Gene (v.497, #2)

Editorial Board (iv-v).

Emerging evidence of the potent immunosuppressive activity of mesenchymal stem cells (MSCs) by modulation of both innate and adaptive immune responses enables MSCs to be developed as a promising therapeutic modality for immune-related or inflammatory diseases. However, it is not clearly understood how MSCs exert their immunosuppressive effects on immune cells under inflammatory conditions. Using human bone marrow (BM)-derived clonal MSCs (hcMSCs), we obtained and analyzed a differentially expressed gene profile when stimulated with the inflammatory cytokines interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) to find novel candidate factors responsible for MSC immunomodulation. Microarray analysis showed that 5650 genes were upregulated and 5862 genes were downregulated with the cutoff of 2-fold expression change. Among these, the ICOSLG and STAT2 genes were drastically upregulated 173-fold and 154-fold, respectively. Reverse transcription-polymerase chain reaction analysis confirmed the microarray data. To evaluate whether their increased expression is related to MSC-mediated immunosuppression, siRNA-induced ICOSLG- or STAT2-knockdown hcMSCs were assessed for their T cell suppressive activity. We demonstrated that STAT2 but not ICOSLG is functionally involved in the immunosuppressive activity of hcMSCs as a novel regulator under inflammatory conditions. Gene ontology and pathway analyses further support the immunomodulatory function of hcMSCs when inflammatory stimulation was provided. Taken together, this study provides an informative genome-wide gene expression profile and molecular evidence for understanding the mechanisms underlying the modulation of immune cells by human BM-derived MSCs under inflammatory conditions.► We examine gene expression profile of MSC in an experimental inflammatory condition. ► STAT2 is highly upregulated when MSC is stimulated by IFN-γ and TNF-α. ► STAT2 knockdown inhibits MSC-mediated T-cell suppression. ► STAT2 is found to be involved in the immunosuppression of MSC.
Keywords: Immunosuppression; Mesenchymal stem cell; STAT2; Microarray; Inflammation;

Chemical genetic profiling of the microtubule-targeting agent peloruside A in budding yeast Saccharomyces cerevisiae by Anja Wilmes; Reem Hanna; Rosemary W. Heathcott; Peter T. Northcote; Paul H. Atkinson; David S. Bellows; John H. Miller (140-146).
Peloruside A, a microtubule-stabilising agent from a New Zealand marine sponge, inhibits mammalian cell division by a similar mechanism to that of the anticancer drug paclitaxel. Wild type budding yeast Saccharomyces cerevisiae (haploid strain BY4741) showed growth sensitivity to peloruside A with an IC50 of 35 μM. Sensitivity was increased in a mad2Δ (Mitotic Arrest Deficient 2) deletion mutant (IC50  = 19 μM). Mad2 is a component of the spindle-assembly checkpoint complex that delays the onset of anaphase in cells with defects in mitotic spindle assembly. Haploid mad2Δ cells were much less sensitive to paclitaxel than to peloruside A, possibly because the peloruside binding site on yeast tubulin is more similar to mammalian tubulin than the taxoid site where paclitaxel binds. In order to obtain information on the primary and secondary targets of peloruside A in yeast, a microarray analysis of yeast heterozygous and homozygous deletion mutant sets was carried out. Haploinsufficiency profiling (HIP) failed to provide hits that could be validated, but homozygous profiling (HOP) generated twelve validated genes that interact with peloruside A in cells. Five of these were particularly significant: RTS1, SAC1, MAD1, MAD2, and LSM1. In addition to its known target tubulin, based on these microarray ‘hits’, peloruside A was seen to interact genetically with other cell proteins involved in the cell cycle, mitosis, RNA splicing, and membrane trafficking.► Peloruside A, a microtubule stabilising agent, inhibits yeast cell division. ► Paclitaxel, another stabilising agent, does not significantly inhibit yeast growth. ► Deletion of the mitotic checkpoint gene MAD2 increases sensitivity to peloruside. ► Using chemical genetics, genetic interactions with peloruside can be identified. ► Peloruside interacts with cell cycle genes, mitosis, RNA splicing, and trafficking.
Keywords: Peloruside; Paclitaxel; Chemical genetics; Yeast; MAD2; Saccharomyces cerevisiae;

A telomerase-associated RecQ protein-like helicase resolves telomeric G-quadruplex structures during replication by Jan Postberg; Maksym Tsytlonok; Daniela Sparvoli; Daniela Rhodes; Hans J. Lipps (147-154).
It is well established that G-quadruplex DNA structures form at ciliate telomeres and their formation throughout the cell-cycle by telomere-end-binding proteins (TEBPs) has been analyzed. During replication telomeric G-quadruplex structure has to be resolved to allow telomere replication by telomerase. It was shown that both phosphorylation of TEBPβ and binding of telomerase are prerequisites for this process, but probably not sufficient to unfold G-quadruplex structure in timely manner to allow replication to proceed. Here we describe a RecQ-like helicase required for unfolding of G-quadruplex structures in vivo. This helicase is highly reminiscent of human RecQ protein-like 4 helicase as well as other RecQ-like helicase found in various eukaryotes and E. coli. In situ analyses combined with specific silencing of either the telomerase or the helicase by RNAi and co-immunoprecipitation experiments demonstrate that this helicase is associated with telomerase during replication and becomes recruited to telomeres by this enzyme. In vitro assays showed that a nuclear extract prepared from cells in S-phase containing both the telomerase as well as the helicase resolves telomeric G-quadruplex structure. This finding can be incorporated into a mechanistic model about the replication of telomeric G-quadruplex structures during the cell cycle. ► Stylonychia StyRecQL is a helicase reminiscent of human RecQ protein-like 4. ► StyRecQL is enriched in replication band of the macronucleus during S-phase. ► StyRecQL colocalizes and is associated with Tert during telomere replication.
Keywords: Macronucleus; Replication band; Tert; Stylonychia lemnae; Ciliates;

A subset of human gliomas shows over-expression of KIT without its amplification by Masum Saini; Ajaya Nand Jha; Andleeb Abrari; Sher Ali (155-163).
Receptor tyrosine kinase (RTK) encoded by proto-oncogene KIT is known to be involved in different types of cancers. Reportedly, KIT expression has been associated with higher grade of gliomas. Initial RT-PCR based KIT expression observed in low grade glioma cases evoked our interest to ascertain its status in glioma patients who underwent resection during 2008–2009. Contrary to earlier reports, over-expression of the RTK was observed in 32.5% glioma cases across low/high grades (n = 40). Using quantitative PCR (qPCR), an up-regulation of the receptor (KIT) and its ligand (KITLG) was detected in most of the immunopositive cases at the transcript level. Sequence analysis of KIT showed two nucleotide substitutions in exons 10 and 17, in 4 and 2 cases, respectively though their pathological significance remained unclear. qPCR detected gene amplification in 2/13 glioma and allele loss in 1/13 glioma cases. This was in accordance with FISH results of these KIT positive neoplastic tissues. The data suggest that deranged expression of KIT is independent of gene amplification (p > 0.05). Aberrant KIT expression is significantly associated with transcriptional up-regulation (p < 0.001), though the precise mechanism(s) for transcriptional activation remain unclear.► Altered KIT expression in gliomas is independent of gene amplification. ► KIT expression is associated with transcriptional up-regulation. ► KIT over-expression in gliomas occurs across low/high grades. ► Most KIT immunopositive gliomas co-express receptor and ligand (KITLG) transcripts.
Keywords: Immunohistochemistry; Relative expression; Copy number variation; FISH; Sequencing;

Identification of two novel splicing variants of murine NTE-related esterase by Ping-An Chang; Zhan-Xiang Wang; Ding-Xin Long; Wen-zhen Qin; Chen-ying Wei; Yi-Jun Wu (164-171).
NTE-related esterase (NRE) is an insulin-regulated lysophospholipase with homology to neuropathy target esterase (NTE), which plays a role in energy metabolism. Here, we reported two alternative splicing variants of the murine NRE (mNRE) gene, termed mNREV1 and mNREV2. Genomic organization analysis indicated that 5′ splice site of mNRE intron 33 was changed in both mNREV1 and mNREV2, and mNRE exon 21 was deleted in mNREV2. mNREV1 had the same protein domains with mNRE, while mNREV2 lacked the patatin domain in the C-terminal catalytic region. Green fluorescent protein-mNREV1 or mNREV2 fusion proteins localized to the endoplasmic reticulum. mNREV1 and mNRE exhibited equal hydrolytic activity to the substrate phenyl valerate, whereas mNREV2 did not have any catalytic activity. The expression profiles of mNRE and its splicing isoforms in white adipose tissue, cardiac muscle, skeletal muscle, and testis tissues were further analyzed by real time quantitative-PCR in fed and fasted states, which indicated that the major isoform of mNRE mRNA generated switched from mNREV2 to mNREV1 during fasting. Thus there was a nutritional regulation of mNRE expression at the mRNA levels via alternative splicing.►mNREV1 and mNREV2, two alternative splice isoforms of mNRE gene were identified. ►mNREV1 had the same protein domains with mNRE, the patatin domain is absent in mNREV2. ►Similar to mNRE, mNREV1 and mNREV2 localized to the endoplasmic reticulum. ►mNREV1, but not mNREV2, exhibited equal enzyme activity of mNRE. ►There was a nutritional regulation of mNRE mRNA levels via splicing.
Keywords: NTE-related esterase; Splice variant; Esterase activity; Endoplasmic reticulum; Nutritional regulation;

Heat shock protein 70 (HSP70) acts mostly as a molecular chaperone and plays a key role in the process of protecting cells by facilitating the folding of nascent peptides and the cellular stress response. The cDNA of the oyster Crassostrea hongkongensis hsp70 (designated chhsp70) was cloned with the techniques of homological cloning and rapid amplification of cDNA ends (RACE). The full-length chhsp70 cDNA was 2251 bp, consisting of a 130 bp 5′-UTR, 216 bp 3′-UTR with a canonical polyadenylation signal sequence AATAAA and a poly (A) tail, and an open reading frame of 1905 bp, which encoded a polypeptide of 634 amino acids. Three classical HSP signature motifs were detected in ChHSP70, i.e., DLGTT-S-V, IFDLGGGTFDVSIL and VVLVGGSTRIPKIQK. BLAST analysis revealed that the ChHSP70 shared high identity with other bivalve HSP70. The phylogenetic analysis indicated that the ChHSP70 was a member of the HSP70 family. The chhsp70 mRNA transcripts were quantified by fluorescent real time RT-PCR under both unstressed and stressed conditions, i. e., heat shock and exposure to Cu2+ and malachite green. Basal expression level was similar in mantle, gill, digestive gland, and heart, but higher in muscle than that in the others. A similar trend showed that the chhsp70 mRNA expression significantly increased at 3–6 h, then dropped and returned to control level at 24 h in the five tissues and organs mentioned above after heat shock. A clearly time-dependent expression pattern of chhsp70 mRNA in digestive gland and gill of the oyster was observed after exposure of Cu2+ and malachite green. In the two tissues, the chhsp70 mRNA level reached the maximum at 6 h after malachite green exposure and on day 4 after Cu2+ exposure, and then decreased progressively to the control level. The results indicated that ChHSP70 of the oyster is an inducible protein, and plays an important role in response to the Cu2+ and malachite green polluted stress, so chhsp70 might be used as a potential molecular biomarker of above pollutants.► The cDNA of Crassostrea hongkongensis inducible hsp70 (chhsp70) was cloned. ► Sequence analysis results indicated that the ChHSP70 belonged to HSP70 family. ► Expression of chhsp70 rose obviously in gill and digestive gland after heat shock. ► Expression of chhsp70 after Cu2+ and malachite green exposure was measured. ► A time-dependent expression pattern was observed in gill and digestive gland.
Keywords: HSP70; Crassostrea hongkongensis; Expression pattern; Heat shock; Cu2+ and malachite green exposure;

Aberrant brain microRNA target and miRISC gene expression in the anx/anx anorexia mouse model by Josep M. Mercader; Juan R. González; Juan José Lozano; Mads Bak; Sakari Kauppinen; Lauro Sumoy; Mara Dierssen; Fernando Fernández-Aranda; Joana Visa; Mònica Gratacòs; Xavier Estivill (181-190).
The anorexia mouse model, anx/anx, carries a spontaneous mutation not yet identified and homozygous mutants are characterized by anorexia–cachexia, hyperactivity, and ataxia. In order to test if the microRNA function was altered in these mice, hypothalamus and cortex transcriptomes were evaluated and the data was analyzed taking into account the presence of microRNA target sites. Subsequent validation of the expression of a subset of miRISC coding genes and microRNA targets was performed by TaqMan real time PCR.In anx/anx hypothalamus we found that predicted microRNA targets were preferentially upregulated in a linearly dependent manner according to the number of microRNA target sites in each mRNA (p = 10− 139). Conversely, we observed that in anx/anx cortex mRNAs predicted to be targeted by microRNAs were preferentially downregulated (p < 10− 74), suggesting a de-regulation of genes targeted by microRNAs in two brain areas in anx/anx mice. A closer look to the mRNA transcriptome allowed us to identify upregulation of five miRISC genes, including Dgcr8 and Fmr1, and Ago2, which were later confirmed by real time PCR.The results suggest alteration of microRNA machinery expression in anx/anx mice and are consistent with its involvement in inflammatory/cancer-associated anorexia–cachexia. The data also support the previously reported link between microRNA machinery and ataxia. Further functional studies and the cloning of the anx gene should be pursued in order to elucidate the causality of microRNA machinery and microRNA target de-regulation, its relationship with the anx/anx phenotype and to propose this mouse as a model for microRNA research.► The microRNA target expression of two brain areas of the anx/anx mouse was analyzed. ► Enrichment of overexpressed miRNA targets in hypothalamus while depletion in cortex. ► mRNA expression was dependent on the number of miRNA target sites in each mRNA. ► Five miRISC genes, including Dgcr8 and Ago2 were upregulated in anx hypothalamus.
Keywords: miRISC; Microarray; Bioinformatics; Dicer; Argonaute; Dgcr8;

In this article, we characterized structure and expression of genes encoding the neuropeptide Corazonin (MdCrz) and its putative receptor (MdCrzR) in the House Fly, Musca domestica. The MdCrz gene contains two introns, one within the 5′ untranslated region and the other within the open reading frame. The 150-amino-acid precursor consists of an N-terminal signal peptide, and mature Crz followed by Crz-associated peptide (CAP). The CAP region is highly diverged from those of other insect precursors, whereas the mature Crz is identical in other dipteran members. In situ hybridization and immunohistochemistry consistently found a group of three MdCrz-producing neurons in the dorso-lateral protocerebrum, and eight pairs of bi-lateral neurons in the ventral nerve cord in the larvae. In adults, the expression was found exclusively in a cluster of five to seven neurons per brain lobe. Comparable expression patterns observed in other dipteran species suggest conserved regulatory mechanisms of Crz expression and functions during the course of evolution. MdCrzR deduced from the full-length cDNA sequence is a 655-amino acid polypeptide that contains seven trans-membrane (TM) domains and other motifs that are characteristics of Class-A G-protein coupled receptors. Although the TMs and loops between the TMs are conserved in other CrzRs, N-terminal extracellular domain is quite dissimilar. Tissue-specific RT-PCR revealed a high level of MdCrzR expression in the larval salivary glands and a moderate level in the CNS. In adults, the receptor was expressed both in the head and body, suggesting multifunctionality of the Crz signaling system.► Characterization of the Corazonin neuropeptide gene in Musca domestica. ► Comparative expression analysis of the Corazonin. ► Evolution of Corazonin gene structure in insects. ► Cloning and expression of the Corazonin receptor in the House Fly. ► Undergraduate research project.
Keywords: Evolution; Diptera; Neuropeptides; GPCR;

Recruitment of Oct-1 protein to the octamer sequence of U6 promoter is critical for optimal transcription by RNA polymerase III. Here we report that p38 kinase inhibitors, SB202190 and SB203580, stimulated U6 promoter activity and this stimulation can be observed only in the presence of octamer sequence. SB202190-treated cell nuclear extract had about 50% increase in Oct-1 binding activity suggesting that the increased U6 promoter activity by p38 kinase inhibitor is mediated through Oct-1. Mutation in octamer sequence significantly reduced the SB202190-stimulated U6 promoter transcription and the distance between octamer and proximal sequence element of U6 promoter is also critical for the p38 kinase inhibitor-stimulated activity. Exogenous Oct-1 expression showed a concentration-dependent activation of U6 promoter that was further stimulated by the p38 kinase inhibitors. When cells were treated with p38 kinase inducer, hydrogen peroxide or phorbol 12-myristate 13-acetate (PMA), U6 promoter activity was down regulated and this inhibition was reversed by p38 kinase inhibitors. Over-expression of p38α kinase down-regulated U6 promoter activity and this inhibition was further enhanced by PMA and p38 kinase inhibitors reversed this inhibition. p38 kinase inhibitor-treated cells had 50% more U6 RNA than the control cells. Taken together, our results show a negative correlation between the p38 kinase levels and Oct-1 binding on U6 promoter, suggesting that U6 promoter is negatively regulated by p38 kinase.► p38 kinase inhibitors, SB202190 and SB203580, enhanced transcription of U6 promoter. ► Stimulation of U6 promoter activity by SB202190 is dependent on octamer sequence. ► SB202190-treated nuclear extract showed increased level of octamer binding activity. ► p38 kinase inhibitor restored down-regulation of U6 promoter by stress, PMA or p38α. ► Cellular U6 RNA was augmented in SB202190-treated Jurkat cells.
Keywords: U6 promoter; Oct-1; p38 kinase; SB202190; MAPK;

A novel ganglioside-induced differentiation-associated protein 1 gene (BmGDAP1) was first cloned and sequenced from silkworm, Bombyx mori using rapid amplification of cDNA ends (RACE). The full-length cDNA of BmGDAP1 was 1514 bp, consisting of a 91 bp 5′ untranslated region (UTR), a 424 bp 3′-UTR and a 999 bp open reading frame (ORF). The ORF encoded a polypeptide of 332 amino acids, which possessed a thioredoxin (TRX)-like domain, a glutathione S-transferase-C (GST-C) family domain and a transmembrane segment. Furthermore, quantitative real-time PCR analysis revealed that BmGDAP1 transcripts were mainly presented in the tissues of hemocytes and midgut of silkworm, and its expression level was down-regulated in the hemocytes, while up-regulated in the midgut. Therefore, it could be concluded that BmGDAP1 plays an important role in the recognition and immune response of silkworm to BmCPV infection.► A novel GDAP1 gene was first cloned and sequenced from silkworm (Bombyx mori). ► BmGDAP1 was presented in all silkworm tissues, but mainly in hemocytes and midgut. ► Expression of BmGDAP1 was down-regulated in hemocytes during BmCPV infection. ► Expression of BmGDAP1 was up-regulated in midgut during BmCPV infection. ► BmGDAP1 may play a role in recognition and response of silkworm to BmCPV infection.
Keywords: Bombyx mori; Cytoplasmic polyhedrosis virus; Midgut; Expression analysis; Ganglioside-induced differentiation-associated protein 1; Rapid amplification of cDNA ends;

Phosphagen (guanidino) kinases (PK) constitute a family of homologous phosphotransferases catalyzing the reversible transfer of the high-energy phosphoryl group of ATP to naturally occurring guanidine compounds. Prior work has shown that PKs can be phylogenetically separated into two distinct groups— an arginine kinase (AK) subfamily and a creatine kinase (CK) subfamily. The latter includes three CK isoforms— cytoplasmic CK (CyCK), mitochondrial CK (MiCK) and three-domain flagellar CK (fCK). In the present study we identified six unique PK genes from the draft genome sequence of the starlet sea anemone Nematostella vectensis, a well-known model organism for understanding metazoan developmental evolution. Using reverse transcription polymerase chain reaction (RTPCR) methods, full length cDNAs were amplified for all of these PKs. These cDNAs were cloned and expressed in Escherichia coli as 6x His-tagged fusion proteins. The six PKs were identified as the three typical CK isoforms (CyCK, MiCK and fCK), two unusual AKs (a two-domain AK (2DAK) and a three-domain AK (3DAK)) and a PK which phosphorylated arginine. The latter enzyme had a very low AK activity (its apparent V max value being less than 0.2% that of 3DAK), lacks several key residues necessary for AK enzyme activity, and was tentatively designated as AK1. As far as we know, this constitutes the first report of an AK with the three fused AK domains. The Bayesian tree suggested that the third domain of 3DAK likely evolved from the gene for domain 2 of typical two-domain AK found widely in cnidarians. Construction of phylogenetic trees and comparison of exon–intron organizations of their respective genes indicated that the N. vectensis three-domain fCK and 3DAK evolved independently, and both enzymes are likely to be targeted to cell membranes since they have a myristoylation signal at their respective N-termini. These results complement prior work on other basal invertebrates showing that multiple CK and AK isoform genes were present at the dawn of the radiation of metazoans. The presence of isoform diversity in an organism lacking in structural complexity reflects an early imperative for targeting of PKs to particular cellular contexts such as muscle fibers, neurons, ciliated/flagellated epithelia and spermatozoa.► We have expressed six unique Phosphagen kinase genes from the Nematostella vectensis. ► The exon/intron organizations of the 78 phosphagen kinases genes were compared. ► Multiple CK and AK isoform genes were present at Nematostella.
Keywords: Guanidino kinase; Phosphagen kinase; Arginine kinase; Creatine kinase; Nematostella vectensis;

mRNA expression is widely used as a proxy for protein expression. However, their true relation is not known and two genes with the same mRNA levels might have different abundances of respective proteins. A related question is whether the coexpression of mRNA for gene pairs is reflected by the corresponding protein pairs.We examined the mRNA–protein correlation for both expression and coexpression. This analysis yielded insights into the relationship between mRNA and protein abundance, and allowed us to identify subsets of greater mRNA–protein coherence.The correlation between mRNA and protein was low for both expression and coexpression, 0.12 and 0.06 respectively. However, applying the best-performing quality measure, high-quality subsets reached a Spearman correlation of 0.31 for expression, 0.34 for coexpression and 0.49 for coexpression when restricted to functionally coupled genes. Our methodology can thus identify subsets for which the mRNA levels are expected to be the strongest correlated with protein levels.► Coherence between mRNA and protein abundances has been shown to be low. ► A low coherence implies that using mRNA as a proxy for protein might be unsuitable. ► We show that subsets of genes with greater mRNA–protein coherence can be identified. ► This is accomplished using quality measures based on expression profiles. ► Using high quality subsets improves mRNA's reliability as a proxy for protein.
Keywords: mRNA expression; mRNA coexpression; Protein expression; Protein coexpression; mRNA–protein expression concordance; Microarray;

The amplification status of proto oncogene HER-2/neu is one of the major molecular prognosis markers in breast cancer and recent adjuvant treatment with Trastuzumab has increased a request for the evaluation of HER-2/neu status in breast cancer. The aim of our study was the evaluation of HER-2/neu amplification status in malignant breast cancer by PCR techniques such as differential PCR and real time PCR and comparison of results of two methods together and with IHC results in some specimens.86 malignant breast cancer tissue specimens were analysed initially by differential PCR and then by real time PCR. Sections from paraffin-embedded or fresh tissue samples were homogenized by squash and then DNA extraction was performed on cell suspension. A standard curve was initially plotted using BioEasy SYBR Green I for using 2− ddct method. A 98 bp fragment of HER-2/neu gene was co-amplified in the same reaction tube with a 150 bp fragment of INFγ gene for differential PCR.The IHC results existed only for 27 of 83 assessed samples by dPCR and for 30 of 86 assessed samples by real time PCR. 29 out of 83 (35%) samples tested by dPCR and 28 out of 86 (32.5%) samples tested by real time PCR have HER-2/neu gene amplification.There was concordance between the results of real time PCR and differential PCR in 61 of 83 specimens (73.5%) tested by both method. Furthermore, in comparison of IHC results with these two methods, 70% concordance between IHC and differential PCR, 63% concordance between IHC and real time PCR and 55.5% concordance between three methods were observed.► The Her2/neu gene amplification has been assessed in 83 breast cancer patients. ► We have used real time PCR, differential PCR and immunohistochemistry. ► We have made a very precise comparison between these three different procedures. ► Very high concordances were seen between IHC and differential PCR or real time PCR. ► 55.5% concordance was seen between these three methods.
Keywords: Breast cancer; Gene amplification; HER-2/neu; Real time PCR; Differential PCR;

Identification of programmed cell death related genes in bamboo by Vineeta Rai; Nrisingha Dey (243-248).
The event of bamboo flowering and subsequent death of bamboo cells, a rare phenomenon is an interesting model to study gene expression/function in the context of the programmed cell death (PCD) in plant. To identify genes involved in autolytic cell death in bamboo (Bambusa arundinacea/Bambusa bambos Voss), a suppressive subtractive cDNA hybridization (SSH) was performed between cDNA isolated from control (healthy), as driver and test internodal tissue (45 days after setting of seeds), as tester. In-silico data revealed that 82% of total ESTs (231) were non-significant (unidentified proteins) while remaining ESTs were classified as protein with known/predicted function/s. Among these, net distribution and differential expression patterns of 11 important B. arundinacea PCD specific ESTs were studied using RNA slot-blot, qRT-PCR and semi-quantitative RT. In-situ localization of mRNA-transcripts for selected bamboo PCD-specific ESTs namely V2Ba48 (Aldehyde dehydrogenase 2) and V2Ba19 (Glycogen phosphorylase) were detected using digoxigenin-labeled corresponding anti-sense RNA probes employing Confocal Laser Scanning Microscope (CLSM). Differential expression-kinetics of the aforementioned genes were confirmed during the progress of PCD after setting of seeds. Global appearance of V2Ba48, V2Ba19, V2Ba95 (Ubiquitin thioesterase) and V2Ba89 (Nebulin isoform 2) genes were identified in monocot (Oryza sativa) and dicots (Arabidopsis thaliana and Nicotiana tabacum). This is the first report on systematic analysis of genes involved in death of bamboo cells that may provide critical information regarding key metabolic/regulatory genes involved in plant PCD.► Identification of genes involved in bamboo PCD after gregarious flowering. ► These genes work in an orchestral fashion. ► Also they are present in other plant species. ► Present study may provide strategies for manipulating PCD in plant.
Keywords: Bambusa arundinacea; Confocal Laser Scanning Microscope (CLSM); In-situ; Programmed cell death (PCD); SSH library;

A novel Acetyl-CoA synthetase short-chain subfamily member 1 (Acss1) gene indicates a dynamic history of paralogue retention and loss in vertebrates by L. Filipe C. Castro; Monica Lopes-Marques; Jonathan M. Wilson; Eduardo Rocha; Maria A. Reis-Henriques; Miguel M. Santos; Isabel Cunha (249-255).
Acetyl-CoA short chain synthetases (ACSSs) are key enzymes in the activation of fatty acids through the formation of thioesters with CoA. Three subfamily members are currently recognized in the human genome, ACSS1, ACSS2 and ACSS3, all single copy genes. The mitochondrial isoform, Acss1, plays a key role in the metabolism of acetate for energy production. While the single copy condition has been accurately established in humans, the evolutionary history of the Acss1 subfamily in vertebrates has yet to be elucidated, in particular, the isoform diversity, origin and function. Through genome database mining we analyzed the diversity of Acss1 isoforms in vertebrate classes. We detected the presence of a novel Acss1 isoform, which we name Acss1B. This new gene, Acss1B, has a curious phylogenetic distribution being found in teleosts (except zebrafish), sauropsids (birds and reptiles) and probably chondrichthyes. In contrast Acss1A is found in all the investigated species, except the teleost medaka. By means of comparative genomics and phylogenetics we show that Acss1A and Acss1B were generated in the quadruplication of the vertebrate genome. In effect, we find that amphioxus, a pre-genome duplication chordate, has a single Acss1 gene in a genomic region equally related to a quadrupled vertebrate genomic set. Consequently, Acss1B has been lost in some teleosts, amphibians and mammals, while Acss1A is probably absent in medaka. The reported findings illustrate an especially dynamic pattern of paralogue retention and independent loss in vertebrate species involving the Acss1 subfamily, whose functional consequences in energy metabolism are as yet unknown.► Acss1 is a key component of the fatty acid metabolism. ► A novel Acss1 gene sheds light into the evolutionary history of vertebrate Acss1. ► Acss1A/Acss1B resulted from genome duplications in vertebrate ancestry. ► The Acss1 gene family underwent events of paralogue loss and retention in vertebrates.
Keywords: ACSS1; Paralogue retention; Gene loss; Genome duplications;

Transcriptome analysis of tree peony during chilling requirement fulfillment: Assembling, annotation and markers discovering by Shupeng Gai; Yuxi Zhang; Ping Mu; Chunying Liu; Shao Liu; Lei Dong; Guosheng Zheng (256-262).
Tree peony (Paeonia suffruticosa Andrews) is a well-known horticultural and medicinal plant. The flower buds must go through a period of endo-dormancy before bud sprouting in winter, but very little information concerned with dormancy release is available. We obtained 625,342 sequencing reads with massive parallel pyrosequencing on the Roche 454 GS FLX platform (mean length: 358.1 bp). De novo assemblies yielded 23,652 contigs and singletons. 15,284 contigs longer than 300 bp were further annotated, among them 12,345 ESTs showed significant similarity with sequences present in public databases (with an E-value < 1e − 10). 484 putative transcription factors were obtained. In addition, 2253 potential Simple Sequence Repeats (SSR) loci were identified in the 454-ESTs. Total 149 pairs of primers were designed, and 121 pairs were amplified successfully in initial screening. In addition, 73 pairs of primers displayed polymorphism. This sequence collection provides a significant resource for gene discovery during endo-dormancy of tree peony.► A total of 23,652 assembled UniGenes were obtained with 15,284 annotated UniGenes with a minimal length of 300 bp. ► Many UniGenes were assigned to putative metabolic pathway. ► 484 putative transcription factors were obtained. ► 2253 potential Simple Sequence Repeats (SSR) loci were identified in the 454-ESTs. ► 73 pairs of primers displayed polymorphism between P. ostti and the two wild species (P. rockii and P. qiui) from 149 pairs designed according to our 454 EST collection.
Keywords: Paeonia suffruticosa Andrews; Transcriptome; Endo-dormancy; Chilling treatment; 454 pyrosequencing; SSR;

Construction and validation of a GFP-based vector for promoter expression analysis in the fish pathogen Flavobacterium psychrophilum by Esther Gómez; David Pérez-Pascual; Lucía Fernández; Jessica Méndez; Pilar Reimundo; Roberto Navais; José A. Guijarro (263-268).
The study of the fish pathogen Flavobacterium psychrophilum has been drastically hampered by the difficulty to perform genetic manipulation of this organism. Although recent publications described the successful transfer of genetic material into this bacterium by transformation and conjugation, additional tools are still needed. This paper reports the construction of vector pCP23-G, which permits for the first time to monitor transcriptional regulation in this pathogen by using a promoterless gfpmut3 gene as a reporter. Additionally, use of pCP23-G enabled the trancriptional analysis of three putative promoter regions of F. psychrophilum, corresponding to genes fpp2fpp1, pdhB and gldJ, under different growth conditions. Overall, the construction of pCP23-G facilitates genetic analysis in F. psychrophilum, by enabling the determination of gene expression both in vitro and in vivo. Furthermore, this would also open the possibility for studies on the location of this bacterium in the fish tissues.► The pCP23-G plasmid was constructed for transcriptional analysis in F. psychrophilum. ► The pCP23-βpth vector was useful for transcriptional analysis in F. psychrophilum. ► GFP fluorescence emission was analyzed by confocal microscopy and flow cytometry. ► fpp2fpp1 promoter was calcium- and temperature-regulated.
Keywords: GFP expression vector; Flavobacterium psycrophilum; Fish pathogen;

Tissue specific genes that contain high GC segments are difficult to amplify by standard PCR. We report an improved method for successful amplification of gene segment that has > 70% GC base pairs. This new method of touch down PCR differed by having an initial annealing temperature (Ta) 1.5 °C below the primers melting temperature that descended 0.2 °C per cycle for 20 cycles and continued thereafter at fixed Ta for next 15 cycles. Different co-solvents were tested with this method to improve the result and betaine proved better than the other co-solvents. This new method is economical, fast and specific in amplifying GC rich region of other genes also.► Initial annealing temperature below the primers Tm. ► Amplicon yield could effectively serve for RFLP, blotting and DNA sequencing. ► It is less time taking. ► Economic, requiring lower volumes of reaction mixture. ► Absence of spurious products that impede during RFLP and DNA sequencing.
Keywords: Touchdown PCR; GC rich; IRS2; Melting temperature (Tm); Co-solvents; Betaine;

Paradoxical role of C1561T glutamate carboxypeptidase II (GCPII) genetic polymorphism in altering disease susceptibility by Shree Divyya; Shaik Mohammad Naushad; Anthony Addlagatta; P.V.L.N. Murthy; Ch Ram Reddy; Raghunadha Rao Digumarti; Suryanarayana Raju Gottumukkala; Ajit Kumar; S. Rammurti; Vijay Kumar Kutala (273-279).
Glutamate carboxypeptidase II (GCPII) is predominantly expressed in brain, intestinal mucosa and prostate cancer in the form of three splice variants i.e. N-acetylated-α-linked acidic dipeptidase (NAALADase), folyl poly-γ-glutamate carboxypeptidase (FGCP) and prostate specific membrane antigen (PSMA) respectively. Its inhibition was found to confer protection against certain neurological disorders and cancer. Despite the pivotal role of this enzyme, the most common polymorphism i.e. H475Y has not been explored comprehensively in all its splice variants. In this study, we have determined the role of this variant in different disease conditions such as breast and prostate cancers, autism, coronary artery disease (CAD) and miscarriages (N = 1561). Genotyping was done by PCR-RFLP and dideoxy sequencing. Plasma folate levels were estimated by Axysm folate kit. GCPII expression was studied by semi-quantitative RT-PCR. In silico model was developed using PYMOL. We observed the protective role of H475Y variant in cancers [breast cancer; OR (95% CI): 0.81 (0.55–1.19), prostate cancer: OR (95% CI): 0.00 (0.00–0.66)], and in autism (OR (95% CI): 0.47 (0.21–1.03), whereas inflated risk was observed in CAD (OR (95% CI): 1.69 (1.20–2.37) and miscarriages [Maternal OR (95% CI): 3.26 (2.11–5.04); Paternal OR(95% CI): 1.99 (1.23–3.21)]. Further, this variant was found to impair the intestinal folate absorption in subjects with dietary folate intake in the lowest tertile (CC vs. CT in lowest tertile; 7.56 ± 0.85 ng/ml vs. 2.73 ± 045 ng/ml, p  = 0.005). In silico model of GCPII showed steric hindrance with H475Y resulting in stereochemical alteration of catalytic site, thus interfering with ligand binding. Statistically significant association was not observed between dietary folate levels and GCPII expression. However, a positive correlation was seen between plasma folate levels and GCPII expression (r = 0.70, p  < 0.05). To conclude, our data suggests that GCPII H475Y variant shows inverse association with autism and cancer while showing positive association with CAD and miscarriages.► GCPII H475Y confers protection against autism and cancer. ► GCPII H475Y inflates the risk for coronary artery disease and miscarriages. ► H475Y impairs intestinal absorption of folate when the dietary folate intake is low. ► GCPII expression correlates positively with plasma folate. ► Steric hindrance in ligand binding in H475Y variant.
Keywords: Glutamate carboxypeptidase II; Cancer; Coronary artery disease; Autism; Polymorphism;

A major imprinted gene involved in hydatidiform mole is not located in 2q31.2-qter or 5q34-qter by Helle Lund; Mette Nyegaard; Tove Svarrer; Anni Grove; Lone Sunde (280-284).
Hydatidiform mole is an abnormal human pregnancy, characterised by absent or abnormal embryonic differentiation, vesicular chorionic villi and trophoblastic hyperplasia. Although the mole phenotype has hereto not been correlated to mutations in the molar genome, the aetiology for hydatidiform moles clearly is genetic: Most molar genomes analysed either have had a relative excess of paternal genome sets relative to maternal genome sets, or a global error in maternally imprinted genes, giving them a “paternal pattern”. However it remains yet to be specified which gene(s) in the molar genome actually causes the molar phenotype when present in a state of “paternal excess” or “maternal deficiency”.A molar pregnancy in a woman with a balanced translocation (t(2;5) was subjected to histopathological evaluation and genetic analyses of ploidy and parental origin of the genome.Morphology: Partial hydatidiform mole. Karyotyping of metaphase chromosomes: 69,XXY,der(5)t(2;5)(q23;q33)mat. SNP array analysis mapped the breakpoints to 2q31.2 (genome position 179 Mb) and 5q34 (genome position 165 Mb). DNA microsatellite marker analysis showed that for the regions not involved in the translocation, the conceptus had two paternal and one maternal allele(s). Telomeric to the breakpoint on chromosome 2, the mole had two paternal and two maternal alleles and telomeric to the breakpoint on chromosome 5 the mole had paternal alleles, exclusively.If the molar phenotype is caused by paternal excess of one gene, only, it is unlikely that this gene is located telomeric to genome position 179 Mb on chromosome 2. And similarly, if the phenotype complete mole is caused by the presence of exclusively paternally imprinted alleles of one gene, this gene is not located telomeric to genome position 165 Mb on chromosome 5.► A triploid partial mole was seen in a women carrying a balanced translocation t(2;5). ► The mole was disomic androgenetic telomeric to the breakpoint on chromosome 5. ► It is unlikely that a gene causing the complete molar type is located in this region. ► On chromosome 2 the mole had balanced biparental genome telomeric to the breakpoint. ► A gene causing the molar phenotype is unlikely to be located in this region.
Keywords: Partial hydatidiform mole; Translocation; Mole gene; SNP analyses; Parental origin;

The aim of our study was to analyze combined impact of 17 polymorphisms at 8 gene regions previously shown to be associated with autoimmunity in diabetes. We hypothesized that the genetic predisposition is multiplicative and joint risk of different diabetic phenotypes forms by distinct combination of susceptibility loci.An ethnically homogenous population of Estonian origin, including 65 LADA patients, 154 patients with T1D, 260 patients with T2D and 229 non-diabetic controls, was genotyped for polymorphisms/haplotypes in HLA-DQB1, insulin gene (rs689, rs3842729), PHTF1–PTPN22 region (rs2476601, rs6679677), CTLA4 region (rs231806, rs16840252, rs5742909, rs231775, rs3087243, rs2033171), ICOS region (rs10932037, rs4675379), CD25 (rs706778), CD226(rs763361), NAA25 (rs17696736).As expected, the risk of T1D was consistently attributed by HLA-DQB1 haplotypes, but also by haplotypes of INS and PHTF1–PTPN22 region, and rs17696736 in NAA25. By contrast, LADA was associated only with T1D-protective HLA haplotypes and with two more frequent haplotypes of the CTLA4. It is of interest, that seldom CT haplotype of PHTF1–PTPN22 region carried the risk for autoantibody-negative T2D. The final best-fitted model for T1D genetic risk contained six gene regions (HLA-DQB1, INS, PHTF1, CTLA4 + 49, CD226 and NAA25) and for LADA only two (HLA-DQB1 and CTLA4 + 49). The AUCs of these models are 0.869 and 0.693, respectively.Classical T1D-risk haplotypes of HLA and some non-HLA loci describe quite well the genetic risk for T1D but not for LADA. The need of further studies should be stressed to discover the real risk factors for slower forms of autoimmune diabetes in adults.► Best-fitted genetic risk model of T1D contains HLA, INS, PHTF1, CTLA4, CD226, NAA25. ► Best-fitted genetic risk model of LADA contains only protective HLA-DQB1 and CTLA4. ► HLA and some non-HLA loci describe quite well genetic risk for T1D but not for LADA. ► LADA and T1D have two different genetic risk profiles. ► The genetic background for LADA may be lower than expected for T1D.
Keywords: Diabetes; T1D; T2D; LADA; Genetic;

Rapp–Hodgkin syndrome and SHFM1 patients: Delineating the p63–Dlx5/Dlx6 pathway by Ascensión Vera-Carbonell; María Rosa Moya-Quiles; María Ballesta-Martínez; Vanesa López-González; Juan Antonio Bafallíu; Encarna Guillén-Navarro; Isabel López-Expósito (292-297).
Rapp–Hodgkin Syndrome (RHS) is a genetic disorder resulting from mutations in the TP63 gene encoding p63 transcription factor. p63 is directly associated with a cis-regulatory element on chromosome 7q21 that controls the expression of DLX5 and DLX6 genes which are involved in craniofacial abnormalities and ectrodactyly or split hand/foot malformation (SHFM). Chromosomal deletions on 7q21 locus can result in loss of DXL5/DLX6 and/or in loss/disruption of cis-regulatory elements, at which p63 binds.We report two patients that have in common a p63–Dlx5/Dlx6 pathway dysregulation. One showed growth retardation, craniofacial dysmorphism, syndactyly, developmental delay and a de novo deletion (~ 8.5 Mb) on chromosome 7q21.13–q21.3, including DLX5 and DLX6. The second patient with a clinical diagnosis of RHS showed a de novo heterozygous missense mutation, c. 401G > A (p.G134D), in TP63 (exon 4). Our findings may contribute to a greater understanding of the pathogenic mechanisms underlying disorders caused by TP63 mutations.► The p63, Dlx5 and Dlx6 transcription factors share a common functional pathway. ► p63–Dlx5/Dlx6 pathway dysregulation responsible for craniofacial and SHFM. ► Patient with deletion on chromosome 7q21.13–q21.3, including DLX5 and DLX6, and SHFM. ► Patient with a heterozygous missense mutation in TP63 and ectodermal dysplasia. ► Different diagnostic strategies to patients with SHFM and/or ectodermal dysplasia.
Keywords: Rapp–Hodgkin syndrome; Split hand/foot malformation; p63-mutation analysis; Array-CGH analysis;

Since vascular risk factors commonly act for susceptibility to Alzheimer's disease (AD) and vascular dementia (VaD) by declining cognitive abilities, we conducted a genetic association study to identify their common underlying genetic factors. We selected single nucleotide polymorphisms (SNPs) which had been previously discovered for association with AD, and case and control associations of VaD were examined with the individual SNPs using 207 patients with VaD and 207 sex- and age-matched control subjects. As a result, no significant associations of susceptibility to VaD with 13 selected SNPs were observed even without employing a multiple test (P  > 0.05). This study suggests that genetics of VaD might be quite different from that of AD, and cautions should be taken especially when inferences about genetic factors are made with patients with mixed dementia.► We examine genetic associations of vascular dementia. ► No common genetic factors are found for vascular dementia and Alzheimer's disease. ► Genetics of vascular dementia might quite differ from that of Alzheimer's disease.
Keywords: Alzheimer's disease; Dementia; Genetic association; Single nucleotide polymorphism; Vascular cognitive impairment;

The present study was designed to investigate the underlying molecular mechanism for Angiotensin II type 1 receptor blockers (ARBs) mediated cardio-protection against pressure overload-induced cardiac remodeling with a focus on Smad7. ROCK-1, Smad3 and fibronectin expressions were increased in male C57BL/6 mice underwent transverse aortic constriction (TAC) for 2 weeks. Treatment with Candesartan (2 mg/kg per day) could effectively downregulate Smad3 and fibronectin accompanied by upregulating of Smad7. Further data showed that Candesartan inhibited TGF-β1 signal-induced epithelial-to-mesenchymal transition (EMT) through attenuating matrix metalloproteinases (MMP-9), such effect was abolished by knocking-down Smad7. Moreover, TAC for 2 weeks caused increased collagen deposition, thickness of left ventricular anterior and posterior wall at end-diastole (LVAWD and LVPWD) and LVEF% reduction, which were reversed by treatment with Candesartan, but failed after knocking-down Smad7. In addition, LV dP/dtmax and dP/dtmin were increased by TAC for 2 weeks, and treatment with Candesartan or Nifedipine effectively depressed the high levels of dP/dtmin induced by TAC. However, only Candesartan-mediated protective role in improving cardiac function was suppressed by tail-vein injection of Smad7 siRNA. This study uncovered a novel role for ARBs in preventing pressure overload-induced cardiac remodeling via Smad7 upregulation, which suppressed MMP-9 expression and TGF-β1 signal-mediated EMT progress.► We uncover the potential mechanism for ARB mediated effect on cardiac remodeling. ► Candesartan prevents cardiac fibrosis via upregulation of Smad7 gene. ► Candesartan induced Smad7 activation inhibits MMP-9 signal and MMP-9 dependent EMT. ► In vivo knock-down Smad7 model confirms the protective role of Candesartan.
Keywords: Candesartan; Smad7; TGF-β1; MMP-9; Cardiac remodeling;

Genetic and transcriptional organization of the groEL operon containing trxA in Gemella morbillorum by Wei-Chun Hung; Hsiao-Jan Chen; Sung-Pin Tseng; Shwu-Jen Liaw; Jui-Chang Tsai; Po-Ren Hsueh; Lee-Jene Teng (307-313).
Gemella morbillorum, a low G + C content Gram-positive bacterium, is considered to be a commensal organism in humans but occasionally causes endocarditis or other diseases. We determined the sequences of groESL, dnaK and their flanking regions in G. morbillorum. Sequence analysis revealed the presence of putative CtsR binding sites in both groE and dnaK operons, but the lack of CIRCE in groE and the presence of CIRCE in dnaK. This finding suggests in addition to the known regulatory systems for the class I heat shock protein genes, there may be another model in G. morbillorum. Furthermore, an unusual organization of the groE operon as groES-groEL-trxA was found. Genome sequence on GenBank database and southern blot indicate that there is only one copy of trxA in G. morbillorum. Sequencing of the groE locus from other Gemella species and clinical isolates revealed the same genetic structure, suggesting the conservation of the structure in Gemella species. Northern hybridization revealed that there were two transcripts, a large transcript, groES-groEL-trxA and a small transcript, trxA, in groE operon. Treatment of heat or diamide increased the transcription level of groES-groEL-trxA, whereas these two stresses did not affect the small trxA transcript. Thus, this study reveals that the trxA is co-transcribed with the groE operon, and most possibly under the control of the CtsR.► A novel genetic structure, groES-groEL-trxA, was found in Gemella morbillorum. ► No CIRCE in upstream region of groE, but there was putative CtsR binding site. ► There were two transcripts, groES-groEL-trxA (large) and trxA only (small). ► Treatment of heat or diamide increased the transcription level of groES-groEL-trxA.
Keywords: groEL; dnaK; Thioredoxin; Heat shock response;

Asthma is a complex multifactorial disease with an obvious genetic predisposition. Polymorphisms of the glutathione-S-transferase (GST) genes are known risk factors for some environmentally-related diseases. The aim of the present study was to investigate the role of polymorphisms in the GSTT1, GSTM1 and GSTP1 genes and asthma susceptibility in Egyptian children, and to analyze their effect on GST activity and lung function.GSTT1 and GSTM1 gene polymorphism was genotyped using the multiplex polymerase chain reaction (PCR) and GSTP1 ILe105Val polymorphism was determined using PCR-restriction fragment length polymorphism (PCR-RFLP) in 168 healthy and 126 asthmatic children (82 atopic and 44 nonatopic). Also GST enzyme activity and lung function were evaluated.Asthmatic children had a significant higher prevalence of the GSTM1 null (P  = 0.003) and significant lower prevalence of GSTP1 Val/Val genotypes (P  = 0.02) than control group. Lung function was significantly decreased in GSTM1 null genotype and GSTP1 Ile/Ile genotype. GSTP1 Val/Val genotypes and GSTM1 null genotype had a significant decrease in plasma GST activity.GST genes polymorphisms may play an important role in pathogenesis and susceptibility to asthma in children.
Keywords: Asthma; Egyptian children; Enzyme activity; Glutathione-S-transferase; Polymerase chain reaction;

A variant of unknown significance in the GLA gene causing diagnostic uncertainty in a young female with isolated hypertrophic cardiomyopathy by Khalid Al-Thihli; Hatim Ebrahim; Derralynn A. Hughes; Millan Patel; Marion Tipple; Ramona Salvarinova; Jane Gardiner; Hilary Vallance; Paula J. Waters (320-322).
Hypertrophic cardiomyopathy (HCM) is genetically heterogeneous, and largely caused by mutations in genes encoding sarcomere proteins. However, GLA mutations causing Fabry disease, an X-linked lysosomal storage disorder, may also present with isolated HCM. As HCM genetic testing panels are increasingly being used clinically, variants of unknown significance (VUS) are encountered, leading to challenges in interpretation. We present an illustrative case: a 10-year-old girl with isolated HCM who, on testing with a HCM multi-gene panel, was found to carry a maternally inherited p.W24R variant in GLA. Attempts to evaluate the significance of this variant, by direct biochemical testing of patient specimens, gave inconclusive results. Subsequent in vitro protein expression studies suggested that the variant is unlikely to be pathogenic. This case highlights diagnostic dilemmas that can be provoked by VUS in general, and specifically raises a question whether GLA sequencing should be included in first-line diagnostic testing for female children with isolated hypertrophic cardiomyopathy.► Functional analysis of a variant of unknown significance (VUS). ► Reviewing the natural history of hypertrophic cardiomyopathy in Fabry disease. ► Discussing challenges created by VUS in clinically variable disorders.
Keywords: GLA; Fabry disease; Hypertrophic cardiomyopathy; Variant of unknown significance; α-Galactosidase A;

SIRT3 gene expression: A link between inherited mitochondrial DNA variants and oxidative stress by Patrizia D'Aquila; Giuseppina Rose; Maria Luisa Panno; Giuseppe Passarino; Dina Bellizzi (323-329).
Signaling pathways between mitochondrial and nuclear genomes are activated to preserve cellular homeostasis, especially in the event of stress. Using cybrid cell lines, we investigated whether inherited mitochondrial DNA (mtDNA) variants modulate the expression profiles of mammalian sirtuins (SIRT1-7) under oxidative stress conditions. We found that the expression of the SIRT3 gene was down-regulated in cybrids harboring mtDNA of the J haplogroup, which correlated with mitochondrial function, resulting in a decline of NAD+/NADH and ATP levels. Overall, the data reported here highlight a link between SIRT3, mitochondrial DNA variability and mitochondrial functionality, three fundamental components of the cellular stress response.
Keywords: Sirtuins; Cybrid cell lines; mtDNA variability; NAD+/NADH; ATP;

Deep sequencing analysis of small non-coding RNAs reveals the diversity of microRNAs and piRNAs in the human epididymis by Yan Li; Hai-Yan Wang; Feng-Chun Wan; Fu-Jun Liu; Juan Liu; Na Zhang; Shao-Hua Jin; Jian-Yuan Li (330-335).
The epididymis plays a crucial role in regulating the development of sperm motility and fertilizing capacity. Small non-coding RNAs (sncRNAs), especially microRNAs (miRNAs), can participate in the regulation of various physiological pathways. However, their abundance and whether they are involved in the regulation of gene expression in the human epididymis are unknown. By adopting the Solexa deep sequencing approach, we systematically investigated the sncRNAs in the adult human epididymis. A total of 4903 unique sequences representing 527 known miRNA were discovered. Eighteen novel miRNA genes encoding 23 mature miRNAs were also identified and the expression of some of them was confirmed by qRT-PCR. The presence of Piwi-interacting RNAs (piRNAs) in the library also adds to the diversity of the sncRNA population in the human epididymis. This research will contribute to a preliminary database for their functional study in male reproductive system.► The sncRNA species were systematically investigated in the human epididymis. ► 527 known and 18 novel miRNA candidates were identified. ► Numerous piRNAs were identified in the human epididymis for the first time. ► This work lays the foundation for clarifying the sncRNA functions in epididymis.
Keywords: Epididymis; Small non-coding RNAs; miRNA; piRNA; Deep sequencing;

Study on the age-dependent tissue expression of FUT1 gene in porcine and its relationship to E. coli F18 receptor by Wen-Bin Bao; Lan Ye; Chen Zi; Xian-Min Su; Zhang-Yuan Pan; Jin Zhu; Guo-Qiang Zhu; Xiao-Guo Huang; Sheng-Long Wu (336-339).
Escherichia coli (E. coli) that produces adhesin F18 is the main pathogen responsible for porcine post-weaning diarrhea and edema disease. The receptor for E. coli F18 has not been described in pigs, however the alpha (1,2)-fucosyltransferase (FUT1) gene on chromosome 6 has been proposed as a candidate. The objective of this study, therefore, was to investigate the relationship between FUT1 gene expression and E. coli F18 receptor in Sutai pigs of different ages (8-, 18-, 30- and 35-day-old). FUT1 gene expression was detected in 11 pig tissues with the highest level in lung, and expressed consistently at the four time points. In most tissues, FUT1 gene expression levels decreased from days 8 to 18, then continually increased on days 30 and 35, with expression around weaning time higher than that on day 8. Gene ontology and pathway analysis showed that FUT1 was involved in 32 biological processes, mainly those integral to the membrane, or involved in glycosylation, as well as regulation of binding, interestingly participating in three pathways related to glycosphingolipid biosynthesis. From this analysis and the high linkage disequilibrium between the FUT1 gene and the E. coli F18 receptor locus, we can speculate that higher expression of the FUT1 gene in small intestine is beneficial to the formation of receptors to the E. coli F18 strain and is related to the sensitivity to the pathogen.► We compared the age-dependent tissue expression of FUT1gene in piglets. ► Expression of FUT1 decreased from days 8 to 18, and then increased to day 35. ► FUT1 higher expression is beneficial to the formation of E. coli F18 receptor. ► Up-regulation of FUT1 was related to the sensitivity to E. coli F18 in piglets.
Keywords: FUT1 gene; Gene expression; Escherichia coli F18; Real-time PCR; Sutai pig;

Saturated fatty acids, acting as ligands for toll-like receptor 4 (TLR4), induce inflammation and mediate the development of insulin resistance. Myeloid differentiation factor 88 (MyD88) is an adaptor protein for TLR4. Previously, we found MyD88-deificient mice fed a high-fat diet (HFD) exhibited a severe diabetic phenotype. Stearoyl-CoA Desaturase 1 (SCD1) is the rate-limiting enzyme in the biosynthesis of monounsaturated fatty acids and known as a risk factor of diabetes. In the present study, we found SCD1 was dramatically increased in HFD-fed MyD88-deficient mice liver. This finding showed the novel linkage between MyD88 and SCD1 in the development of diabetes mellitus.► We investigated mechanisms of diabetes observed in MyD88-deificient mice fed a HFD. ► MyD88-deficiency did not affect expression levels of G6Pase. ► MyD88-deficiency did not affect expression levels of GLUT2. ► SCD1 was up-regulated on the liver samples of MyD88-deificient mice fed a HFD. ► These results suggest MyD88 and SCD1 would link for the development of diabetes.
Keywords: MyD88; Diabetes; Obesity; SCD1; G6Pase; GLUT2;

Gene expression profile of the cynobacterium synechocystis genome by Shibsankar Das; Uttam Roymondal; Brajadulal Chottopadhyay; Satyabrata Sahoo (344-352).
The expression of functional proteins plays a crucial role in modern biotechnology. The free-living cynobacterium Synechocystis PCC 6803 is an interesting model organism to study oxygenic photosynthesis as well as other metabolic processes. Here we analyze a gene expression profiling methodology, RCBS (the scores of relative codon usage bias) to elucidate expression patterns of genes in the Synechocystis genome. To assess the predictive performance of the methodology, we propose a simple algorithm to calculate the threshold score to identify the highly expressed genes in a genome. Analysis of differential expression of the genes of this genome reveals that most of the genes in photosynthesis and respiration belong to the highly expressed category. The other genes with the higher predicted expression level include ribosomal proteins, translation processing factors and many hypothetical proteins. Only 9.5% genes are identified as highly expressed genes and we observe that highly expressed genes in Synechocystis genome often have strong compositional bias in terms of codon usage. An important application concerns the automatic detection of a set of impact codons and genes that are highly expressed tend to use this narrow set of preferred codons and display high codon bias .We further observe a strong correlation between RCBS and protein length indicating natural selection in favor of shorter genes to be expressed at higher level. The better correlations of RCBS with 2D electrophoresis and microarray data for heat shock proteins compared to the expression measure based on codon usage difference, E(g) and codon adaptive index, CAI indicate that the genomic expression profile available in our method can be applied in a meaningful way to study the mRNA expression patterns, which are by themselves necessary for the quantitative description of the biological states.► We analyze a gene expression profiling methodology in the Synechocystis genome. ► Most of the genes in photosynthesis and respiration belong to the highly expressed category. ► An automatic detection of a set of impact codons. ► A strong correlation between RCBS and protein length. ► The better correlations of RCBS with 2D electrophoresis and microarray data.
Keywords: Codon usage; Impact codons; Codon adaptation index; Gene expression; Predicted highly expressed genes; Synechocystis;