A study of candidate genes for day blindness in the standard wire haired dachshund

Background

A genetic study was performed to identify candidate genes associated with day blindness in the standard wire haired dachshund. Based on a literature review of diseases in dogs and human with phenotypes similar to day blindness, ten genes were selected and evaluated as potential candidate genes associated with day blindness in the breed.

Results

Three of the genes, CNGB3, CNGA3 and GNAT2, involved in cone degeneration and seven genes and loci, ABCA4, RDH5, CORD8, CORD9, RPGRIP1, GUCY2D and CRX, reported to be involved in cone-rod dystrophies were studied. Polymorphic markers at each of the candidate loci were studied in a family with 36 informative offspring. The study revealed a high frequency of recombinations between the candidate marker alleles and the disease.

Conclusion

Since all of the markers were at the exact position of the candidate loci, and several recombinations were detected for each of the loci, all ten genes were excluded as causal for this canine, early onset cone-rod dystrophy. The described markers may, however, be useful to screen other canine resource families segregating eye diseases for association to the ten genes.

     

Low-dose lymphocyte immunotherapy rebalances the peripheral blood Th1/Th2/Treg paradigm in patients with unexplained recurrent miscarriage

Background

The published results regarding lymphocytes immunotherapy for unexplained recurrent miscarriage (uRM) patients are conflicting due to different screening criteria and therapeutic protocols. The objective of the present study is to evaluate the effectiveness of immunotherapy using low-dose lymphocytes in patients with uRM and Th1/Th2/Treg paradigm disorders.

Methods

Sixty-four uRM patients who received low-dose lymphocytes immunotherapy served as the immunotherapy group, while the other 35 women who did not receive the treatment served as the control group. The proportions of peripheral blood Th1 cells, Th2 cells and Treg cells; and the concentration of TGF-β1 in serum were detected by flow cytometry and enzyme-linked immunosorbent assay (ELISA), respectively, before and after the immunotherapy.

Results

The proportion of Th1 cells was significantly decreased while the proportions of Th2 cells and Treg cells were significantly increased in immunotherapy group patients after treatment. In addition, the concentration of TGF-β1 in serum was significantly higher after immunotherapy than before. Forty-three uRM patients achieved pregnancy after receiving immunotherapy and 5 patients underwent miscarriages in the immunotherapy group (11.6%, 5/43), while 8 of the 23 pregnant patients experienced a miscarriage in the control group (34.8%, 8/23; p?<?0.05).

Conclusions

Low-dose lymphocyte immunotherapy is beneficial for restoring balance in the Th1/Th2/Treg paradigm and improving pregnancy outcome in uRM patients.

Trial registration

NCT03081325. ClinicalTrials.gov. Retrospectively registered July 2015.

     

Identification of hub genes with diagnostic values in pancreatic cancer by bioinformatics analyses and supervised learning methods

Background

Pancreatic cancer is one of the most lethal tumors with poor prognosis, and lacks of effective biomarkers in diagnosis and treatment. The aim of this investigation was to identify hub genes in pancreatic cancer, which would serve as potential biomarkers for cancer diagnosis and therapy in the future.

Methods

Combination of two expression profiles of GSE16515 and GSE22780 from Gene Expression Omnibus (GEO) database was served as training set. Differentially expressed genes (DEGs) with top 25% variance followed by protein-protein interaction (PPI) network were performed to find candidate genes. Then, hub genes were further screened by survival and cox analyses in The Cancer Genome Atlas (TCGA) database. Finally, hub genes were validated in GSE15471 dataset from GEO by supervised learning methods k-nearest neighbor (kNN) and random forest algorithms.

Results

After quality control and batch effect elimination of training set, 181 DEGs bearing top 25% variance were identified as candidate genes. Then, two hub genes, MMP7 and ITGA2, correlating with diagnosis and prognosis of pancreatic cancer were screened as hub genes according to above-mentioned bioinformatics methods. Finally, hub genes were demonstrated to successfully differ tumor samples from normal tissues with predictive accuracies reached to 93.59 and 81.31% by using kNN and random forest algorithms, respectively.

Conclusions

All the hub genes were associated with the regulation of tumor microenvironment, which implicated in tumor proliferation, progression, migration, and metastasis. Our results provide a novel prospect for diagnosis and treatment of pancreatic cancer, which may have a further application in clinical.

     

Study of the tumor microenvironment during breast cancer progression

Background

Different cells and mediators in the tumor microenvironment play important roles in the progression of breast cancer. The aim of this study was to determine the composition of the microenvironment during tumor progression in order to discover new related biomarkers and potentials for targeted therapy.

Methods

In this study, breast cancer biopsies from four different stages, and control breast biopsies were collected. Then, the mRNA expression of several markers related to different CD4⁺ T cell subsets including regulatory T cells (Treg), T helper (Th) type 1, 2 and 17 were determined. In addition, we investigated the expression of two inflammatory cytokines (TNF-α and IL-6) and inflammatory mediators including FASL, IDO, SOCS1, VEGF, and CCR7.

Results

The results showed that the expression of Th1 and Th17 genes was decreased in tumor tissues compared to control tissues. In addition, we found that the gene expression related to these two cell subsets decreased during cancer progression. Moreover, the expression level of TNF-α increased with tumor progression.

Conclusion

We conclude that the expression of genes related to immune response and inflammation is different between tumor tissues and control tissues. In addition, this difference was perpetuated through the different stages of cancer.

     

Time-course microarray analysis for identifying candidate genes involved in obesity-associated pathological changes in the mouse colon

Background

Obesity is known to increase the risk of colorectal cancer. However, mechanisms underlying the pathogenesis of obesity-induced colorectal cancer are not completely understood. The purposes of this study were to identify differentially expressed genes in the colon of mice with diet-induced obesity and to select candidate genes as early markers of obesity-associated abnormal cell growth in the colon.

Methods

C57BL/6N mice were fed normal diet (11% fat energy) or high-fat diet (40% fat energy) and were euthanized at different time points. Genome-wide expression profiles of the colon were determined at 2, 4, 8, and 12 weeks. Cluster analysis was performed using expression data of genes showing log₂ fold change of ≥1 or ≤?1 (twofold change), based on time-dependent expression patterns, followed by virtual network analysis.

Results

High-fat diet-fed mice showed significant increase in body weight and total visceral fat weight over 12 weeks. Time-course microarray analysis showed that 50, 47, 36, and 411 genes were differentially expressed at 2, 4, 8, and 12 weeks, respectively. Ten cluster profiles representing distinguishable patterns of genes differentially expressed over time were determined. Cluster 4, which consisted of genes showing the most significant alterations in expression in response to high-fat diet over 12 weeks, included Apoa4 (apolipoprotein A-IV), Ppap2b (phosphatidic acid phosphatase type 2B), Cel (carboxyl ester lipase), and Clps (colipase, pancreatic), which interacted strongly with surrounding genes associated with colorectal cancer or obesity.

Conclusions

Our data indicate that Apoa4, Ppap2b, Cel, and Clps are candidate early marker genes associated with obesity-related pathological changes in the colon. Genome-wide analyses performed in the present study provide new insights on selecting novel genes that may be associated with the development of diseases of the colon.

     

Iridoid and phenylethanoid/phenylpropanoid metabolite profiles of <Emphasis Type="Italic">Scrophularia</Emphasis> and <Emphasis Type="Italic">Verbascum</Emphasis> species used medicinally in North America

Introduction

Botanicals containing iridoid and phenylethanoid/phenylpropanoid glycosides are used worldwide for the treatment of inflammatory musculoskeletal conditions that are primary causes of human years lived with disability, such as arthritis and lower back pain.

Objectives

We report the analysis of candidate anti-inflammatory metabolites of several endemic Scrophularia species and Verbascum thapsus used medicinally by peoples of North America.

Methods

Leaves, stems, and roots were analyzed by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) and partial least squares-discriminant analysis (PLS-DA) was performed in MetaboAnalyst 3.0 after processing the datasets in Progenesis QI.

Results

Comparison of the datasets revealed significant and differential accumulation of iridoid and phenylethanoid/phenylpropanoid glycosides in the tissues of the endemic Scrophularia species and Verbascum thapsus.

Conclusions

Our investigation identified several species of pharmacological interest as good sources for harpagoside and other important anti-inflammatory metabolites.

     

integRATE: a desirability-based data integration framework for the prioritization of candidate genes across heterogeneous omics and its application to preterm birth

Background

The integration of high-quality, genome-wide analyses offers a robust approach to elucidating genetic factors involved in complex human diseases. Even though several methods exist to integrate heterogeneous omics data, most biologists still manually select candidate genes by examining the intersection of lists of candidates stemming from analyses of different types of omics data that have been generated by imposing hard (strict) thresholds on quantitative variables, such as P-values and fold changes, increasing the chance of missing potentially important candidates.

Methods

To better facilitate the unbiased integration of heterogeneous omics data collected from diverse platforms and samples, we propose a desirability function framework for identifying candidate genes with strong evidence across data types as targets for follow-up functional analysis. Our approach is targeted towards disease systems with sparse, heterogeneous omics data, so we tested it on one such pathology: spontaneous preterm birth (sPTB).

Results

We developed the software integRATE, which uses desirability functions to rank genes both within and across studies, identifying well-supported candidate genes according to the cumulative weight of biological evidence rather than based on imposition of hard thresholds of key variables. Integrating 10 sPTB omics studies identified both genes in pathways previously suspected to be involved in sPTB as well as novel genes never before linked to this syndrome. integRATE is available as an R package on GitHub (https://github.com/haleyeidem/integRATE).

Conclusions

Desirability-based data integration is a solution most applicable in biological research areas where omics data is especially heterogeneous and sparse, allowing for the prioritization of candidate genes that can be used to inform more targeted downstream functional analyses.

     

SNP markers associated with body size and pelt length in American mink (<Emphasis Type="Italic">Neovison vison</Emphasis>)

Background

Identification of genes underlying production traits is a key aim of the mink research community. Recent availability of genomic tools have opened the possibility for faster genetic progress in mink breeding. Availability of mink genome assembly allows genome-wide association studies in mink.

Results

In this study, we used genotyping-by-sequencing to obtain single nucleotide polymorphism (SNP) genotypes of 2496 mink. After multiple rounds of filtering, we retained 28,336 high quality SNPs and 2352 individuals for a genome-wide association study (GWAS). We performed the first GWAS for body weight, behavior, along with 10 traits related to fur quality in mink.

Conclusions

Combining association results with existing functional information of genes and mammalian phenotype databases, we proposed WWC3, MAP2K4, SLC7A1 and USP22 as candidate genes for body weight and pelt length in mink.

     

Selection and validation of reliable reference genes in <Emphasis Type="Italic">Gossypium raimondii</Emphasis>

Objective

To identify reliable reference genes for gene expression analysis in Gossypium raimondii.

Results

Five different software tools, geNorm, NormFinder, BestKeeper, ReFinder and ?Ct method were employed to analyze the qRT-PCR data systematically of 12 housekeeping genes. SAD and TUA11 showed relatively stable expression levels in all tissues (i.e. leaves, shoots, buds, and sepals). We then limited our analysis to each plant part and identified tissue-specific reference genes. Our results showed TUA11, TUB6 and EF1a, EF1a, MZA and GAPC2, MZA, GAPC2, SAD and TUA11, and UBQ and MZA were reliable reference genes in leaves, shoots, buds, and sepals, respectively.

Conclusion

Some genes were commonly identified as candidate reference genes in more than two tissue, while others were tissue-specific. Thus, our study allows choosing an appropriate control gene based on sampling for gene expression analysis.

     

Using marker gene analysis instead of mixed lymphocyte reaction assay for identification of functional CD4+FOXP3+ regulatory T cells

Objective

To establish a quick analytical method using quantitative PCR for marker gene analysis to identify the functions of iTreg cells and subsequently curtail the harvest time for iTreg cells.

Results

The data from the marker gene analysis indicated that varying proportions of iTreg cells could reveal the various expression levels of these genes. FoxP3 expression increased to a considerable degree. By using the same iTreg population, the mixed lymphocyte reaction assay was conducted for 5 days. The suppression percentage of T-cells was dependent on the proportion of iTreg cells, indicating that gene expression levels can represent the biological functions of iTreg cells. By using human peripheral blood mononuclear cells for Treg cell induction, the marker gene expression analysis showed a difference between iTreg cells and uninduced T cells.

Conclusion

Marker gene analysis requires only 1 day to identify the functions of human iTreg cells can save time in clinical application and might prevent graft-versus-host disease occurrence effectively.

     

New eight genes identified at the clinical multidrug-resistant <Emphasis Type="Italic">Acinetobacter baumannii</Emphasis> DMS06669 strain in a Vietnam hospital

Background

Acinetobacter baumannii is an important nosocomial pathogen that can develop multidrug resistance. In this study, we characterized the genome of the A. baumannii strain DMS06669 (isolated from the sputum of a male patient with hospital-acquired pneumonia) and focused on identification of genes relevant to antibiotic resistance.

Methods

Whole genome analysis of A. baumannii DMS06669 from hospital-acquired pneumonia patients included de novo assembly; gene prediction; functional annotation to public databases; phylogenetics tree construction and antibiotics genes identification.

Results

After sequencing the A. baumannii DMS06669 genome and performing quality control, de novo genome assembly was carried out, producing 24 scaffolds. Public databases were used for gene prediction and functional annotation to construct a phylogenetic tree of the DMS06669 strain with 21 other A. baumannii strains. A total of 18 possible antibiotic resistance genes, conferring resistance to eight distinct classes of antibiotics, were identified. Eight of these genes have not previously been reported to occur in A. baumannii.

Conclusions

Our results provide important information regarding mechanisms that may contribute to antibiotic resistance in the DMS06669 strain, and have implications for treatment of patients infected with A. baumannii.

     

Identification of suitable reference genes for real-time quantitative PCR analysis of hydrogen peroxide-treated human umbilical vein endothelial cells

Background

Oxidative stress can induce cell injury in vascular endothelial cells, which is the initial event in the development of atherosclerosis. Although quantitative real-time polymerase chain reaction (qRT-PCR) has been widely used in gene expression studies in oxidative stress injuries, using carefully validated reference genes has not received sufficient attention in related studies. The objective of this study, therefore, was to select a set of stably expressed reference genes for use in qRT-PCR normalization in oxidative stress injuries in human umbilical vein endothelial cells (HUVECs) induced by hydrogen peroxide (H₂O₂).

Results

Using geNorm analysis, we found that five stably expressed reference genes were sufficient for normalization in qRT-PCR analysis in HUVECs treated with H₂O₂. Genes with the most stable expression according to geNorm were U6, TFRC, RPLP0, GAPDH, and ACTB, and according to NormFinder were ALAS1, TFRC, U6, GAPDH, and ACTB.

Conclusion

Taken together, our study demonstrated that the expression stability of reference genes may differ according to the statistical program used. U6, TFRC, RPLP0, GAPDH, and ACTB was the optimal set of reference genes for studies on gene expression performed by qRT-PCR assays in HUVECs under oxidative stress study.

     

Simultaneous Cr(VI) reduction and Zn(II) biosorption by <Emphasis Type="Italic">Stenotrophomonas</Emphasis> sp. and constitutive expression of related genes

Objectives

To investigate simultaneous Cr(VI) reduction and Zn(II) biosorption by a microorganism.

Results

A new isolate, Stenotrophomonas sp. TD3, simultaneously reduced Cr(VI) and achieved Zn(II) biosorption. The strain was resistant to other metals and salts, and its Zn biosorption could be stimulated greatly by CaCl₂ and MgCl₂, which has not been reported previously. Three putative Cr- or Zn-related genes, chrR, zupT and hmrE, were amplified and measured using quantitative real-time PCR to evaluate their mRNA expression levels in Stenotrophomonas sp. The three putative genes were not sensitive to Cr⁶⁺, Zn²⁺, Ca²⁺, and Mg²⁺, which suggested that they were constitutively expressed in Stenotrophomonas sp. TD3.

Conclusions

These results improve our understanding of the mechanism and suggest that the strain is a potential candidate for facilitating remediation of Cr(VI) and Zn contaminated sites.