Selection of suitable reference genes for gene expression studies in HMC3 cell line by quantitative real-time RT-PCR.

Microglia represent the primary immune defense system within the central nervous system and play a role in the inflammatory processes occurring in numerous disorders, such as Parkinson's disease (PD). PD onset and progression are associated with factors considered possible causes of neuroinflammation, i.e. genetic mutations. In vitro models of microglial cells were established to identify specific molecular targets in PD through the analysis of gene expression data. Recently, the Human Microglial Clone 3 cell line (HMC3) has been characterized and a new human microglia model has emerged. Here we perform RT-qPCR analyses to evaluate the expression of ten reference genes in HMC3, untreated or stimulated to a pro-inflammatory status. The comparative ∆CT method, BestKeeper, Normfinder, geNorm and RefFinder algorithms were used to assess the stability of the candidate genes. The results showed that the most suitable internal controls are HPRT1, RPS18 and B2M genes. In addition, the most stable and unstable reference genes were used to normalize the expression of a gene of interest in HMC3, resulting in a difference in the statistical significance in cells treated with Rotenone. This is the first reference gene validation study in HMC3 cell line in pro-inflammatory status and can contribute to more reliable gene expression analysis in the field of neurodegenerative and neuroinflammatory research.

Cytochalasin B Modulates Nanomechanical Patterning and Fate in Human Adipose-Derived Stem Cells.

Cytoskeletal proteins provide architectural and signaling cues within cells. They are able to reorganize themselves in response to mechanical forces, converting the stimuli received into specific cellular responses. Thus, the cytoskeleton influences cell shape, proliferation, and even differentiation. In particular, the cytoskeleton affects the fate of mesenchymal stem cells (MSCs), which are highly attractive candidates for cell therapy approaches due to their capacity for self-renewal and multi-lineage differentiation. Cytochalasin B (CB), a cyto-permeable mycotoxin, is able to inhibit the formation of actin microfilaments, resulting in direct effects on cell biological properties. Here, we investigated for the first time the effects of different concentrations of CB (0.1-10 µM) on human adipose-derived stem cells (hASCs) both after 24 h (h) of CB treatment and 24 h after CB wash-out. CB influenced the metabolism, proliferation, and morphology of hASCs in a dose-dependent manner, in association with progressive disorganization of actin microfilaments. Furthermore, the removal of CB highlighted the ability of cells to restore their cytoskeletal organization. Finally, atomic force microscopy (AFM) revealed that cytoskeletal changes induced by CB modulated the viscoelastic properties of hASCs, influencing their stiffness and viscosity, thereby affecting adipogenic fate.

The crucial role of non-enzymatic NO-production in plants. An EPR study.

Polyamines and polyamides have a fundamental role in the biology of plants, and the presence of NO seems compulsory to account for their actions. In general, the NO production has claimed to occur through an enzymatic process, but not involving polyamines and polyamides. Nevertheless, a non-enzymatic mechanism, such as an electron transfer process among polyamines or polyamides and an acid nitrite solution, could account for rapid production of NO, even in anoxic conditions. EPR experiments, carried out with these substrates, proved the formation of NO. This evidence supports a non-enzymatic mechanism as an alternative source of NO, even in plants. So, since the NO production seems directly dependent on polyamines or polyamides presence, and these responsible for many activities in plants, it comes plausibly to consider crucial the involvement of NO in their actions. Furthermore, as for mammals, these results would confirm that, even in plants, NO production can occur through both enzymatic and non-enzymatic mechanisms.

Herb-Derived Products: Natural Tools to Delay and Counteract Stem Cell Senescence.

Cellular senescence plays a very important role in organismal aging increasing with age and in age-related diseases (ARDs). This process involves physiological, structural, biochemical, and molecular changes of cells, leading to a characteristic trait referred to "senescence-associated secretory phenotype (SASP)." In particular, with aging, stem cells (SCs) in situ exhibit a diminished capacity of self-renewal and show a decline in their functionality. The identification of interventions able to prevent the accumulation of senescent SCs in the organism or to pretreat cultured multipotent mesenchymal stromal cells (MSCs) prior to employing them for cell therapy is a main purpose of medical research. Many approaches have been investigated and resulted effective to prevent or counteract SC senescence in humans, as well as other animal models. In this work, we have reviewed the chance of using a number of herb-derived products as novel tools in the treatment of cell senescence, highlighting the efficacy of these agents, often still far from being clearly understood.

Sex-Specific Transcriptome Differences in Human Adipose Mesenchymal Stem Cells.

In humans, sexual dimorphism can manifest in many ways and it is widely studied in several knowledge fields. It is increasing the evidence that also cells differ according to sex, a correlation still little studied and poorly considered when cells are used in scientific research. Specifically, our interest is on the sex-related dimorphism on the human mesenchymal stem cells (hMSCs) transcriptome. A systematic meta-analysis of hMSC microarrays was performed by using the Transcriptome Mapper (TRAM) software. This bioinformatic tool was used to integrate and normalize datasets from multiple sources and allowed us to highlight chromosomal segments and genes differently expressed in hMSCs derived from adipose tissue (hADSCs) of male and female donors. Chromosomal segments and differentially expressed genes in male and female hADSCs resulted to be related to several processes as inflammation, adipogenic and neurogenic differentiation and cell communication. Obtained results lead us to hypothesize that the donor sex of hADSCs is a variable influencing a wide range of stem cell biologic processes. We believe that it should be considered in biologic research and stem cell therapy.

Correction to "Discovery of the First-in-Class GSK-3ß/HDAC Dual Inhibitor as Disease-Modifying Agent To Combat Alzheimer's Disease".

[This corrects the article DOI: 10.1021/acsmedchemlett.8b00507.].

Early Developmental Zebrafish Embryo Extract to Modulate Senescence in Multisource Human Mesenchymal Stem Cells.

Stem cells undergo senescence both in vivo, contributing to the progressive decline in self-healing mechanisms, and in vitro during prolonged expansion. Here, we show that an early developmental zebrafish embryo extract (ZF1) could act as a modulator of senescence in human mesenchymal stem cells (hMSCs) isolated from both adult tissues, including adipose tissue (hASCs), bone marrow (hBM-MSCs), dental pulp (hDP-MSCs), and a perinatal tissue such as the Wharton's Jelly (hWJ-MSCs). In all the investigated hMSCs, ZF1 decreased senescence-associated ß-galactosidase (SA ß-gal) activity and enhanced the transcription of TERT, encoding the catalytic telomerase core. In addition, it was associated, only in hASCs, with a transcriptional induction of BMI1, a pleiotropic repressor of senescence. In hBM-MSCs, hDP-MSCs, and hWJ-MSCs, TERT over-expression was concomitant with a down-regulation of two repressors of TERT, TP53 (p53), and CDKN1A (p21). Furthermore, ZF1 increased the natural ability of hASCs to perform adipogenesis. These results indicate the chance of using ZF1 to modulate stem cell senescence in a source-related manner, to be potentially used as a tool to affect stem cell senescence in vitro. In addition, its anti-senescence action could also set the basis for future in vivo approaches promoting tissue rejuvenation bypassing stem cell transplantation.

Discovery of the First-in-Class GSK-3ß/HDAC Dual Inhibitor as Disease-Modifying Agent To Combat Alzheimer's Disease.

Several evidence pointed out the role of epigenetics in Alzheimer's disease (AD) revealing strictly relationships between epigenetic and "classical" AD targets. Based on the reported connection among histone deacetylases (HDACs) and glycogen synthase kinase 3ß (GSK-3ß), herein we present the discovery and the biochemical characterization of the first-in-class hit compound able to exert promising anti-AD effects by modulating the targeted proteins in the low micromolar range of concentration. Compound 11 induces an increase in histone acetylation and a reduction of tau phosphorylation. It is nontoxic and protective against H2O2 and 6-OHDA stimuli in SH-SY5Y and in CGN cell lines, respectively. Moreover, it promotes neurogenesis and displays immunomodulatory effects. Compound 11 shows no lethality in a wt-zebrafish model (<100 µM) and high water solubility.

Sex-Specific Transcriptome Differences in Substantia Nigra Tissue: A Meta-Analysis of Parkinson's Disease Data.

Parkinson's disease (PD) is one of the most common progressive neurodegenerative diseases. Clinical and epidemiological studies indicate that sex differences, as well as genetic components and ageing, can influence the prevalence, age at onset and symptomatology of PD. This study undertook a systematic meta-analysis of substantia nigra microarray data using the Transcriptome Mapper (TRAM) software to integrate and normalize a total of 10 suitable datasets from multiple sources. Four different analyses were performed according to default parameters, to better define the segments differentially expressed between PD patients and healthy controls, when comparing men and women data sets. The results suggest a possible regulation of specific sex-biased systems in PD susceptibility. TRAM software allowed us to highlight the different activation of some genomic regions and loci involved in molecular pathways related to neurodegeneration and neuroinflammatory mechanisms.

Difficulty in obtaining the complete mRNA coding sequence at 5' region (5' end mRNA artifact): Causes, consequences in biology and medicine and possible solutions for obtaining the actual amino acid sequence of proteins (Review).

The known difficulty in obtaining the actual full length, complete sequence of a messenger RNA (mRNA) may lead to the erroneous determination of its coding sequence at the 5' region (5' end mRNA artifact), and consequently to the wrong assignment of the translation start codon, leading to the inaccurate prediction of the encoded polypeptide at its amino terminus. Among the known human genes whose study was affected by this artifact, we can include disco interacting protein 2 homolog A (DIP2A; KIAA0184), Down syndrome critical region 1 (DSCR1), SON DNA binding protein (SON), trefoil factor 3 (TFF3) and URB1 ribosome biogenesis 1 homolog (URB1; KIAA0539) on chromosome 21, as well as receptor for activated C kinase 1 (RACK1, also known as GNB2L1), glutaminyl­tRNA synthetase (QARS) and tyrosyl-DNA phosphodiesterase 2 (TDP2) along with another 474 loci, including interleukin 16 (IL16). In this review, we discuss the causes of this issue, its quantitative incidence in biomedical research, the consequences in biology and medicine, and the possible solutions for obtaining the actual amino acid sequence of proteins in the post-genomics era.

Meta-Analysis of Parkinson's Disease Transcriptome Data Using TRAM Software: Whole Substantia Nigra Tissue and Single Dopamine Neuron Differential Gene Expression.

The understanding of the genetic basis of the Parkinson's disease (PD) and the correlation between genotype and phenotype has revolutionized our knowledge about the pathogenetic mechanisms of neurodegeneration, opening up exciting new therapeutic and neuroprotective perspectives. Genomic knowledge of PD is still in its early stages and can provide a good start for studies of the molecular mechanisms that underlie the gene expression variations and the epigenetic mechanisms that may contribute to the complex and characteristic phenotype of PD. In this study we used the software TRAM (Transcriptome Mapper) to analyse publicly available microarray data of a total of 151 PD patients and 130 healthy controls substantia nigra (SN) samples, to identify chromosomal segments and gene loci differential expression. In particular, we separately analyzed PD patients and controls data from post-mortem snap-frozen SN whole tissue and from laser microdissected midbrain dopamine (DA) neurons, to better characterize the specific DA neuronal expression profile associated with the late-stage Parkinson's condition. The default "Map" mode analysis resulted in 10 significantly over/under-expressed segments, mapping on 8 different chromosomes for SN whole tissue and in 4 segments mapping on 4 different chromosomes for DA neurons. In conclusion, TRAM software allowed us to confirm the deregulation of some genomic regions and loci involved in key molecular pathways related to neurodegeneration, as well as to provide new insights about genes and non-coding RNA transcripts not yet associated with the disease.

Characterization of human gene locus CYYR1: a complex multi-transcript system.

Cysteine/tyrosine-rich 1 (CYYR1) is a gene we previously identified on human chromosome 21 starting from an in-depth bioinformatics analysis of chromosome 21 segment 40/105 (21q21.3), where no coding region had previously been predicted. CYYR1 was initially characterized as a four-exon gene, whose brain-derived cDNA sequencing predicts a 154-amino acid product. In this study we provide, with in silico and in vitro analyses, the first detailed description of the human CYYR1 locus. The analysis of this locus revealed that it is composed of a multi-transcript system, which includes at least seven CYYR1 alternative spliced isoforms and a new CYYR1 antisense gene (named CYYR1-AS1). In particular, we cloned, for the first time, the following isoforms: CYYR1-1,2,3,4b and CYYR1-1,2,3b, which present a different 3' transcribed region, with a consequent different carboxy-terminus of the predicted proteins; CYYR1-1,2,4 lacks exon 3; CYYR1-1,2,2bis,3,4 presents an additional exon between exon 2 and exon 3; CYYR1-1b,2,3,4 presents a different 5' untranslated region when compared to CYYR1. The complexity of the locus is enriched by the presence of an antisense transcript. We have cloned a long transcript overlapping with CYYR1 as an antisense RNA, probably a non-coding RNA. Expression analysis performed in different normal tissues, tumour cell lines as well as in trisomy 21 and euploid fibroblasts has confirmed a quantitative and qualitative variability in the expression pattern of the multi-transcript locus, suggesting a possible role in complex diseases that should be further investigated.

Improving mRNA 5' coding sequence determination in the mouse genome.

The incomplete determination of the mRNA 5' end sequence may lead to the incorrect assignment of the first AUG codon and to errors in the prediction of the encoded protein product. Due to the significance of the mouse as a model organism in biomedical research, we performed a systematic identification of coding regions at the 5' end of all known mouse mRNAs, using an automated expressed sequence tag (EST)-based approach which we have previously described. By parsing almost 4 million BLAT alignments we found 351 mouse loci, out of 20,221 analyzed, in which an extension of the mRNA 5' coding region was identified. Proof-of-concept confirmation was obtained by in vitro cloning and sequencing for Apc2 and Mknk2 cDNAs. We also generated a list of 16,330 mouse mRNAs where the presence of an in-frame stop codon upstream of the known start codon indicates completeness of the coding sequence at 5' end in the current form. Systematic searches in the main mouse genome databases and genome browsers showed that 82% of our results are original and have not been identified by their annotation pipelines. Moreover, the same information is not easily derivable from RNA-Seq data, due to short sequence length and laboriousness in building full-length transcript structures. In conclusion, our results improve the determination of full-length 5' coding sequences and might be useful in order to reduce errors when studying mouse gene structure and function in biomedical research.

An estimation of the number of cells in the human body.

BACKGROUND: All living organisms are made of individual and identifiable cells, whose number, together with their size and type, ultimately defines the structure and functions of an organism. While the total cell number of lower organisms is often known, it has not yet been defined in higher organisms. In particular, the reported total cell number of a human being ranges between 10(12) and 10(16) and it is widely mentioned without a proper reference. AIM: To study and discuss the theoretical issue of the total number of cells that compose the standard human adult organism. SUBJECTS AND METHODS: A systematic calculation of the total cell number of the whole human body and of the single organs was carried out using bibliographical and/or mathematical approaches. RESULTS: A current estimation of human total cell number calculated for a variety of organs and cell types is presented. These partial data correspond to a total number of 3.72 × 10(13). CONCLUSIONS: Knowing the total cell number of the human body as well as of individual organs is important from a cultural, biological, medical and comparative modelling point of view. The presented cell count could be a starting point for a common effort to complete the total calculation.

Genome-scale analysis of human mRNA 5' coding sequences based on expressed sequence tag (EST) database.

The "5' end mRNA artifact" issue refers to the incorrect assignment of the first AUG codon in an mRNA, due to the incomplete determination of its 5' end sequence. We performed a systematic identification of coding regions at the 5' end of all human known mRNAs, using an automated expressed sequence tag (EST)-based approach. Following parsing of more than 7 million BLAT alignments, we found 477 human loci, out of 18,665 analyzed, in which an extension of the mRNA 5' coding region was identified. Proof-of-concept confirmation was obtained by in vitro cloning and sequencing for GNB2L1, QARS and TDP2 cDNAs, and the consequences for the functional studies of these loci are discussed. We also generated a list of 20,775 human mRNAs where the presence of an in-frame stop codon upstream of the known start codon indicates completeness of the coding sequence at 5' in the current form.

Complexity of bidirectional transcription and alternative splicing at human RCAN3 locus.

Human RCAN3 (regulator of calcineurin 3) belongs to the human RCAN gene family.In this study we provide, with in silico and in vitro analyses, the first detailed description of the human multi-transcript RCAN3 locus. Its analysis revealed that it is composed of a multigene system that includes at least 21 RCAN3 alternative spliced isoforms (16 of them identified here for the first time) and a new RCAN3 antisense gene (RCAN3AS). In particular, we cloned RCAN3-1,3,4,5 (lacking exon 2), RCAN3-1a,2,3,4,5, RCAN3-1a,3,4,5, RCAN3-1b,2,3,4,5, RCAN3-1c,2,3,4,5, RCAN3-1c,2,4,5 and RCAN3-1c,3,4,5, isoforms that present a different 5' untranslated region when compared to RCAN3. Moreover, in order to verify the possible 5' incompleteness of previously identified cDNA isoforms with the reference exon 1, ten more alternative isoforms were retrieved. Bioinformatic searches allowed us to identify RCAN3AS, which overlaps in part with exon 1a, on the opposite strand, for which four different RCAN3AS isoforms were cloned.In order to analyze the different expression patterns of RCAN3 alternative first exons and of RCAN3AS mRNA isoforms, RT-PCR was performed in 17 human tissues. Finally, analyses of RCAN3 and RCAN3AS genomic sequences were performed to identify possible promoter regions, to examine donor and acceptor splice sequences and to compare evolutionary conservation, in particular of alternative exon 1 or 1c--exon 2 junctions in different species.The description of its number of transcripts, of their expression patterns and of their regulatory regions can be important to clarify the functions of RCAN3 gene in different pathways and cellular processes.

TRAM (Transcriptome Mapper): database-driven creation and analysis of transcriptome maps from multiple sources.

BACKGROUND: Several tools have been developed to perform global gene expression profile data analysis, to search for specific chromosomal regions whose features meet defined criteria as well as to study neighbouring gene expression. However, most of these tools are tailored for a specific use in a particular context (e.g. they are species-specific, or limited to a particular data format) and they typically accept only gene lists as input. RESULTS: TRAM (Transcriptome Mapper) is a new general tool that allows the simple generation and analysis of quantitative transcriptome maps, starting from any source listing gene expression values for a given gene set (e.g. expression microarrays), implemented as a relational database. It includes a parser able to assign univocal and updated gene symbols to gene identifiers from different data sources. Moreover, TRAM is able to perform intra-sample and inter-sample data normalization, including an original variant of quantile normalization (scaled quantile), useful to normalize data from platforms with highly different numbers of investigated genes. When in 'Map' mode, the software generates a quantitative representation of the transcriptome of a sample (or of a pool of samples) and identifies if segments of defined lengths are over/under-expressed compared to the desired threshold. When in 'Cluster' mode, the software searches for a set of over/under-expressed consecutive genes. Statistical significance for all results is calculated with respect to genes localized on the same chromosome or to all genome genes. Transcriptome maps, showing differential expression between two sample groups, relative to two different biological conditions, may be easily generated. We present the results of a biological model test, based on a meta-analysis comparison between a sample pool of human CD34+ hematopoietic progenitor cells and a sample pool of megakaryocytic cells. Biologically relevant chromosomal segments and gene clusters with differential expression during the differentiation toward megakaryocyte were identified. CONCLUSIONS: TRAM is designed to create, and statistically analyze, quantitative transcriptome maps, based on gene expression data from multiple sources. The release includes FileMaker Pro database management runtime application and it is freely available at http://apollo11.isto.unibo.it/software/, along with preconfigured implementations for mapping of human, mouse and zebrafish transcriptomes.

Identification of housekeeping genes suitable for gene expression analysis in the zebrafish.

Housekeeping (HK) genes are constitutively expressed in order to maintain cellular function. They produce the minimal essential transcripts necessary for normal cellular physiology. Wide range expression, stable expression level and high expression level are independent features of a single gene expression and are all desirable for the definition of an "ideal" HK. Recent studies have questioned the possible existence of "ideal" HK mRNAs, mainly because of the wide expression conditions variability. This would imply that for each investigated organism the suitability of a putative HK should be verified. We perform a systematic analysis to identify "optimal" HK genes in Danio rerio (zebrafish), to be used in expression analyses conducted on embryos/larvae at different developmental stages, as well as on differentiated adult tissues from single donors. The expression pattern of candidate genes, selected on the basis of the literature available and of ad hoc bioinformatics analysis, was assessed by quantitative relative RT-PCR in an RNA panel, including six different embryo/larvae developmental stages and six adult tissues. Statistical analysis was performed to identify genes with the lowest expression standard deviation in the studied panel. Our results showed that beta-actin 2 (bactin2) is the mRNA with the lowest variability of expression.

Identification and analysis of human RCAN3 (DSCR1L2) mRNA and protein isoforms.

Human RCAN3 (Regulator of calcineurin 3; previously known as DSCR1L2, Down syndrome critical region gene 1-like 2) is a five-exon gene mapped on chromosome 1 and belongs to the human RCAN gene family which also includes RCAN1 and RCAN2. The novel denomination RCAN for genes and proteins, instead of DSCR1L (Down syndrome critical region gene 1-like) has recently been widely discussed. The aim of the present work was to perform a multiple approach analysis of five RCAN3 mRNA and encoded protein isoforms, two of which have been identified for the first time in this research. The two new RCAN3 mRNA isoforms, RCAN3-2,4,5, which lacks exon 3, and RCAN3-2,3,5, which lacks exon 4, were identified during RCAN3 RT-PCR (reverse transcription-polymerase chain reaction) cloning, the product of which unexpectedly revealed the presence of five isoforms as opposed to the three previously known. In order to analyze the expression pattern of the five RCAN3 mRNA isoforms in seven different human tissues, a quantitative relative RT-PCR was performed: interestingly, all isoforms are present in all tissues investigated, with a statistically significant constant prevalence of RCAN3 isoform (the most complete, "reference" isoform). The RCAN3 locus expression level was comparable in all seven tissues analyzed, considering all isoforms, which indicates a ubiquitous expression of this human RCAN family member. To date two possible interactors have been described for this protein: human cardiac troponin I (TNNI3) and calcineurin. Here we report the interaction between the new RCAN3 variants and TNNI3, demonstrated by both yeast cotransformation and by the GST (glutathione-sepharose transferase) fusion protein assay, as was to be expected from the presence of exon 2 whose product has been seen to be sufficient for binding to TNNI3.

Systematic analysis of mRNA 5' coding sequence incompleteness in Danio rerio: an automated EST-based approach.

BACKGROUND: All standard methods for cDNA cloning are affected by a potential inability to effectively clone the 5' region of mRNA. The aim of this work was to estimate mRNA open reading frame (ORF) 5' region sequence completeness in the model organism Danio rerio (zebrafish). RESULTS: We implemented a novel automated approach (5'_ORF_Extender) that systematically compares available expressed sequence tags (ESTs) with all the zebrafish experimentally determined mRNA sequences, identifies additional sequence stretches at 5' region and scans for the presence of all conditions needed to define a new, extended putative ORF. Our software was able to identify 285 (3.3%) mRNAs with putatively incomplete ORFs at 5' region and, in three example cases selected (selt1a, unc119.2, nppa), the extended coding region at 5' end was cloned by reverse transcription-polymerase chain reaction (RT-PCR). CONCLUSION: The implemented method, which could also be useful for the analysis of other genomes, allowed us to describe the relevance of the "5' end mRNA artifact" problem for genomic annotation and functional genomic experiment design in zebrafish.