforementioned comparison groups. GO category indicated these DEGs were involved in a series of functional terms relevant to biological process, cellular component, and molecular function, such as metabolic process, cell part, and catalytic activity. Additionally, KEGG pathway analysis suggested that the DEGs were engaged in an array of pathways of great importance, such as metabolic pathway, glycolysis, and biosynthesis of secondary metabolites. Further, expression clustering analysis demonstrated that majority of genes encoding virulence factors such as ricin B lectins and polar tube proteins displayed apparent up-regulation, whereas a few virulence factor-associated genes such as hexokinase gene and 6-phosphofructokinase gene presented down-regulation during the fungal infection. Finally, the expression trend of 14 DEGs was confirmed by RT-qPCR, validating the reliability of our transcriptome datasets. These results together demonstrated that an overall alteration of the transcriptome of N. ceranae occurred during the infection of A. c. ceranae workers, and most of virulence factor-related genes were induced to activation to promote the fungal invasion. Our findings not only lay a foundation for clarifying the molecular mechanism underlying N. ceranae infection of eastern honeybee workers, but also shed light on developing novel targets for microsporidiosis control.

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In-silico analysis: common biomarkers of NDs

10.1101/2021.09.25.461779 ◽

2021 ◽

Author(s):

Deepanjan Sarkar ◽

Souvik Chakraborty ◽

Tarasankar Maiti ◽

Sushmita Bhowmick

Keyword(s):

Protein Interactions ◽

Neurological Disorders ◽

Biological Process ◽

Kegg Pathway ◽

In Silico Analysis ◽

Molecular Function ◽

Hub Genes ◽

Parkinsons Disease ◽

Online Tool ◽

Silico Analysis

Neurodegenerative disorders (NDs) are a class of rapidly rising devastating diseases and the reason behind are might be an improper function of related genes or a mutation in a particular gene or even could be autoimmune also. Parkinsons disease (PD), Multiple sclerosis (MS), Huntingtons disease (HD) are some of the NDs, and still, incurable fully. Apart from the similarities in symptoms, there are common genes that express somehow a differential manner in patients of PDs, MSs, and HDs. A total of 1197 differentially expressed genes (DEGs) are obtained by analyzing the chosen datasets. The protein interactions by STRING online tool and degree sorted hubs obtained through a plug-in in Cytoscape; Cyto-Hubba. Among the sorted hubs KRAS, CREB1, PIK3CA, JAK2 are the ones that are not only common to all the studied datasets of NDs but also in other neurological disorders like Alzheimers. The enriched pathways with biological process, molecular function, cellular component, and KEGG pathway details are obtained and analyzed using Enricher. This paper frames that the obtained hub genes could be potential biomarkers also and a need for further drug design for finding a possible cure.

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GOLink: Finding Cooccurring Terms across Gene Ontology Namespaces

International Journal of Genomics ◽

10.1155/2013/594528 ◽

2013 ◽

Vol 2013 ◽

pp. 1-10 ◽

Cited By ~ 3

Author(s):

Richard W. Francis

Keyword(s):

Gene Ontology ◽

Standard Method ◽

Biological Process ◽

Cellular Component ◽

Molecular Function ◽

Gene Products ◽

Go Annotation ◽

Or Gene ◽

Public Repositories ◽

Sensitivity Specificity

The Gene Ontology (GO) provides a resource for consistent annotation of genes and gene products that is extensively used by numerous large public repositories. The GO is constructed of three subontologies describing the cellular component of action, molecular function, and overall biological process of a gene or gene product. Querying across the subontologies is problematic and no standard method exists to, for example, find all molecular functions occurring in a particular cellular component. GOLink addresses this problem by finding terms from all subontologies cooccurring with a term of interest in annotation across the entire GO database. Genes annotated with this term are exported and their GO annotation is assigned to three separate GOLink terms lists based on specific criteria. The software was used to predict the most likely Biological Process for a group of genes using just their Molecular Function terms giving sensitivity, specificity, and accuracy between 80 and 90% across all the terms lists. GOLink is made freely available for noncommercial use and can be downloaded from the project website.

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Microarray Profiling of Bone Marrow Long Non-Coding RNA Expression in Multiple Myeloma

Blood ◽

10.1182/blood.v128.22.5617.5617 ◽

2016 ◽

Vol 128 (22) ◽

pp. 5617-5617

Author(s):

Ying Shen ◽

Jing Liu ◽

Yun Yang ◽

Ju Bai ◽

Yue Peng ◽

...

Keyword(s):

Cell Cycle ◽

Bone Marrow ◽

Gene Ontology ◽

Iron Deficiency ◽

Iron Deficiency Anemia ◽

Pathway Analysis ◽

Biological Process ◽

Kegg Pathway ◽

Go Analysis ◽

Kegg Pathway Analysis

Abstract Introduction: Multiple myeloma (MM), the second most frequent hematologic malignancy, is characterized with clonal proliferation of malignant plasma cells in the bone marrow, and usually monoclonal protein in the blood and/or urine. Its clinical manifestations are associated with end-organ damage consisting of anaemia, renal insufficiency, bone lesions and hypercalcaemia. Long non-coding RNAs (lncRNAs) represent more than half of the mammalian noncoding transcriptome and are involved in many biological processes, including transcription regulation, competition with other linear RNAs and involvement in tumorigenesis or oncogenic pathways. Accumulating studies have revealed the important role of lncRNAs in the hematological tumors, especially MM. In order to investigate the further relationship between MM and lncRNAs, we performed global lncRNA profiling in both incipient MM patients and iron deficiency anemia (IDA) patients. We assumed that the bunch of deregulated lncRNAs in MM patients would broaden current understanding of progression of MM, and present a new approach to novel therapeutic targets. Methods: Bone marrow mononuclear cells were obtained from five MM patients with normal karyotype at the Department of Hematology, Second Affiliated Hospital, Xi'an Jiaotong University, in 2016. Additionally, bone marrow samples from five patients with IDA were used as controls. One sample in five MM patients was rejected because of its disqualification in clustering analysis. Arraystar Human LncRNA Array V4.0 was used to profile expression of lncRNAs, which was performed by KangChen Bio-tech (Shanghai, China). The array detects a total of 40,173 lncRNAs probes and an entire collection of 20,730 protein coding mRNAs. Gene Ontology (GO) analysis (http://david.abcc.ncifcrf.gov/summary.jsp) was used to analyze differentially expressed transcripts. Pathway analysis of differentially expressed transcripts were accomplished using Kyoto Encyclopedia of Genes and Genomes (KEGG) database (http://www.genome.jp/kegg). Results: Bioinformatic analysis of the lncRNA expression identified a total of 2445 lncRNAs were upregulated and 1519 lncRNAs were downregulated in four MM samples, among them, 199 lncRNAs were significantly upregulated and 27 lncRNAs were notably downregulated more than 10-fold in MM samples compared with IDA (Figure 1). GO analysis by Database for Annotation, Visualization and Integrated Discovery (DAVID ) showed that the top ten enriched biological process (BP) by upregulated transcripts were involved in multicellular organismal development and cell-cell adhesion, while downregulated transcripts were dragged in mitotic cell cycle and cell cycle. KEGG pathway analysis of the top ten enriched pathways included ECM-receptor interaction and protein processing in endoplasmic reticulum in upregulated transcript, while cell cycle and DNA replication were the top two enriched pathways in downregulated transcript (Figure 2). Among them, mitotic cell cycle was the most enriched pathway with p-value 3.86E-35. These results suggested that the dysregulated lncRNAs were related to MM cells adhesion and proliferation or differentiation closely. Conclusions: We have identified a group of dysregulated lncRNAs and mRNAs in bone marrow samples obtained from MM patients versus IDA controls. These lncRNAs participate in MM cell growth, migration and invasion by regulating cell adhesion and cell cycle, playing an important role in the development and progression of MM. Further investigation is still required to detect the underlying functions of these lncRNAs in MM pathogenesis and whether these lncRNAs may serve as new diagnostic biomarkers and therapeutic targets for MM. Figure 1 Clustering analysis of four mulitiple myeloma (MM) samples and five iron deficiency anemia (IDA) controls. Figure 1. Clustering analysis of four mulitiple myeloma (MM) samples and five iron deficiency anemia (IDA) controls. Figure 2 Gene Ontology(GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of dysregulated transcripts. (A) The top ten enriched biological process (BP) by upregulated transcripts. (B) The top ten enriched BP by downregulated transcripts. (C) The top ten enriched pathways in upregulated transcripts. (D) The top ten enriched pathways in downregulated transcripts. Figure 2. Gene Ontology(GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of dysregulated transcripts. (A) The top ten enriched biological process (BP) by upregulated transcripts. (B) The top ten enriched BP by downregulated transcripts. (C) The top ten enriched pathways in upregulated transcripts. (D) The top ten enriched pathways in downregulated transcripts. Disclosures No relevant conflicts of interest to declare.

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The Effect of Lysophosphatidic Acid-supplemented Culture Medium on Human Immature Oocytes Matured in Vitro

10.21203/rs.3.rs-196908/v1 ◽

2021 ◽

Author(s):

Qigui Xie ◽

Yaxin Xing ◽

Jianhong Zhou ◽

Ling Wang ◽

Jie Wu ◽

...

Keyword(s):

Gene Ontology ◽

Treatment Group ◽

Oocyte Maturation ◽

Lysophosphatidic Acid ◽

Culture Medium ◽

Biological Process ◽

Cellular Component ◽

Molecular Function ◽

Upregulated Genes

Abstract Background: Lysophosphatidic acid-supplemented culture medium significantly increases the oocyte maturation rate in vitro. However, potential targets and pathways involved remain unknown.Methods: A total of 43 women, who underwent cesarean section and aged between 18-35 years with good health, were included in this study. Immature oocytes were obtained and cultured with 10 mM lysophosphatidic acid. After culture, oocyte maturation was assessed and oocytes and cumulus cells were collected for RNA sequencing. HISAT2 was used to align clean reads to the human genome. The featureCounts and edgeR package were used to calculate gene expression and analyze differences between groups respectively. ClusterProfiler program was used to perform Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analysis.Results: Oocyte maturation rate increased significantly after 48 h treatment with lysophosphatidic acid. In cumulus cells, 259 genes were upregulated and 237 were downregulated in the treatment group; Gene Ontology analysis revealed that 653 biological process, 61 cellular component, and 86 molecular function items were enriched by upregulated genes; 379 biological process, 46 cellular component, and 97 molecular function items were enriched by downregulated genes; Kyoto Encyclopedia of Genes and Genomes analysis showed that upregulated genes in the treatment group were enriched in TNF signaling and insulin secretion pathways and downregulated genes were enriched in TNF signaling and cell adhesion molecules. In oocytes, 128 genes were upregulated and downregulated in the treatment group; Gene Ontology analysis revealed that 300 biological process items, 44 cellular component items, and 93 molecular function items were enriched by upregulated genes; 268 biological process items, 22 cellular component items, and 72 molecular function items were enriched by downregulated genes; Kyoto Encyclopedia of Genes and Genomes analysis showed that upregulated genes in the treatment group were enriched in MAPK signaling, gap junction, and cell cycle pathways and downregulated genes were enriched in MAPK signaling, estrogen signaling, RAP1 signaling, and gap junction pathways.Conclusions: Our study indicates that lysophosphatidic acid in culture medium enhances human oocyte maturation in vitro and identifies some potential targets and pathways associated with oocyte maturation for the first time and are important in clinical settings.

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Functional and Molecular Characterization for the Damp-Obstructed Rat Model in Chinese Medicine

The American Journal of Chinese Medicine ◽

10.1142/s0192415x06003862 ◽

2006 ◽

Vol 34 (02) ◽

pp. 323-340 ◽

Cited By ~ 3

Author(s):

G. Steven Huang ◽

Meng-Yen Hong

Keyword(s):

Signal Transduction ◽

Real Time ◽

Molecular Characterization ◽

Biological Process ◽

Cellular Component ◽

Molecular Function ◽

Channel Activity ◽

Rt Pcr ◽

Melanin Biosynthesis ◽

Atp Metabolism

Functional and molecular characterization was performed on the major organs of damp-obstructed rats by applying expression datasets of microarray experiments and real-time RT-PCR. Gene ontology repertoires, i.e. cellular component, molecular function, and biological process were used to classify differentially expressed genes in the major organs of rats upon treatment of dampness. As to the cellular component, over-expression of genes associated with the plasma membrane was observed in the stomach, spleen, kidney, heart, liver, and lung. Genes associated with translational machinery, endoplasmic recticulum membrane, Golgi apparatus, and nuclear envelope were down-regulated in the stomach. Concerning the molecular function, genes associated with oxidoreductase activity were up-regulated in the stomach, spleen, kidney, lung, and brain. Channel activity, membrane receptor, and electron transporter activity were up-regulated in stomach, kidney, and lung. Regarding the biological process, genes associated with signal transduction were up-regulated in the stomach, while genes associated with biosynthesis and ATP metabolism were down-regulated. In the spleen, melanin biosynthesis was up-regulated while hormone-related activities were down-regulated. In the kidney, genes associated with nucleotide biosynthesis and ATP metabolism were depressed. In the heart and liver, apoptosis was up-regulated while immune response and RAS signal transduction were down-regulated. Interestingly, genes associated with oncogenesis were up-regulated in the stomach and kidney. Functional fingerprints indicated that dampness weakened membrane structures, depressed metabolic activity (especially ATP metabolism), damaged matrix proteins, enhanced signal transduction, and revealed a positive association with oncogenesis. To quantify the functional impact at the molecular level, mRNA levels of key genes were determined by real-time RT-PCR. The results indicated that ATP storage in kidney, spleen, and stomach was depleted in damp-obstructed rats. We propose that oxidative stress, membrane integrity, melanin biosynthesis, ion channel activity, and ATP metabolism might be hallmarks for damp-obstructed rats. Our results also suggested dampness as a pathogenic factor in rats which is possibly associated with enhanced liabilities of cancer.

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