A comparative study of Gaussian Graphical Model approaches for genomic data

The inference of networks of dependencies by Gaussian Graphical models on high-throughput data is an open issue in modern molecular biology. In this paper we provide a comparative study of three methods to obtain small sample and high dimension estimates of partial correlation coefficients: the Moore-Penrose pseudoinverse (PINV), residual correlation (RCM) and covariance-regularized method $(\ell_{2C})$. We first compare them on simulated datasets and we find that PINV is less stable in terms of AUC performance when the number of variables changes. The two regularized methods have comparable performances but $\ell_{2C}$ is much faster than RCM. Finally, we present the results of an application of $\ell_{2C}$ for the inference of a gene network for isoprenoid biosynthesis pathways in Arabidopsis thaliana.Comment: 7 pages, 1 figure, RevTex4, version to appear in the proceedings of 1st International Workshop on Pattern Recognition, Proteomics, Structural Biology and Bioinformatics: PR PS BB 2011, Ravenna, Italy, 13 September 201

A comparative study of covariance selection models for the inference of gene regulatory networks

Display Omitted Three different models for inferring gene networks from microarray data are proposed.The most sensitive approach is selected by an exhaustive simulation study.The method reveals a cross-talk between the isoprenoid biosynthesis pathways in Arabidopsis thaliana.The method highlights 9 genes in HRAS signature regulated by the transcription factor RREB1. MotivationThe inference, or 'reverse-engineering', of gene regulatory networks from expression data and the description of the complex dependency structures among genes are open issues in modern molecular biology. ResultsIn this paper we compared three regularized methods of covariance selection for the inference of gene regulatory networks, developed to circumvent the problems raising when the number of observations n is smaller than the number of genes p. The examined approaches provided three alternative estimates of the inverse covariance matrix: (a) the 'PINV' method is based on the Moore-Penrose pseudoinverse, (b) the 'RCM' method performs correlation between regression residuals and (c) '?2C' method maximizes a properly regularized log-likelihood function. Our extensive simulation studies showed that ?2C outperformed the other two methods having the most predictive partial correlation estimates and the highest values of sensitivity to infer conditional dependencies between genes even when a few number of observations was available. The application of this method for inferring gene networks of the isoprenoid biosynthesis pathways in Arabidopsis thaliana allowed to enlighten a negative partial correlation coefficient between the two hubs in the two isoprenoid pathways and, more importantly, provided an evidence of cross-talk between genes in the plastidial and the cytosolic pathways. When applied to gene expression data relative to a signature of HRAS oncogene in human cell cultures, the method revealed 9 genes (p-value<0.0005) directly interacting with HRAS, sharing the same Ras-responsive binding site for the transcription factor RREB1. This result suggests that the transcriptional activation of these genes is mediated by a common transcription factor downstream of Ras signaling. AvailabilitySoftware implementing the methods in the form of Matlab scripts are available at: http://users.ba.cnr.it/issia/iesina18/CovSelModelsCodes.zip

hybrid x space a new approach for mpi reconstruction

Magnetic particle imaging (MPI) is a new medical imaging technique capable of recovering the distribution of superparamagnetic particles from their measured induced signals. In literature there are two main MPI reconstruction techniques: measurement-based (MB) and x-space (XS). The MB method is expensive because it requires a long calibration procedure as well as a reconstruction phase that can be numerically costly. On the other side, the XS method is simpler than MB but the exact knowledge of the field free point (FFP) motion is essential for its implementation. Our simulation work focuses on the implementation of a new approach for MPI reconstruction: it is called hybrid x-space (HXS), representing a combination of the previous methods. Specifically, our approach is based on XS reconstruction because it requires the knowledge of the FFP position and velocity at each time instant. The difference with respect to the original XS formulation is how the FFP velocity is computed: we estimate it from the experimental measurements of the calibration scans, typical of the MB approach. Moreover, a compressive sensing technique is applied in order to reduce the calibration time, setting a fewer number of sampling positions. Simulations highlight that HXS and XS methods give similar results. Furthermore, an appropriate use of compressive sensing is crucial for obtaining a good balance between time reduction and reconstructed image quality. Our proposal is suitable for open geometry configurations of human size devices, where incidental factors could make the currents, the fields and the FFP trajectory irregular

Karyopherins: potential biological elements involved in the delayed graft function in renal transplant recipients.

Background: Immediately after renal transplantation, patients experience rapid and significant improvement of their clinical conditions and undergo considerable systemic and cellular modifications. However, some patients present a slow recovery of the renal function commonly defined as delayed graft function (DGF). Although clinically well characterized, the molecular mechanisms underlying this condition are not totally defined, thus, we are currently missing specific clinical markers to predict and to make early diagnosis of this event.Methods: We investigated, using a pathway analysis approach, the transcriptomic profile of peripheral blood mononuclear cells (PBMC) from renal transplant recipients with DGF and with early graft function (EGF), before (T0) and 24 hours (T24) after transplantation.Results: Bioinformatics/statistical analysis showed that 15 pathways (8 up-regulated and 7 down-regulated) and 11 pathways (5 up-regulated and 6 down-regulated) were able to identify DGF patients at T0 and T24, respectively. Interestingly, the most up-regulated pathway at both time points was NLS-bearing substrate import into nucleus, which includes genes encoding for several subtypes of karyopherins, a group of proteins involved in nucleocytoplasmic transport. Signal transducers and activators of transcription (STAT) utilize karyopherins-alpha (KPNA) for their passage from cytoplasm into the nucleus. In vitro functional analysis demonstrated that in PBMCs of DGF patients, there was a significant KPNA-mediated nuclear translocation of the phosphorylated form of STAT3 (pSTAT3) after short-time stimulation (2 and 5 minutes) with interleukin-6.Conclusions: Our study suggests the involvement, immediately before transplantation, of karyopherin-mediated nuclear transport in the onset and development of DGF. Additionally, it reveals that karyopherins could be good candidates as potential DGF predictive clinical biomarkers and targets for pharmacological interventions in renal transplantation. However, because of the low number of patients analyzed and some methodological limitations, additional studies are needed to validate and to better address these points

Hyperactivation of WNT-\u3b2-Catenin and PI3K/Akt Pathways in Monocytes and B Lymphocytes of IgA Nephropathy (IgAN) Patients Could Lead to a Defect in Antigen Handling

A whole genomic screening was carried out to identify genes and pathways differently modulated in peripheral blood leukocytes isolated from 12 IgAN patients and 8 healthy subjects (HS). Bionformatic analysis revealed 210 genes discriminating IgAN pts from HS, these genes were also able to discriminate IgAN pts from other glomerular diseases. The identified set of genes generated highly significant networks involving WNT-b-catenin and PI3K/Akt. These pathways were further tested on peripheral blood mononuclear cells (PBMC) isolated from an independent group of 16 IgAN pts and 16 HS. Low protein levels of inversin and phosphatase and tensin homolog, two modulators of the WNT-b-catenin and PI3K/Akt pathways, were found in IgAN pts suggesting a hyperactivation of these pathways. Furthermore, we found increased phospho-Akt protein levels, nuclear b-catenin accumulation with enhanced PBMC proliferation in IgAN pts. Our next aim was to investigate which PBMC subpopulation of IgAN pts maybe principally involved in the WNT signaling alteration. We isolated T, B lymphocytes and monocytes and used a WNT pathway PCR Array to compare the transcript levels of 84 genes in IgAN pts and HS. Monocytes from IgAN pts showed an hyperactivation of the WNT pathway, in particular we found 24 significantly up regulated genes. B lymphocytes, showed a more blunt modulation since only 8 genes were found significantly up regulated, these genes overlapped with altered monocyte genes. T lymphocytes displayed only 5 deregulated genes. These findings highlight the emerging role of the WNT pathway in orchestrating the immune system in response to microbial stimulation of the immune cells in IgAN pts. Thus, the hyperactivation of this pathway could explain the abnormal systemic response to mucosal encountered antigens responsible for the onset of the disease. These newly identified pathways could be exploited for the identification of specific targets for the treatment

Hyperactivation of WNT-β-Catenin and PI3K/Akt Pathways in Monocytes and B Lymphocytes of IgA Nephropathy (IgAN) Patients Could Lead to a Defect in Antigen Handling

A whole genomic screening was carried out to identify genes and pathways differently modulated in peripheral blood leukocytes isolated from 12 IgAN patients and 8 healthy subjects (HS). Bionformatic analysis revealed 210 genes discriminating IgAN pts from HS, these genes were also able to discriminate IgAN pts from other glomerular diseases. The identified set of genes generated highly significant networks involving WNT-b-catenin and PI3K/Akt. These pathways were further tested on peripheral blood mononuclear cells (PBMC) isolated from an independent group of 16 IgAN pts and 16 HS. Low protein levels of inversin and phosphatase and tensin homolog, two modulators of the WNT-b-catenin and PI3K/Akt pathways, were found in IgAN pts suggesting a hyperactivation of these pathways. Furthermore, we found increased phospho-Akt protein levels, nuclear b-catenin accumulation with enhanced PBMC proliferation in IgAN pts. Our next aim was to investigate which PBMC subpopulation of IgAN pts maybe principally involved in the WNT signaling alteration. We isolated T, B lymphocytes and monocytes and used a WNT pathway PCR Array to compare the transcript levels of 84 genes in IgAN pts and HS. Monocytes from IgAN pts showed an hyperactivation of the WNT pathway, in particular we found 24 significantly up regulated genes. B lymphocytes, showed a more blunt modulation since only 8 genes were found significantly up regulated, these genes overlapped with altered monocyte genes. T lymphocytes displayed only 5 deregulated genes. These findings highlight the emerging role of the WNT pathway in orchestrating the immune system in response to microbial stimulation of the immune cells in IgAN pts. Thus, the hyperactivation of this pathway could explain the abnormal systemic response to mucosal encountered antigens responsible for the onset of the disease. These newly identified pathways could be exploited for the identification of specific targets for the treatment

ALTERED ANTIGEN HANDLING AND INVOLVEMENT OF THE CX3CR1-FKN AXIS IN PROMOTING HEMATURIA IN IGA NEPHROPATHY PATIENTS

Introduction and Aims: The hallmark of IgA nephropathy (IgAN) is gross hematuria (GH) coinciding with or immediately following an upper respiratory or gastrointestinal tract infection; thus a dysregulation of innate immunity has been extensively postulated in IgAN. Oral immunization with a variety of common pathogens and alimentary components is able to reproduce IgAN in experimental mice models. Furthermore, some of these exogenous antigens can be detected in the renal tissue of IgAN patients. Since GH can represent a disease triggering event, we decided to study this important clinical time point in order to unravel the link between mucosal encountered antigens and the occurrence of glomerular hematuria. Methods: The first step of the study was to perform an exploratory/hypothesis-generating analysis using microarray technology in order to identify genes/pathways modulated in the acute phase of the disease. For this aim we analyzed the genomic profile of PBMCs from 3 IgAN patients at two different clinical time points, the first sample during the GH episode and the second during the remission phase of the disease characterized by persistent microscopic hematuria (MH). Identified genes and pathways were then validated with classical biomolecular tools on a large cohort of 79 biopsy-proven IgAN patients, 5 membranous nephropathy (MN), 3 membranoproliferative glomerulonephritis (MPGN) patients and 10 Healthy blood donors (HBD). Results: The modulated genes during GH showed a clear involvement of the interferon signalling (e.g. STAT1), antigen presenting pathway (HLA-E, TAPBP) and the immuno-proteasome (e.g. PSMB8, PSMB9, PSMB10). The gene characterizing cytotoxic effector lymphocytes (CX3CR1), implicated in vascular endothelial damage, was found up-regulated at both mRNA and protein level. Furthermore, a significant increase of CX3CR1 surface expression was found in cell subsets with well known cytotoxic effector functions. In vitro antigenic stimulation of PBMCs on an independent set of IgAN patients demonstrated that patients up- regulate specifically CX3CR1 and STAT1 in an enhanced and dose dependant manner, while an expected down-regulation occurred in HBD and in other glomerulonephritis control groups. This enhanced activation not only occurred in patients characterized by recurrent GH or by permanent microscopic hematuria (MH) but also in IgAN patients without hematuria. These results confirm that the deregulation of the interferon signaling pathway through STAT1 and the enhanced CX3CR1 expression during endotoxin challenge is specific to IgAN patients, however, the enhanced receptor expression is not sufficient to cause GH. Then we analyzed glomerular fractalkine (FKN) expression, since this ligand, is involved in the vascular gateway for CX3CR1+ cells towards the inflamed tissues and induces vascular injury. A significantly higher glomerular and urinary FKN level was found in IgAN patients with recurrent GH episodes compared to permanent MH patients and MPGN and MN patients, suggesting a predisposition for cytotoxic cell extravasation only in recurrent GH patients. Conclusions: Taken together, our findings demonstrate, for the first time, a defect in antigen handling in PBMCs of IgAN patients with a specific up-regulation of CX3CR1 and STAT-1. Furthermore, the constitutive up regulation of glomerular FKN, suggests an involvement of the CX3CR1-FKN axis in the exacerbation of GH

ALTERED ANTIGEN HANDLING AND INVOLVEMENT OF THE CX3CR1-FKN AXIS IN PROMOTING HEMATURIA IN IGA NEPHROPATHY PATIENTS

Introduction and Aims: The hallmark of IgA nephropathy (IgAN) is gross hematuria (GH) coinciding with or immediately following an upper respiratory or gastrointestinal tract infection; thus a dysregulation of innate immunity has been extensively postulated in IgAN. Oral immunization with a variety of common pathogens and alimentary components is able to reproduce IgAN in experimental mice models. Furthermore, some of these exogenous antigens can be detected in the renal tissue of IgAN patients. Since GH can represent a disease triggering event, we decided to study this important clinical time point in order to unravel the link between mucosal encountered antigens and the occurrence of glomerular hematuria. Methods: The first step of the study was to perform an exploratory/hypothesis-generating analysis using microarray technology in order to identify genes/pathways modulated in the acute phase of the disease. For this aim we analyzed the genomic profile of PBMCs from 3 IgAN patients at two different clinical time points, the first sample during the GH episode and the second during the remission phase of the disease characterized by persistent microscopic hematuria (MH). Identified genes and pathways were then validated with classical biomolecular tools on a large cohort of 79 biopsy-proven IgAN patients, 5 membranous nephropathy (MN), 3 membranoproliferative glomerulonephritis (MPGN) patients and 10 Healthy blood donors (HBD). Results: The modulated genes during GH showed a clear involvement of the interferon signalling (e.g. STAT1), antigen presenting pathway (HLA-E, TAPBP) and the immuno-proteasome (e.g. PSMB8, PSMB9, PSMB10). The gene characterizing cytotoxic effector lymphocytes (CX3CR1), implicated in vascular endothelial damage, was found up-regulated at both mRNA and protein level. Furthermore, a significant increase of CX3CR1 surface expression was found in cell subsets with well known cytotoxic effector functions. In vitro antigenic stimulation of PBMCs on an independent set of IgAN patients demonstrated that patients up- regulate specifically CX3CR1 and STAT1 in an enhanced and dose dependant manner, while an expected down-regulation occurred in HBD and in other glomerulonephritis control groups. This enhanced activation not only occurred in patients characterized by recurrent GH or by permanent microscopic hematuria (MH) but also in IgAN patients without hematuria. These results confirm that the deregulation of the interferon signaling pathway through STAT1 and the enhanced CX3CR1 expression during endotoxin challenge is specific to IgAN patients, however, the enhanced receptor expression is not sufficient to cause GH. Then we analyzed glomerular fractalkine (FKN) expression, since this ligand, is involved in the vascular gateway for CX3CR1+ cells towards the inflamed tissues and induces vascular injury. A significantly higher glomerular and urinary FKN level was found in IgAN patients with recurrent GH episodes compared to permanent MH patients and MPGN and MN patients, suggesting a predisposition for cytotoxic cell extravasation only in recurrent GH patients. Conclusions: Taken together, our findings demonstrate, for the first time, a defect in antigen handling in PBMCs of IgAN patients with a specific up-regulation of CX3CR1 and STAT-1. Furthermore, the constitutive up regulation of glomerular FKN, suggests an involvement of the CX3CR1-FKN axis in the exacerbation of GH

Karyopherins: potential biological elements involved in the delayed graft function in renal transplant recipients.

BACKGROUND: Immediately after renal transplantation, patients experience rapid and significant improvement of their clinical conditions and undergo considerable systemic and cellular modifications. However, some patients present a slow recovery of the renal function commonly defined as delayed graft function (DGF). Although clinically well characterized, the molecular mechanisms underlying this condition are not totally defined, thus, we are currently missing specific clinical markers to predict and to make early diagnosis of this event. METHODS: We investigated, using a pathway analysis approach, the transcriptomic profile of peripheral blood mononuclear cells (PBMC) from renal transplant recipients with DGF and with early graft function (EGF), before (T0) and 24 hours (T24) after transplantation. RESULTS: Bioinformatics/statistical analysis showed that 15 pathways (8 up-regulated and 7 down-regulated) and 11 pathways (5 up-regulated and 6 down-regulated) were able to identify DGF patients at T0 and T24, respectively. Interestingly, the most up-regulated pathway at both time points was NLS-bearing substrate import into nucleus, which includes genes encoding for several subtypes of karyopherins, a group of proteins involved in nucleocytoplasmic transport. Signal transducers and activators of transcription (STAT) utilize karyopherins-alpha (KPNA) for their passage from cytoplasm into the nucleus. In vitro functional analysis demonstrated that in PBMCs of DGF patients, there was a significant KPNA-mediated nuclear translocation of the phosphorylated form of STAT3 (pSTAT3) after short-time stimulation (2 and 5 minutes) with interleukin-6. CONCLUSIONS: Our study suggests the involvement, immediately before transplantation, of karyopherin-mediated nuclear transport in the onset and development of DGF. Additionally, it reveals that karyopherins could be good candidates as potential DGF predictive clinical biomarkers and targets for pharmacological interventions in renal transplantation. However, because of the low number of patients analyzed and some methodological limitations, additional studies are needed to validate and to better address these points