Henoch-Schönlein purpura (HSP) is a commonest systemic vasculitis in childhood. The long-term prognosis of HSP is determined by the degree of renal involvement. The aim of this study is to search novel clinically applicable biomarkers to evaluate renal involvement in HSP patients. 20 bio-indexes in urine samples were simultaneously screened by antibody array assay. We indicated that urinary levels of cystatin C (Cys C) and neutrophil gelatinase-associated lipocalin (NGAL) in HSP patients with renal involvement were significantly higher than those without renal involvement and healthy controls. Furthermore, ELISA was used to analyze urinary Cys C and NGAL levels in HSP patients with or without renal involvement, atopic dermatitis (AD) patients and healthy controls. Our results demonstrated that urinary Cys C and NGAL levels in HSP patients with renal involvement were significantly elevated, when compared with those without renal involvement, AD patients and control subjects. In addition, by receiver operating characteristic (ROC) curve analysis, we demonstrated that the area under the ROC curve of NGAL (0.789) was larger than that of Cys C (0.692). Taken together, we show firstly that urinary Cys C and NGAL levels is abnormally elevated in HSP patients with renal involvement. We suggest that urinary Cys C and NGAL are novel useful biomarkers of renal involvement in HSP patients. Henoch-Schönlein purpura (HSP) is a commonest systemic vasculitis in childhood with purpuric rash, arthritis, renal involvement and abdominal pain. It has also been known as an immune complex-mediated disease characterized by circulating immune complexes containing immunoglobulin A (IgA) predominantly depositing on small vessel wall.The most frequent complication of HSP is renal involvement, which can appear at onset or during the course of this disease. Although most of HSP patients with renal involvement have a good prognosis, some patients do develop end-stage renal disease (ESRD) Thus, it would be interesting to search novel clinically applicable biomarkers to evaluate renal involvement in HSP patients. In this study, urine samples from HSP patients with or without renal involvement were simultaneously screened by using an commercially available antibody array consisting of 20 bio-indexes, which have been identified as potential biomarkers of acute kidney injury. We found that urinary levels of cystatin C (Cys C) and neutrophil gelatinase-associated lipocalin (NGAL) in HSP patients with renal involvement were significantly higher than those without renal involvement and healthy controls. Then the levels of urinary Cys C and NGAL in HSP patients and healthy controls were quantitatively analyzed by enzyme-linked immunosorbent assay (ELISA). Patients with atopic dermatitis (AD), an inflammatory skin diseases without vasculitis, were also enrolled into this study as disease control.Seventy-two patients with HSP who met the diagnostic criteria for HSP Bio-indexes present in the urinary samples from part of HSP1 group, HSP2 group and control group  were analyzed by using the Human Acute Kidney Injury Antibody Array  following the protocols of the manufacturers.Urinary levels of Cys C and NGAL  in different groups were quantitated with commercially available ELISA kits according to the manufacturer’s instructions.Each experiment was performed at least three times. All results are expressed as mean ± SD, Statistical differences between groups were determined according to one-way analysis of variance (ANOVA), Kruskal-Wallis test and Mann-Whitney U Test. Correlation coefficients were obtained by Spearman tests. Receiver operating characteristic (ROC) curve was generated and the area under the curve was calculated to analyze and describe the accuracy of Cys C and NGAL as biomarker. P<0.05 was considered statistically significant.First, 20 bio-indexes were simultaneously detected by using Human Acute Kidney Injury Antibody Array. In order to further establish the results from antibody array assay, ELISA was used to analyze Cys C and NGAL levels in the urinary samples from HSP1 group, HSP2 group, AD group and control group. As shown in We also investigated the relationship of Cys C and NGAL urinary levels with different clinical manifestation in HSP patients (HSP1 and HSP2). However, there were no significant difference in Cys C and NGAL urinary levels between HSP patients with or without arthritis, abdominal pain, and upper respiratory tract infection (data not shown).Furthermore, correlation analysis was used to investigate the relationships of Cys C and NGAL. As presented in The ROC curve was used to evaluate the sensitivity and specificity to discriminate renal involvement for various cut-off values of urinary Cys C and NGAL. ROC curves for Cys C and NGAL are shown in HSP is an IgA related immune complex-mediated disease in childhood and is also known as a classic leucocytoclastic vasculitis characterized by perivascular leukocyte infiltrates. The long-term prognosis of HSP is determined by the degree of renal involvement, which can manifest with haematuria, with or without proteinuria, and occasionally with nephritic or nephrotic syndrome, or with renal failure.. NGAL is a 25-kDa protein produced by neutrophils and various epithelial cells, including renal tubular cells. It is up-regulated in the urine after ischemic renal injury and has been identified as a powerful early marker of acute kidney injury Over the years, a great deal of research has been preformed to investigate urinary biomarkers for the diagnosis or prediction of renal injury Taken together, this study provides first observations on the changes of urinary Cys C and NGAL levels in patients HSP. We suggest that urinary Cys C and NGAL are promising as novel non-invasive biomarkers useful for early diagnosis of renal involvement in HSP patients. A limitation of our study was the relatively small sample size. Although our results have significant implications, further research with large sample size is still needed. Also, it is necessary to investigate whether the urinary levels of Cys C and NGAL are restored to normal levels in convalescent stage of HSP.


These observations support a novel “serial progression” model of TEC development, in which progenitors serially acquire cTEC lineage markers, prior to their commitment to the mTEC differentiation pathway. Gaining a better understanding of the phenotypic properties of early stages in TEC progenitor development should help in determining the mechanisms regulating cTEC/mTEC lineage development, and in strategies aimed at thymus reconstitution involving TEC therapy.Thymic epithelial cells (TECs) provide key instructive signals for T-cell differentiation. Thymic cortical (cTECs) and medullary (mTECs) epithelial cells constitute two functionally distinct microenvironments for T-cell development, which derive from a common bipotent TEC progenitor. While seminal studies have partially elucidated events downstream of bipotent TECs in relation to the emergence of mTECs and their progenitors, the control and timing of the emergence of the cTEC lineage, particularly in relation to that of mTEC progenitors, has remained elusive. In this review, we describe distinct models that explain cTEC/mTEC lineage divergence from common bipotent progenitors. In particular, we summarize recent studies in mice providing evidence that mTECs, including the auto-immune regulator The thymus is dedicated to the generation of functional self-tolerant T lymphocytes, a chief effector arm of immune responses. Within thymic niches, hematopoietic progenitors, arriving from the fetal liver and bone marrow, differentiate primarily into T cells with diverse αβTCR specificities that are restricted to self-MHCs and tolerant to self-antigens  comprise a chief component. TECs constitute specialized structural and functional microenvironments that support critical steps of T-cell differentiation, by providing multiple cytokines, chemokines, lineage inductive ligands, and selective self-antigens that control T-cell commitment, migration, survival, proliferation, and selection  reside, and the peripheral cortex area in which thymic cortical epithelial cells (cTECs) reside. These areas and the cells therein also define functionally distinct niches. cTECs have an important role during the early stages of T-cell development, driving the commitment and expansion of early T-cell progenitors via the expression of Notch ligand DLL4 −) thymus fraction Ulex europaeus agglutinin 1 Despite being fundamentally different in their anatomical location and functions, cTECs and mTECs share some phenotypic markers; for example, both are routinely defined by the expression of epithelial cell adhesion molecule 1/CD326 and MHC class II (MHCII) within the nonhematopoietic  present within the embryonic The discrimination between cTECs and mTECs, although particularly evident in the postnatal adult thymus, is less conspicuous at early stages of thymic organogenesis. This perhaps results from their common ancestry and the dynamic nature of TEC patterning, which is initiated during fetal development and continues throughout postnatal life loCD80loAire− (mTEClo), mature MHCIIhiCD80hiAirehi (mTEChi), and recently identified terminally differentiated MHCIIloCD80loAireloInvolucrin+  + mTEC subset  and Cld4 has been shown to mark a minor subset of TECs at E13 in mice, representing an mTEC progenitor subset at this developmental stage that is able to generate Aire-expressing mTECs hi cells of the nude mouse relate to functionally identified mTEC progenitors − TECs expressing high levels of CD40 and a panel of mTEC-associated genes has been reported to arise at E14.5 Several studies in mice have examined the development of distinct lineages downstream of bipotent TEPs. While Rodewald et al. In contrast to an emerging picture of discrete stages in the mTEC lineage, events occurring in the cTEC developmental pathway are less clear. However, recent studies in mice suggested the existence of cTEC-specific progenitors identified by the expression of CD205 and β5t, both hallmarks of the cTEC lineage, which appear as early as E12.5 and are absent in nude mouse embryos Based on current findings, a simple model of TEC lineage development from bipotent TEPs can be proposed Fig.A, in whiThe identification of bipotent TEPs and evidence for the existence of compartment-restricted progenitors has cemented the concept that cTECs and mTECs follow independent differentiation pathways. Interestingly, despite several studies demonstrating the presence of mTEC progenitors +CD40− TECs, displaying molecular traits associated with cTECs + and CD205− compartments, demonstrating that mTEC progenitors exist within the TEC subset defined by expression of the cTEC marker CD205 − subset are derived from initial CD205+ progeny, or whether additional RANK+CD205− cells at early embryonic stages represent a separate stream of mTEC progenitors that do not pass through a CD205+ stage. Whatever the case, such observations demonstrate a “blurring” of cTEC/mTEC properties at initial stages of development in TEC populations of the embryonic thymus, and argue against the synchronous emergence of cTEC progenitors and mTEC progenitors from a common TEP pool.Using a cellular approach that combines reaggregate organ cultures and ectopic thymic transplantation of phenotypically defined embryonic putative TEC progenitor populations, Baik et al. demonstrated that purified CD205Il7YFP+) Il7YFP+ TECs represent a particular subset of CD205+Ly51+ cTECs throughout fetal development and perinatal life. Of note, IL-7 expression is also detected in mTECs, albeit at significantly lower levels compared to Il7YFP+ TECs Il7YFP+ TECs emerge as early as E12.5 Il7YFP+ TECs can give rise to both Ly51+CD205+ cTECs and CD80+ mTECs Il7YFP+ cells give rise to mTECs in a stepwise differentiation process via an intermediate CD80lo immature mTEC stage. Still, Il7YFP+ cells do not exclusively form the entire TEC compartment at E13–14, and a smaller fraction of YFP− cells is detected at this period, which steadily accumulates medullary traits as TEC maturation proceeds, including responsiveness to RANK stimulation. Similarly to the E13 CD205− progenitors detected by Baik et al. − TECs found within the E13 thymus have a direct lineage relationship with Il7YFP+ cells or represent an alternative pathway of mTEC development. Although both studies indicate that embryonic TEC progenitors with cTEC features have the potential to generate mTECs, these studies do not determine to what degree such progenitors contribute to thymus medulla formation within the embryonic and adult thymus.In a complementary study, Ribeiro et al. + subset, throughout ontogeny In this respect, using a knock-in mouse strategy and lineage tracing experiments, Ohigashi et al. Collectively, these observations demonstrate that the medullary lineage is derived from progenitors defined by a cTEC “footprint,” thereby arguing against a model of TEC development involving the synchronous emergence of distinct pools of cTEC progenitors and mTEC progenitors from bipotent TEPs. Based on these findings, we propose an alternative model of TEC development, referred to here as the “serial progression” model Fig.B, to expThe recent developments discussed in this review provide an important change in the understanding of TEC lineage specification. The identification of transitional progenitors with cortical traits indicates that cTECs and mTECs might share a more intricate lineage relationship downstream of bipotent TEPs than previously recognized. This raises more general questions of how TEC differentiation is balanced to create functionally diverse epithelial microenvironments. Given the considerable plasticity in the lineage potential of transitional TEPs, it will be important to determine whether these cells exist in the adult thymus. Additionally, we still lack evidence on how transcriptional and epigenetic changes contribute to the specification of TEC lineages.The “asymmetrical serial progression” model implies that the specification into the cortical lineage is the default pathway downstream of the TEC bipotent progenitors. From an evolutionary point of view, one can envisage that TECs from ancient vertebrates initially evolved with the functional capacity to commit thymic seeding precursors into the T-cell lineage and to restrict the immense diversity of generated TCR specificities to self-MHC molecules. In this scenario, the emergence of the mTEC lineage is a sophisticated event that evolved later to guarantee self-tolerance, either through the purging of autoreactive T cells or the generation of T regulatory cells Interestingly, the concept presented here seems to extend to other cell types and tissues, in which the indistinctness between progenitors and lineage-specific phenotypes is observed. For example, during the development of the central nervous system, both glial and neuronal cell lineages emerge from a common progenitor that expresses proteolipid protein, a predominant myelin neuronal-restricted component Taken together, the recent studies discussed here provide a clearer definition of TEC progenitors capable of giving rise to both cTECs and mTECs. This could have important implications in relation to cellular immunotherapy approaches involving thymus regeneration/replacement


Increasing evidence suggests long non-coding RNAs (lncRNAs) are frequently aberrantly expressed in cancers, however, few related lncRNA signatures have been established for prediction of cancer prognosis. We aimed to develop a lncRNA signature to improve prognosis prediction of colorectal cancer (CRC). Using a lncRNA-mining approach, we performed lncRNA expression profiling in large CRC cohorts from Gene Expression Ominus (GEO), including GSE39582 test series(N=436), internal validation series (N=117); and two independent validation series GSE14333 (N=197) and GSE17536(N=145). We established a set of six lncRNAs that were significantly correlated with the disease free survival (DFS) in the test series. Based on this six-lncRNA signature, the test series patients could be classified into high-risk and low-risk subgroups with significantly different DFS . The prognostic value of this six-lncRNA signature was confirmed in the internal validation series and another two independent CRC sets. Gene set enrichment analysis (GSEA) analysis suggested that risk score positively correlated with several cancer metastasis related pathways. Functional experiments demonstrated three dysregulated lncRNAs, AK123657, BX648207 and BX649059 were required for efficient invasion and proliferation suppression in CRC cell lines. Our results might provide an efficient classification tool for clinical prognosis evaluation of CRC. As the third leading culprit in cancer incidence worldwide , CRC conMany studies have exploited microarray technology to investigate gene expression profiles (GEPs) in CRC in recent years, but only a small subset demonstrates clear prognostic significance -10. MolePresently, a large group of lncRNA-specific probes were fortuitously represented on the commonly used microarray platform (Affymetrix HG-U133 plus 2.0), so we initially mined previously published gene expression microarray data from the Gene Expression Omnibus (GEO), and conducted lncRNA profiling on large cohorts of CRC patients. By using the sample-splitting method and Cox regression analysis, we identified a prognostic, six-lncRNA signature from the GSE39582 test series patients, and validated it in the internal validation series and another two independent GEO cohorts. To further confirm the reliability of the new signature we conducted a series of experiments to investigate three of six lncRNAs' biological behavior in CRC cell lines.CRC data sets and corresponding clinical data were downloaded from the publicly available GEO databases. After removal of the samples without survival status, a total of 895 patients were analyzed, as detailed in Supplementary The 553 GSE39582 CRC patients were assigned to a test series (N=436) or a validation series N=117). The test data set was used for the detection of prognostic lncRNAs. By subjecting the lncRNA expression data of the test series to univariable Cox proportional hazards regression analysis using the BRB-Array Tools, we identified a set of six lncRNAs that were significantly correlated with patients' DFS  , as follows: Risk score=(0.07826*expression level of AK024680)+(-0.14300* expression level of AK123657)+(-0.19355* expression level of CR622106)+(-0.00172* expression level of BX649059)+(-0.20855* expression level of BX648207)+(0.24326* expression level of AK026784). We then calculated the six-lncRNA signature risk score for each patient in the test series, and ranked them according to their risk score. As such, patients were divided into a high-risk group (N=218) or a low-risk group (N=218) using the median risk score of the test series as the cutoff point. Patients in the high-risk group had significantly shorter median DFS than those in the low-risk group (log-rank test ) Figure . In detaP=0.0212)  and a low-risk group (N=73) using the median score of the test series as the cutoff point as for the validation series. In consistence with the findings described above, patients in the high-risk group had significantly shorter median DFS than those in the low-risk group (log-rank test ) Figure . Risk sc) Figure . The DFSP=0.0019). For the GSE17536 set, patients could similarly be segregated into a high-risk group and a low-risk group  as covariables. The results showed that the six-lncRNA risk score remained to be significantly associated with DFS when adjusted by AJCC stage and other variables in every cohort Table . Data stPrognostic value of the signature for the patients with or without postoperative chemotherapy was also assessed. In the combined GSE39582 and GSE14333 series (when chemotherapy information available), high-risk score significantly correlated with an unfavourable DFS in those with and without postoperative chemotherapy Figure , resultsP<0.0001), results of separate series see Supplementary We performed GSEA to identify associated biological processes and signaling pathways using the lncRNA signature based risk score for classification. Significant gene sets  were visualized as interaction networks with Cytoscape and Enrichment Map Figure . The risP<0.0001), BX648207 (P=0.0188) and BX649059 (P<0.0001) expression were decreased in CRC cancer  were employed to knockdown AK123657, BX648207 and BX649059 expression in human CRC cell lines, HCT116 and SW1116. In case of target-off effect, two small interfering RNAs were used for each lncRNA Figure . Cell-coThe conventional view of gene regulation in biology has centered around protein-coding genes until the discovery of thousands of lncRNAs. Numerous reports of dysregulated lncRNA expression across numerous cancer types suggest that abnormal lncRNA expression may be a major contributor to tumorigenesis. The aberrant expressions of specific lncRNAs in cancer could mark the spectrum of disease progression and these lnRNAs may serve as independent biomarkers for diagnosis and prognosis , 28. MorBy applying the six-lncRNA signature to the GSE39582 test series patients, a clear separation was observed in the survival curves between patients with low- or high-risk signatures. Patients with a low-risk six-lncRNA signature in their tumor specimens tended to have prolonged overall survival, whereas patients with a high-risk signature tended to have shortened survival. The association between the lncRNA signature and survival was significant regardless of the former was evaluated as a continuous variable or category variable (dividing by the median cutoff). The usefulness of this lncRNA signature could be internally validated in the non-overlapping GSE39582 patients  and two independent cohorts of GSE14333 and GSE17536 that were profiled through the same platform of GSE39582 patients, indicating good reproducibility of this lncRNA signature in CRC.Further analysis revealed that the prognostic value of the six-lncRNA signature was independent of one of the main prognostic factors-AJCC stage. Currently, AJCC stage has been widely accepted as a powerful predictor of treatment response and survival in CRC. In general, stage I and II shows better survival, whereas stage III and IV is correlated with worse survival in a treatment-independent manner. In accordance with other studies, AJCC stage was a significant prognostic factor in our study when assessed in the univariable Cox regression analysis Table . TherefoOur signature predicted an unfavourable prognosis of those with or without postoperative chemotherapy in combined cohorts, indicating that some signature lncRNAs might involve in chemotherapy response and contribute to therapeutic outcome, thus needed to be studied further.Examination of associated molecular pathways revealed that the six-lncRNA signature was more likely to involve with Integrin pathway, extracelluar matrix pathways and focal adhesion. Extracellular matrix via its receptors, the integrins, and kinases downstream of β1 integri-focal adhesion kinase (FAK) have emerged as a major pathway leading to several cellular responses including migration, differentiation, and proliferation , 32. FurHowever, gene signatures attempted to serve as prognostic and/or predictive markers for CRC hardly achieve widespread use despite accumulating signatures have been explored -9, 35. TThe limitations should be acknowledged for this study. In particular, treatment response was not included because this information was not available for a substantial proportion of cases. Thus, the significance and robustness of the signature as a prognostic classification requires further confirmation, ideally with large prospective patient cohorts included in adjuvant trials.The present study demonstrated the associations between the expressions of these lncRNAs and DFS of CRC patients. The findings may identify high-risk patients for more intensive adjuvant therapy in addition to the standard regimen; low-risk patients, on the other hand, may not need to receive intensive, and potentially toxic, therapies.Biological behavior of three lncRNAs from the gene signature was studied because we supposed gene signature consisted of functional lncRNAs tended to have better prediction. However, the roles of the other lncRNAs in CRC pathogenesis are presently unclear, and our findings suggest that they deserve further studied. Additional functional investigations of these lncRNAs on cancer cell lines and xenograft models may increase our outstanding of their roles in determining CRC prognosis. Last but not least, all the lncRNAs were derived through the re-annotation algorithm in this study and should be validated in the further studies.In conclusion, this study presents a powerful lncRNA signature by probing and integrating currently available microarray data. This innovative lncRNA signature showed independence of one of the main prognostic factors-AJCC stage and the signature might help personalize prediction of CRC prognosis. The GSEA analysis suggested that the signature might involve with several cancer pathways, most likely with cancer metastasis, which provided support for DFS predictive ability of the signature. These identified signature lncRNAs played vital roles in the CRC cell lines, which might serve as alternative biomarkers and therapeutic targets for CRC.The raw CEL files were downloaded from GEO database and background were adjusted using Robust Multichip Average. GATExplorer was used to process microarrays on a local computer for gene expressions of lncRNAs . This GAhttp://www.broadinstitute.org/gsea) using MSigDB C2 CP: Canonical pathways gene set collection(1320 gene sets available).The GSEA, visualized in Cytoscape (version 2.8.2), and the Enrichment Map software[GSEA was performed by the JAVA program  and disease specific survival (DSS) was assessed by univariable Cox regression analysis along with a permutation test using Biometric Research Branch-Array (BRB-Array) Tools . Genes wU (MWU) test was used to determine the significance of the comparisons.A total of 402 of the 553 primary CRC samples from the entire GSE39582 cohort could be analyzed for array-based comparative genomic hybridization (CGH). To exam whether lncRNAs within amplified (or deleted) regions have increased (or decreased) expression levels, Mann-Whitney Cell viability was assessed by the Cell Counting Kit 8  as described previously , 41. BriAK123657 siRNA1 CCUCCAGACUGUGAGUAAUTT AUUACUCACAGUCUGGAGGTTsiRNA2 GGAGGUGCAUGACUAACAATT UUGUUAGUCAUGCACCUCCTTBX648207 siRNA1 GGCCUGAAUUUGGUUACAUTT AUGUAACCAAAUUCAGGCCTTsiRNA2 GCCACUUGCAAGUGGAAUATT UAUUCCACUUGCAAGUGGCTTBX649059 siRNA1 GGCUUAAAGUAGGUAUUUATT UAAAUACCUACUUUAAGCCTTsiRNA2 CCUGAAGAGUACAGAUAAATT UUUAUCUGUACUCUUCAGGTT5 cells after transfected with lncRNA siRNAs or control siRNA were seeded in the upper chamber, while the RPMI-1640 medium (Invitrogen) supplemented with 30% fetal bovine serum was placed in the lower chamber. The plates were incubated for 48h. Then the cells were fixed in 4% formaldehyde and stained with 0.05% crystal violet for 20min at room temperature. Cells on the upper side of the filters were removed by cotton-tipped swabs, and the filters were washed with PBS. The cells on the lower side of the filters were defined as invasive cells.Tumor cell invasion assays were performed using Boyden chambers with filter inserts  coated with Matrigel  in 24-well dishes (Corning) as described previously . Briefly


Widespread tau labelling was identified in all IBNC brains examined and with each of seven tau antibodies recognising different hyperphosphorylated sites. Labelling with each antibody was associated with dendrites, neuronal perikarya and glia. Thus IBNC is a sporadic, progressive neurological disease predominantly affecting aged cattle that occurs throughout the UK and is associated with hyperphosphorylation of tau, a rare example of a naturally-occurring tauopathy in a non-primate species. Secondary accumulation of alpha synuclein and ubiquitin was also present. The neuropathology does not precisely correspond with any human tauopathy. The cause of IBNC remains undetermined but environmental factors and exposure to agrochemicals needs to be considered in future aetiological investigations.Many human neurodegenerative diseases are associated with hyperphosphorylation and widespread intra-neuronal and glial associated aggregation of the microtubule associated protein tau. In contrast, animal tauopathies are not reported with only senescent animals showing inconspicuous tau labelling of fine processes albeit significant tau aggregation may occur in some experimental animal disease. Since 1986, an idiopathic neurological condition of adult cattle has been recognised in the UK as a sub-set of cattle slaughtered as suspect bovine spongiform encephalopathy cases. This disorder is characterised by brainstem neuronal chromatolysis and degeneration with variable hippocampal sclerosis and spongiform change. Selected cases of idiopathic brainstem neuronal chromatolysis (IBNC) were identified from archive material and characterised using antibodies specific to several tau hyperphosphorylation sites or different isoforms of the tau microtubule binding region. Labelling was also carried out for alpha synuclein, ubiquitin, TDP43, Aβ Idiopathic brainstem neuronal chromatolysis (IBNC) with variable hippocampal sclerosis is a chronic neurodegenerative disease of adult cattle. It was first reported in 1992 as a distinct pathological subset of clinical bovine spongiform encephalopathy (BSE) suspects occurring in older cows . The proIBNC was originally identified through heightened awareness of adult cattle neurological disease associated with the UK BSE epidemic in the late 1980’s. Cows presenting with clinical neurological disease resembling BSE in this period were examined in the UK under the BSE Orders ,5. In thIn the 1990s several pilot studies were undertaken to investigate possible causes of IBNC. In situ hybridisation or immunocytochemical testing for Borna disease virus (two brains), or for Aujeszky’s, Louping-ill or bovine viral diarrhoea virus (five brains) was carried out but antigens to these viruses were not detected (unpublished data). Serum or plasma samples from 5–10 cases of IBNC were examined for levels of Vitamin E, Vitamin B1 and the trace elements and minerals Selenium, Magnesium, Zinc, Iron and Copper. No significant abnormalities were detected (unpublished data). Immunohistochemical labelling for the prion protein (PrP), the protein which accumulates in BSE and other prion diseases, has detected abnormal PrP within the brains of IBNC cases ; howeverTau is a microtubule associated protein that occurs predominantly in axons and is central to the assembly of tubulin monomers into microtubules. Tau controls microtubule stability through two key molecular mechanisms. Firstly, an increase in phosphorylation of tau favours de-polymerisation of microtubules ; secondlDespite the well recognised and wide ranging spectrum of human disorders associated with P-tau, no generalised tauopathy has yet been described in association with discrete neurological deficits in domestic animals. P-tau with neurofibrillary tangles are reported in aged simian species, rabbit, bear, guanaco, reindeer and bison at sites that may correspond to the initial pathology of Alzheimer’s disease , but theNo animal was killed for the purposes of this study. All tissues were obtained from cows suspected of clinical BSE and killed as notifiable disease suspects under the UK government BSE orders  Statutory Instrument No’s 1049 and N0 2299).Between 1988 and 1995, 101 cases of neuropathologically defined IBNC were recognised amongst brains from clinical BSE suspects submitted to our laboratory. Cases of IBNC have continued to occur up to the present day but the means of reaching diagnosis on BSE suspects has changed making it problematic to be sure of the actual numbers and proportions of IBNC cases submitted relative to the BSE suspects. Formalin fixed- paraffin embedded blocks from confirmed cases of IBNC were retrieved from archive. Twelve cases were selected for further immunohistochemical analysis, representing brains with a range of pathological features, . Additional samples of BSE and other unrelated bovine neurological diseases such as encephalic listeriosis, malignant catarrhal fever, and brains from cows aged 5 to 16 years with no histological lesions that were contemporary to the IBNC brains, were also sourced and included as controls.Frozen tissues from the majority of IBNC brains were not routinely retained. However, following the recognition of P-tau by immunohistochemistry, three IBNC cases with frozen brain samples were obtained from within the resources of the Biological Archive Group at the Animal and Plant Health Agency, Weybridge. Immunohistochemistry using the AT8 antibody confirmed the presence of P-tau in brainstem in all three cases and 3–5g of frozen tissue from cerebellum hippocampus and thalamus was retrieved from them for Western blotting. Additionally frozen brain tissues from two adult cattle with no significant histological lesions and a case of encephalic listeriosis were sought as controls. These controls were AT8 P-tau negative by immunohistochemistry.1–42 and, Aβ1–40 ; the Parkinson’s disease associated protein alpha synuclein ; the hyper-phosphorylated, ubiquitinated and 43 kDa cleaved form of the transactive response (TAR) DNA-binding protein known as pathologic TDP43 , which is a major disease protein of frontotemporal dementia and motor neurone disease; and Ubiquitin  which is abnormally accumulated in the presence of aggregated tau, alpha synuclein and other misfolded proteins.For histology and immunohistochemistry tissue sections were cut at 5μm according to standard procedures for light microscopy and stained by Haematoxylin and Eosin (HE) or Bielschowsky silver stain for detection of neurofibrillary tangles. Immunohistochemistry was performed on sections of cerebral cortex, striatum, hippocampus, thalamus, midbrain, cerebellum and brainstem using primary Tau antibody AT8 , and tau antibodies specific to hyperphosphorylated threonine (T) T205, T212, T231 , and serine (S) S214 , S396, S404  residues and also to RD3 and RD4  which recognise three (3R) and four repeat (4R) binding domains of the microtubule binding region. Labelling on selected sections was also carried out for: the Alzheimer’s associated proteins AβSections were labelled according to the protocol in routine use in our laboratory , with thet al ))6]).P-tau labelling was identified in all 15 IBNC brains examined and with all tau antibodies except RD4 . LabelliNo labelling for tau or for alpha synuclein was detected in control brains of adult, aged cows lacking histological changes or from cows with neurological conditions other than IBNC. Ubiquitin labelled occasional neuronal nuclei in control cow brains but did not label dendrites, neuronal perikarya or glial cells.Glial cell—associated P-tau labelling appeared as punctuate neuropil labelling surrounding astrocytic nuclei in grey and white matter . AlternaP-tau immunolabelling was found on dendrites and in a peri-neuronal pattern of labelling . The labOnly in a single case were intracytoplasmic neuronal inclusions of olivary neurons labelled for tau (not shown). On HE sections the P-tau positive inclusions corresponded to areas of cytoplasm with slightly refractile granules.No labelling for P-tau of glia or dendrites was seen in control cattle brains .The range of severity of lesions in IBNC varies considerably. In approximately half the cases lesions are confined to the neuraxis. In these cases P-tau labelling was mostly confined to glial associated patterns. The most widespread and intense P-tau accumulations were found in those cases which showed the additional lesions of hippocampal sclerosis and spongiform changes.In cases of IBNC in which hippocampal neuronal loss were minimal or absent, glial associated tau labelling markedly predominated over dendritic and peri-neuronal patterns. Glial associated patterns of labelling occurred throughout the neuraxis, in the cerebellum, hippocampus and neocortex. In some brains with minimal histologically evident morphological lesions, glial associated labelling alone was found in hippocampus while in other brains both dendritic and glial labelling was found. Dendrite and perineuronal labelling of the hippocampus mainly involved the CA1 neuronal layer, the stratum oriens and dentate gyrus. Glial associated P-tau labelling was also present in dentate gyrus but mainly involved the stratum radiatum of the CA1 sector. In IBNC brains in which hippocampal lesions were conspicuous, glial associated tau labelling was retained throughout the neuraxis but dendrite and peri-neuronal labelling predominated in the thalamus, neocortex, and hippocampus. Where hippocampal sclerosis and atrophy occurred there was a decrease in labelling intensity corresponding to the loss of neurons and processes from the CA1 and dentate sectors. In the cerebral cortex, dendrite and perineuronal patterns of labelling were most consistently found in the entorhinal cortex but sometimes involved other cortical regions. P-tau labelling of the cerebral cortex was mainly found in the superficial and deep pyramidal neuron layers with sparing of the mid cortical layers.Glial associated P-tau labelling was present throughout grey matter nuclei of the neuraxis, cerebellum and hippocampus. The extent of glial cell tau labelling also varied in proportion to the magnitude of hippocampal change observed in HE stained sections. The number of grey matter locations in which glial labelling was found increased in line with the severity of hippocampal changes. Sites showing glial associated labelling in minimally lesioned brains included, the cuneate nuclei, the spinal tract of the trigeminal nerve, red nucleus, substantia nigra, dorsal thalamic nuclei, hypothalamus, globus pallidus and internal capsule of the striatum. As described above glial tau labelling was also prominent in the hippocampus of minimally lesioned brains suggesting that a glial tauopathy precedes the dendritic and peri-neuronal tauopathy.Degenerate and chromatolytic neurons of the neuraxis were not labelled for P-tau but vacuolation in thalamus, hippocampus and cerebral cortex was related to dendritic and perineuronal patterns of labelling . It is lNo labelling was detected with TDP-43 or with either of the Aβ peptide specific antibodies used and thus P-tau immunolablleing in IBNC cattle is not correlated with either TDP-43 or Aβ.Alpha synuclein labelling was also conspicuous in the hippocampus. Punctuate alpha synuclein labelling was present in neuropil surrounding neurons and their primary dendrites in the dentate gyrus . LabelliAt other neuroanatomic sites a pattern of parallel granular labelling occurred surrounding dendrites of the substantia nigra , the gloAlthough amphophilic intracytoplasmic neuronal inclusion bodies are an infrequent feature of HE stained sections of IBNC we did not find evidence that these inclusions were labelled with alpha synuclein nor were typical alpha synuclein labelled Lewy bodies or neurites identified in this series.No labelling for alpha synuclein was seen in control cattle brain tissues .Punctate ubiquitin labelling was present along neuronal dendrites in the dentate gyrus and in the stratum radiatum and stratum oriens of CA1 sector of the hippocampus. This pattern corresponded to the labelling found on tau positive dendrites at this site. Punctuate ubiquitin labelling was in found in association with glia of the cortical white matter, and a small proportion of dendritic processes in the deeper cortex. There was no association with vacuolation.A punctuate perineuronal ubiquitin labelling pattern was conspicuous in brainstem, mid-brain and thalamus  which alUbiquitin labelling was not found in degenerate neuronal cytoplasm though occasional intra-nuclear inclusions were ubiquitinated.Punctuate labelling of dendrites or perikarya was not found in control cattle tissues Intra-cytoplasmic labelling suggestive of neurofibrillary tangles was not identified with Bielschowsky stain. Electron microscopy revealed abundant florid bundles of intermediate filaments that were in frequent astrocytes but clear evidence for paired and twisted filaments consistent with intra-neuronal neurofibrillary tangles were not identified.Western blots showed the presence of multiple tau immuno-reactive bands in the brains of IBNC cattle. Similar tau signals were also obtained for controls .Tau is phosphorylated at about 30–40 sites in normal human brains and is also minimally phosphorylated in normal adult mammalian brains. In some human disease states tau may become hyperphosphorylated with up to 60 phosphorylation sites. In animals, P- tau has been described as thread like processes in the brains of clinically normal aged animals  and is iP-tau may aggregate into intra-cellular fibrillar structures. In Alzheimer’s disease aggregated tau takes the form of paired helical filaments. These may be identified by silver impregnation methods and are usually referred to as neurofibrillary tangles. Fibrils composed of aggregated tau molecules are not confined to Alzheimer’s disease but may occur as straight or random filaments in some other human tauopathies . IBNC apMany human neurodegenerative diseases show abnormalities of tau and microtubules and although most are considered complex proteinopathies, some familial diseases with mutations of the microtubule associated protein gene, such as familial fronto-temporal dementia are considered pure tauopathies ,25. No gIBNC shows hyperphosphorylation at numerous sites across the tau molecule, albeit of those sites tested some were more conspicuous than others. 3R forms of tau were recognised but not the 4R forms. Labelling of 3R tau is known to require specific pre-treatments  and labe1–40 or Aβ1–42 were absent from IBNC brains. Hippocampal sclerosis in man may be associated with TDP-43 immunolabelling, but this too was absent in IBNC. The predominant glial tau association of IBNC has similarities to the tau gliopathy reported for argyrophilic grain disease, progressive supranuclear palsy and corticobasal degeneration [Tauopathies in man are classified according to the physical state of aggregation of abnormal tau, by the molecular size and nature of P-tau, by the neuroanatomical distribution of lesions and by additional neurodegenerative features ,23,27,28neration  but, in neration . Howeverneration  and TablIBNC is a chronic progressive neurological illness that is widespread and occurs sporadically throughout the UK in aged cattle. Clinical signs of IBNC include behavioural changes, increased apprehension and locomotor problems such as tremor and stiffness of gait, and weight loss ,3. Diffi


Hyponatraemia is one of the major adverse effects of thiazide and thiazide-like diuretics and the leading cause of drug-induced hyponatraemia requiring hospital admission. We sought to review and analyze all published cases of this important condition.Ovid Medline, Embase, Web of Science and PubMed electronic databases were searched to identify all relevant articles published before October 2013. A proportions meta-analysis was undertaken.−2. Presentation with thiazide-induced hyponatraemia occurred a mean of 19  days after starting treatment, with mean trough serum sodium concentration of 116  mm and serum potassium of 3.3  mm. Mean urinary sodium concentration was 64 mm . The most frequently reported drugs were hydrochlorothiazide, indapamide and bendroflumethiazide.One hundred and two articles were identified of which 49 were single patient case reports. Meta-analysis showed that mean age was 75  years, 79% were women  and mean body mass index was 25  kg mPatients with thiazide-induced hyponatraemia were characterized by advanced age, female gender, inappropriate saliuresis and mild hypokalaemia. Low BMI was not found to be a significant risk factor, despite previous suggestions. The time from thiazide initiation to presentation with hyponatraemia suggests that the recommended practice of performing a single investigation of serum biochemistry 7–14 days after thiazide initiation may be insufficient or suboptimal. Further larger and more systematic studies of thiazide-induced hyponatraemia are required. Thiazide and thiazide-like diuretics, although they differ in chemical structure, all inhibit the thiazide-sensitive sodium–chloride co-transporter, NCC, in the distal convoluted tubule of the kidney . Since tHowever thiazide diuretics often cause adverse effects, of which thiazide-induced hyponatraemia is amongst the most clinically important . ThiazidThiazide diuretics are the most common cause of drug-induced hyponatraemia in secondary care . Severe m was used [Medline, Embase, Web of Science and PubMed databases were searched on 1 October 2013 without limitation on language. The Ovid interface was used to search Medline and Embase using the terms ‘thiazide AND hyponatr$emia’, ‘thiazide-induced hyponatr$emia’ and ‘thiazide-associated hyponatr$emia’. Web of Science and PubMed were searched using the terms ‘(thiazide AND (hyponatraemia OR hyponatraemia)’, ‘(thiazide-induced AND (hyponatraemia OR hyponatraemia)’, ‘(thiazide-associated AND (hyponatraemia OR hyponatraemia)’. Database searches were also undertaken with the term ‘thiazide’ replaced alternatively by either ‘indapamide’ or ‘chlortalidone’. Articles that cited or were cited by the included studies were also screened to identify any further relevant studies. Duplicated results and studies containing no primary data or non-human data only were removed. The conventional definition for hyponatraemia of serum sodium concentration ≤135 mwas used ,12.Two authors (JB and MG) independently reviewed the titles, abstracts and full text of identified papers. References of all full text papers were searched to identify any additional pertinent papers. Disagreements were resolved by discussion. Data extraction was performed using a structured template to collect information on study design (including location of study and year of publication) and thiazide-induced hyponatraemia phenotype including age, gender, presenting symptoms, drug history including concomitant drug use and laboratory findings. Methodological quality was independently rated by two authors (JB and MG) using a modified version scale developed for observational studies . The ran2 scores. Causes of high levels of heterogeneity were explored by dividing the following variables at the median level: quality score, year of publication, and age of study population. Evidence for the possibility of publication bias was assessed by funnel plots. Single case reports were simply summarized.Study parameters which were reported in more than 1% of patients are presented. We excluded from the analysis any measurement given qualitatively as ‘normal’ without any indication of the value itself or the reference range. For publications in which more than one patient was reported a proportions meta-analysis was conducted to look at the weighted frequency of clinical phenotype, drug history and laboratory findings for the combined number of papers contributing to each separate analysis. A random effects model was used to determine 95% confidence intervals (CI), using the DerSimonian & Laird method to calculate weights . Study hThe presentation of the meta-analyses adhered to the Meta-analysis of Observational Studies in Epidemiology (MOOSE) consensus statement . All proDatabase searches resulted in 1359 citations. After exclusion of duplicates and articles where data were non-human or irrelevant to thiazide-induced hyponatraemia in adults, 102 articles remained  and were analyzed Figure  and the The mean value for quality score was 4.0 (range 1–8) for studies included in the meta-analysis and 3.2 for single case reports (range 1–6). The main reasons for low quality scores were a lack of clearly stated inclusion and exclusion criteria, absence of documented patient consent and/or ethical approval, a lack of inclusion of patient perception, a lack of clarity regarding the name, dose and duration of thiazide therapy and whether thiazide-induced hyponatraemia patients represented a consecutive series treated consistently by the same physician(s) or at a single institution.2 = 65%, based on 43 studies and 3269 patients, −2 , fatigue , weakness , confusion  nausea  and vomiting . Also reported were other neurological symptoms, dizziness, unconsciousness and seizures  or in combination with amiloride  or triamterene . Other thiazide/thiazide-like drugs implicated were indapamide, bendroflumethiazide (bendrofluazide), and chlortalidone. Analyses of which individual thiazide/thiazide-like drugs were associated with hyponatraemia revealed high levels of heterogeneity between studies and this was not explained by quality score, year of publication or age of patient , angiotensin converting enzyme (ACE) inhibitors , non-thiazide diuretics (e.g. loop and potassium sparing diuretics) , non-steroidal anti-inflammatory drugs (NSAIDs)  and anti-depressants . While selective serotonin re-uptake inhibitors (SSRIs) are associated with hyponatraemia, there was insufficient data to determine what proportion of the anti-depressant medication reported were SSRIs. Analyses of polypharmacy demonstrated high levels of heterogeneity between studies, which was not explained by quality score, year of publication or age of patient , mild hypokalaemia, with serum potassium 3.3 mm  and normal renal function, serum creatinine 76.8 μmol l−1 . Corresponding urinary electrolyte data indicated inappropriate saliuresis, with urinary sodium concentration 64 mm . Mean serum and urine osmolalities were 240 mosm kg−1  and 402 mosm kg−1 , respectively. In most analyses of laboratory characteristics there were high levels of heterogeneity between studies, which were not explained by quality score, year of publication or age of patient  aldosterone activation of the distal nephron, as is to be expected and not uncommonly seen with thiazide-induced hypokalaemia and hyperaldosteronism , or (ii)vs. non-symptomatic patients.Hyperlipidaemia was the most common comorbidity. Whilst severe hyperlipidaemia is a well-recognized cause of pseudo-hyponatraemia in older studies that relied on methods such as flame photometry to measure serum sodium concentration, most dyslipidaemia is very unlikely to affect modern assay methods. The second most common comorbidity was diabetes mellitus. Plasma glucose concentration was reported infrequently and it is possible that hyperglycaemia could have contributed to hyponatraemia in some cases. Since for the vast majority of patients reported the indication for thiazide prescription was hypertension and presentation was with symptomatic hyponatraemia it is not possible to make meaningful conclusions either regarding thiazide-induced hyponatraemia when thiazide prescriptions were for indications other than hypertension or the differences between symptomatic 1 receptor antagonists, NSAIDS and some anti-depressants, are associated with hyponatraemia through well-described mechanisms. This raises the possibility that some cases of apparent thiazide-induced hyponatraemia could result from pharmacodynamic interactions of these drugs with thiazides. It was also notable that hypokalaemia was seen despite the frequency of concurrent therapy with ACE inhibitors, AT1 receptor antagonists and potassium supplements.Several of the medicines most commonly co-prescribed with thiazides among studies included in this meta-analysis, such as ACE inhibitors, ATThe finding of a normal serum creatinine concentration is unexpected in such an elderly cohort at higher than average vascular risk, many of whom also took ACE inhibitors, angiotensin receptor blockers (ARBs) and/or NSAIDs. BMI was normal, so low muscle mass is unlikely to predominantly account for this raising the possibility of a dilutional component to the observed serum creatinine concentrations. Thus in addition to inappropriate saliuresis and inappropriately high urinary osmolarity, inappropriately low serum creatinine concentration is consistent with volume expansion and the overall phenotype could therefore be alternatively described as having much in common with thiazide-induced syndrome of inappropriate anti-diuretic hormone secretion (SIADH) .There are significant limitations to this systematic review. Included studies were very heterogenous with respect to the detail of their description, specific parameters recorded and laboratory methods used and this did not appear to be explained by either individual study quality, median date of publication or whether the study pertained to a particular ‘specialist group’. One possible explanation for the high level of heterogeneity found in the analyses may be the local prescribing habits of the areas in which these often small studies were undertaken given that case series usually focused on small numbers from a single institution over a relatively short project interval. Case reports were excluded from our meta-analysis but we accept that a case report may describe unusual but important presenting clinical and laboratory characteristics of patients with thiazide-induced hyponatraemia. For this reason we have presented a summary of the case report data alongside that for the meta-analysis in Tables There are also specific issues with respect to the phenotypic parameters measured by observational studies. Advanced age is confounded by the prescribing of thiazides to older patients and the over-representation of females may be confounded by the shorter life expectancy of males. Although oestrogens do affect sodium transport via the thiazide-sensitive NCC , the ageIt is not possible to draw meaningful conclusions regarding the prevalence of thiazide-induced hyponatraemia with respect to individual thiazide drugs from the available data, apart from observing that thiazide-induced hyponatraemia is reported with many thiazides including indapamide and chlortalidone, the two currently recommended thiazide-like diuretics for hypertension in the UK .The detailed study of the phenotype and pathophysiology of patients with thiazide-induced hyponatraemia has the potential not only to improve clinical care of patients prescribed thiazides but also potentially to uncover novel pathophysiological insights into salt and water handling in the kidney which are clearly disturbed in these individuals. In the absence of a prospective trial exposing thousands of patients to thiazides with extensive follow-up , larger scale prospective observational studies with detailed phenotyping of thiazide-induced hyponatraemia patients is required. Such a study is already taking place across several acute hospitals throughout the UK (NIHR CRN portfolio identity 10795).In conclusion this study found that patients with thiazide-induced hyponatraemia were characterized by advanced age, female gender, inappropriate saliuresis and mild hypokalaemia. Such patients had a normal BMI and were diagnosed later than the usual serum electrolyte monitoring interval of 7–14 days after thiazide commencement. Until further studies determine the optimal timing and frequency of electrolyte monitoring regimens it would seem prudent to be mindful of the development of hyponatraemia outside of the first fortnight of thiazide therapy.


Genomic alterations affecting drug target proteins occur in several tumor types and are prime candidates for patient-specific tailored treatments. Increasingly, patients likely to benefit from targeted cancer therapy are selected based on molecular alterations. The selection of a precision therapy benefiting most patients is challenging but can be enhanced with integration of multiple types of molecular data. Data integration approaches for drug prioritization have successfully integrated diverse molecular data but do not take full advantage of existing data and literature.We have built a knowledge-base which connects data from public databases with molecular results from over 2200 tumors, signaling pathways and drug-target databases. Moreover, we have developed a data mining algorithm to effectively utilize this heterogeneous knowledge-base. Our algorithm is designed to facilitate retargeting of existing drugs by stratifying samples and prioritizing drug targets. We analyzed 797 primary tumors from The Cancer Genome Atlas breast and ovarian cancer cohorts using our framework. FGFR, CDK and HER2 inhibitors were prioritized in breast and ovarian data sets. Estrogen receptor positive breast tumors showed potential sensitivity to targeted inhibitors of FGFR due to activation of FGFR3.http://csblcanges.fimm.fi/GOPredict/.Our results suggest that computational sample stratification selects potentially sensitive samples for targeted therapies and can aid in precision medicine drug repositioning. Source code is available from The online version of this article (doi:10.1186/s13040-016-0097-1) contains supplementary material, which is available to authorized users. Finding the right drug for the right patient is an integral part of precision medicine and computational methods to facilitate matching patients to drugs are urgently needed . PatientThe molecular landscape of a tumor affects the efficacy of several drugs and is central for clinical trial design for targeted therapies . In partThe large variety of molecular level alterations in cancers calls for computational data integration methods to enable precision medicine via improved patient stratification and drug repositioning –12. MostWe introduce here a computational algorithm called GOPredict that allows patient stratification and drug repositioning via comprehensive integration of genomics data, signaling pathway information, drug target databases and curated knowledge in databases. We demonstrate the utility of GOPredict by stratifying Cancer Genome Atlas (TCGA) breast and ovarian cancer samples and prioritizing drugs in these two cohorts –19.Our data integration approach consists of two major steps. First, we have developed a knowledge-base that contains molecular, drug information and analysis results from multiple public databases and private sources. Second, we have developed an algorithm (GOPredict) to mine the knowledge-base. An overall schematic of the approach is given in Fig. The major design principles of the knowledge-base are as follows. First, the knowledge-base is gene-centric (meaning that the information in the knowledge-base is associated to a gene identifier) because this allows taking into account published results that are mostly gene-centric. This design also allows automated analysis of drug-gene target-pairs. Second, results in the knowledge-base are stored as ranks. The use of ranks enables comparing and combining data over multiple data sets, data types, and measurement technologies as well as between numerical, ordinal and categorical data . In addiGOPredict uses signaling pathway information defined with Gene Ontology (GO) biological processes . The GenThe underlying modeling question for GOPredict is “what is the best drug to target proteins affected by genomic aberrations and driving tumorigenic signaling in tumors”. The GOPredict algorithm is described in detail in Additional file in-house data and curated data. In-house data comprise raw data that we downloaded and analyzed. Curated data comprise analyzed gene-level result data that we downloaded from the source databases and did not process further. A short description of studies is in Table We gathered results of genomic, transcriptomic, and epigenomic (DNA methylation), and descriptive (gene-phenotype connections) analysis data from nine public cancer data sets. Each data set consisted of one or more of these data levels. There are two sources of data which we call The in-house data comprise four Cancer Genome Atlas (TCGA) primary tumor data sets totaling approximately 2,200 samples of breast, ovarian, colorectal and glioblastoma brain cancer –19, 25. studies in the knowledge-base. A study is a ranked list of genes that are ranked based on a statistical analysis of a molecular data type in a specific cancer or literature source. All studies are stored gene-wise and for each gene the database contains its rank order in each study . Full dactivating , inactivating , or survival-associated . A gene may belong to one or several studies and study sets. A full list of studies in each default study set is in Additional file Studies can be combined into study sets. Users can tailor and modify study sets flexibly to suit different research questions. We provide three default study sets: http://geneontology.org/, downloaded August 2013). This compound design enables rapid integration, combination and comparison of data over multiple data sets, data types, and measurement technologies. KEGGDrug, DrugBank and Gene Ontology are stored in the database as described in Additional file In addition to gene ranks in studies, the knowledge-base contains drug gene-target information from KEGGDrug  and DrugThe overall goals of the GOPredict algorithm are to prioritize drugs with known protein targets, characterize genes, and stratify samples. GOPredict works in four steps  has a rank in two studies which are used to calculate its K-rank. The two studies for FGFR3 are a curated study (activating mutations in the Cancer Gene Census) and an in-house study . Each K-rank quantifies the cancer-essentiality of a gene.First, gene ranks are extracted from the database for each study and used to calculate normalized gene ranks called the K-ranks are used to calculate GOPredict cancer-essentiality scores for GO processes . The K-ranks of these genes from step one are summed up to produce the cancer-essentiality score for ‘cell development’ and statistical significance is assessed with a permutation test  and 390 ovarian adenocarcinoma (OVCA) tumors from the Cancer Genome Atlas , 19. We A byproduct of the knowledge-base design is that it allows defining hypothesis-driven selection of study sets and calculating cancer-essentiality in the study sets. A full list of studies in each study set is in Additional file K-rank. The genes, which GOPredict characterized to be cancer-essential, include known cancer genes such as EGFR, ERBB2 and FGFR3, tumor suppressors such as RB1, TP53 and PTEN as well as genes not previously associated with cancer  are ranked the highest in the entire sample set , all FGFR family members (FGFR1-4) and possible confounders . In addition, the immunohistochemical staining status of estrogen receptor, progesterone receptor and HER2 receptor are shown. Samples sensitive to the three drugs were assigned almost exclusively according to FGFR3 activation status . The proportion of sensitive samples in these three subtypes differed significantly from luminal A (Fisher’s exact test P=0.0006) and luminal B proportions (Fisher’s exact test P=0.00001). In addition, FGFR3 inhibitor sensitive samples were enriched in luminal B samples when compared directly with luminal A (Fisher’s exact test P=0.004). In summary, luminal subtypes in general and preferentially luminal B breast cancer samples were significantly enriched for FGFR inhibitor sensitive samples according to activity patterns of FGFR inhibitor targets.To further characterize the sensitive samples, we compared GOPredict’s strata to the PAM50 subtypes. PAM50 is a gene expression based molecular subtyping method for breast cancer and is well established . FGFR inIn addition to breast cancer, we tested GOPredict in an independent set of ovarian adenocarcinoma primary tumors. In OVCA . Many of our predicted sensitive samples harbored genomic alterations in FGFRs. One of the first clinical breast cancer studies where the selection of patients was based on FGFR1 amplification status, found dovitinib to reduce tumor size more in FGFR1 amplified than non-amplified patients [Five of the top ten prioritized drugs for breast cancer were FGFR inhibitors. All five are in Phase 2 or 3 trials for multiple cancers  and pazopatients .FGFR3 activated and were enriched for PAM50 luminal A and B breast cancer subtypes. Luminal breast cancers are characterized by estrogen receptor (ER) positivity [FGFR4 predicts poor response to tamoxifen therapy in primary tumors [The samples predicted to be FGFR inhibitor sensitive were almost exclusively sitivity . Tamoxifsitivity . Interessitivity  and highy tumors . FurtherTamoxifen resistant breast tumors have been found to be sensitive to vismodegib (Smoothened antagonist) in xenograft mice . In our FGFR3. According to current guidelines, HER2 positive patients are pharmacologically treated with HER2 inhibitors [In our breast cancer data, roughly 20 % of HER2 positive tumors had potentially activating alterations in hibitors . Our resAmiloride was the highest ranked drug in HER2 positive tumors. Amiloride is a pyrazine compound used to treat hypertension and heart failure. Interestingly, amiloride and its derivatives have been recently suggested to have anti-cancer effects in breast cancer cells independent of subtype , 54. An CDK4 in ovarian cancer [https://clinicaltrials.gov/, Accessed 25 Jan 2015). Moreover, dinaciclib, the highest scoring CDK inhibitor by GOPredict, has been shown to sensitize ovarian cancer cell lines to platinum drugs via downregulation of BRCA1 [In ovarian cancer, seven out of ten top priority drugs are cyclin-dependent kinase (CDK) inhibitors. CDKs are a family of protein kinases that participate in the cell cycle and are targeted by several inhibitors . Severaln cancer . To daten cancer  and of tof BRCA1 , 60. TheOur knowledge-base contains both in-house and curated microarray data sets from multiple microarray platforms and sources. Since GOPredict is designed to be extendable and to contain data sets from multiple sources, preprocessing steps such as normalization cannot be fully standardized and can therefore induce some noise. Nonetheless, our results with varying study sets indicate that GOPredict scoring is robust to noise in the study data.GOPredict is dependent on centralized and frequently maintained databases such as GO and Ensembl. Many databases, however, undergo changes in both data content and database interfaces. These changes increase the maintenance burden of tools such as GOPredict. GOPredict could be improved in the future in three ways: (1) the addition of binding-affinity of drug-target pairs to weight each drug target gene; (2) inclusion of data on drug combinations and synthetic lethal interactions and; (3) addition of more result databases. Data on the first two points are currently scattered and automated retrieval is infeasible. GOPredict utilizes many result databases but this list is incomplete. For example, the Comparative Toxicogenomics Database (CTD) contains special disease related GO annotations as well as breast and ovarian cancer marker gene studies which could be incorporated into GOPredict .GOPredict results can in future work guide experimental design. For example, top scoring drugs from our GOPredict analysis could be administered to breast cells with suitable genetic profiles to test their efficacy in vitro.SLC25A32 received high cancer-essentiality scores through alterations in both ovarian and breast cancer study sets which could indicate a role for SLC25A32 in these cancers. Accordingly, we built a multivariate Cox survival model in TCGA OVCA data and found that the overexpression of SLC25A32 (ANOVA P=0.003) and lack of residual tumor (ANOVA P=0.02) were significant independent predictors of poor survival .GOPredict produces several by-product results when prioritizing drugs. For example, Here we present GOPredict, which is a novel approach to integrate information from multiple public sources, signaling pathway and drug target data with local genomic data in cancer. Our results suggest that GOPredict can augment current pathology-based definitions of patient groups for targeted drug therapies which can potentially benefit many cancer patients. A practical application for GOPredict is to screen genomic measurements of cancer model systems for previously overlooked druggable genomic alterations and simultaneously prioritize which drugs to screen.Our approach is able to infer kinase inhibitors as highly relevant drugs in breast and ovarian cancer based solely on signaling pathway information, pre-existing genomic result data and molecular measurement data. These inhibitors are prime candidates for further testing in drug repositioning experiments. Furthermore, our results highlight none-cancer drugs such as amiloride which have only recently been tested for anti-cancer efficacy with promising results. Our primary results indicate that FGFR inhibitors in breast cancer and CDK inhibitors in ovarian cancer as well as pazopanib in both cancers are predicted to have the largest proportion of putatively sensitive samples.


Regenerative failure remains a significant barrier for functional recovery after central nervous system (CNS) injury. As such, understanding the physiological processes that regulate axon regeneration is a central focus of regenerative medicine. Studying the gene transcription responses to axon injury of regeneration competent neurons, such as those of the peripheral nervous system (PNS), has provided insight into the genes associated with regeneration. Though several individual “regeneration-associated genes” (RAGs) have been identified from these studies, the response to injury likely regulates the expression of functionally coordinated and complementary gene groups. For instance, successful regeneration would require the induction of genes that drive the intrinsic growth capacity of neurons, while simultaneously downregulating the genes that convey environmental inhibitory cues. Thus, this view emphasizes the transcriptional regulation of gene “programs” that contribute to the overall goal of axonal regeneration. Here, we review the known RAGs, focusing on how their transcriptional regulation can reveal the underlying gene programs that drive a regenerative phenotype. Finally, we will discuss paradigms under which we can determine whether these genes are injury-associated, or indeed necessary for regeneration. It likely comes as no surprise the striking dichotomy that exists between peripheral nervous system (PNS) and central nervous system (CNS) neurons following injury. While PNS neurons show robust regenerative capacity, CNS neurons exhibit negligible capacity. This difference has been known and intensively studied since the time of Ramón y Cajal, and in those years many reasons have been postulated for this fundamental difference . Axotomy of PNS neurons induces broad and coordinated gene transcription, a response that is lacking following CNS injury  environment  levels can increase RAG expression and allow modest CNS axon regeneration in in vivo platform to test whether RAG induction allows regeneration of CNS axons. These sensory neurons possess pseudounipolar axons that extend in the periphery and into the spinal cord; a subset of these axons ascend the dorsal column of the spinal cord  neurons have provided an important in vivo imaging, and complement those studies performed in mammals. They offer insights into how injury-induced gene expression both recapitulates and differs from pathways involved during development. For example, screening 654 conserved genes in an axotomy model of mechanosensory neurons in C. elegans identified clusters of genes that promote or repress axon growth. Many of these are components of pathways critical for neuronal plasticity of both development and regeneration; however, some clusters are only required for regeneration  leads to more dramatic effects in vivo. For example, genetic knockout of c-Jun, the first identified transcription factor RAG, reduced re-innervation of peripheral targets by 4-fold after facial nerve axotomy and was associated with decreased induction of its downstream target RAGs α7 integrin, CD44, and galanin  non-transcription factor “terminal” RAGs span many functional categories, including genes that encode adhesion/guidance molecules  axon regeneration in vivo. Further, ATF3 overexpression did not allow axon growth on inhibitory substrates in vitro, suggesting that ATF3 increases the intrinsic growth of axons, but does not alter its response to the inhibitory environment of the CNS  deletion or Rheb activation] or transcriptional regulation of anabolic processes , all of which increase CNS axon growth after injury , which “opens” the chromatin to allow access to the associated genes for transcription. By contrast, histones are deacetylated by histone deacetylases (HDACs), which “closes” the chromatin and is typically repressive  and their role in RNA interference have added greatly to our understanding of regulation of gene expression. Based on bioinformatic predictions, miRNAs likely regulate >30% of all mammalian protein coding genes  contained high levels of cAMP and retained the ability to regenerate and overcome extrinsic axon growth inhibitors. This is in contrast to mature neurons , which are incapable of regeneration and have markedly lower cAMP. In these studies, the regenerative competence of embryonic neurons in vitro effect of the conditioning lesion are indeed dependent on PKA ; however, this was PKA-independent if the neurons were harvested 6 days after injection. These results indicated that the transient elevation of cAMP set forth longer-lived changes in neuronal function, which persist past the point of elevated cAMP levels. These findings were seminal as they identified a relevant, injury-induced second messenger that could initiate the regenerative process  enzymes. Membrane-bound ACs are regulated by the canonical heterotrimeric G-proteins, whereas soluble isoforms are activated by bicarbonate. Both soluble- and some isoforms of membrane-bound AC are also activated/regulated by Ca2+-dependent regulation of AC activity. cAMP likely exerts its effects locally, affecting nearby target proteins. As such cAMP can function in both the axon and cell body compartments of neurons to regulate axon growth. In the axon/growth cone, cAMP regulates axonal guidance through the PKA-mediated inactivation of RhoA signaling and by the phosphorylation of other targets, which allows the cytoskeletal rearrangement that supports growth cone motility  of RAGs on their own may not be optimal for promoting regeneration. For instance, accumulating data suggests that peripheral injury triggers concerted responses that Previous studies have suggested that cAMP can play a facilitative role in mediating CNS axon regeneration. In the optic nerve crush model, RGC axonal regeneration is stimulated by the release of oncomodulin by macrophages that are recruited by lens injury. cAMP is required for oncomodulin’s actions on axon growth, likely through facilitation of receptor binding  downstream of CREB and cAMP that mediated axon growth. Using arginase 1 (Arg1) as a model RAG, we identified an AP1 site in the proximal promoter region. Blocking AP1 activity with a dominant-negative Fos protein (which inhibits AP1 binding to DNA) blocked both CREB-CA and cAMP-mediated axon growth; which was mirrored by Arg1 expression levels. Importantly, a constitutive-active Fos showed only modest increases in axon growth, suggesting that the AP1-controlled genes cooperate with other CREB targets to stimulate regeneration  with the pharmacological upregulation of relevant signaling pathways  to activate regeneration-associated transcription Figure . AdditioThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The immunological mechanisms that evolved for host defense against pathogens and injury are also responsible for transplant rejection. Host rejection of foreign tissue was originally thought to be mediated mainly by T cell recognition of foreign MHC alleles. Management of solid organ transplant rejection has thus focused mainly on inhibition of T cell function and matching MHC alleles between donor and host. Recently, however, it has been demonstrated that the magnitude of the initial innate immune responses upon transplantation has a decisive impact on rejection. The exact mechanisms underlying this phenomenon have yet to be characterized. Ischemic cell death and inflammation that occur upon transplantation are synonymous with extracellular release of various heat shock proteins (Hsps), many of which have been shown to have immune-modulatory properties. Here, we review the impact of Hsps upon alloimmunity and discuss the potential use of Hsps as accessory agents to improve solid organ transplant outcomes. During organ transplantation, tissues are injured as a consequence of ischemia and reperfusion. Upon organ harvest, ischemia ensues and continues during organ preservation. Reperfusion happens upon anastomosis of graft vessels. Ischemia–reperfusion injury (IRI) induces cell death by necrosis and apoptosis leading to production of molecules denominated damage-associated molecular pattern (DAMPs) or alarmins . These aTo improve the longevity of solid organ transplants, it is desirable to develop methods to limit IRI-induced inflammation and the ensuing alloreactive immune response. This review will discuss how heat shock proteins (Hsps) can modulate inflammatory and allogeneic immune responses, and how this can be harnessed to manage graft rejection in solid organ transplantation.c release (Hsp27), mitochondrial release of Smac (Hsp27), nuclear translocation of apoptosis-inducing factor (AIF) (Hsp70), and cleavage of procaspase 3 (Hsp70)  –12. Hsps (Hsp70) , 14. Fun (Hsp70) .Increased levels of Hsps in transplant organ cells either by treatment or genetic manipulation have been demonstrated to be beneficial for transplant longevity . Hsps prRecently, several studies have highlighted a previously overlooked importance of innate cells in shaping T cell-mediated responses to alloantigens . Indeed,During IRI, an important DAMP released by injured cells is the nucleotide adenosine triphosphate (ATP). Extracellular ATP (eATP) is recognized by purinergic receptors expressed by immune cells. Once eATP engages such receptors, it can trigger innate inflammatory responses and activation and proliferation of T cells. This can lead to further inflammation and cell damage, contributing to rejection . AdHeat shock proteins have been suggested to act as DAMPs . InitialAside from being passively released, Hsps can reach the extracellular milieu through different active pathways. Hsp70 can be exported by an active non-classical secretory pathway, which cannot be blocked by inhibitors of the ER–Golgi pathway . Also, HThus, a scenario in which Hsps are found extracellularly during transplantation is likely, independently of passive or active release. More recent studies suggested that extracellular Hsps in transplants play additional immune roles – triggering anti-inflammatory responses and acting as immune modulators. In contrast to the DAMPs hypothesis, it was proposed that Hsps could belong to a group of molecules denominated resolution-associated molecular patterns (RAMPs) . RAMPs aResolution-associated molecular patterns are proposed to counterbalance acute inflammation and restore immune homeostasis by modulating innate cells. After tissue damage, they can modulate acute inflammation by inducing the production of IL-10 . IL-10 hex vivo and stimulated a reduced allogeneic inflammatory response compared with hearts transplanted from littermate controls (A member of the Hsp70 family that has immunomodulatory effects and acts as a RAMP is the endoplasmic reticulum (ER) protein GRP78 or BiP . When iscontrols . Markerscontrols . Hsp70 ocontrols , 45. Concontrols , 47.In summary, increased levels of Hsps can be beneficial through the prevention of cell death, precluding the release of DAMPs. It is also possible that in HspTg mice, after tissue damage is induced by transplantation, the concentration of extracellular Hsps released from damaged cells is higher, resulting in induction of tolerogenic responses and the dampening of inflammation.Regulatory T cells (Tregs) can suppress excessive effector immune responses that are harmful to the host . Tregs cRecently, it was shown that heat pre-conditioning of the organ had protective effects in acute kidney injury induced by IRI, and that protection was mediated by a direct immunomodulatory response of Hsp70-specific Tregs .Mycobacterium tuberculosis  was found to prolong skin graft acceptance . Interesrculosis . The autrculosis . Consistrculosis . In thisin situ delivery of recombinant Hsp10 improved skin allograft survival in rats (Heat shock protein 10 is an Hsp60 co-chaperone and was first described as early pregnancy factor (EPF) . This pr in rats . The aut in rats .M. tuberculosis can improve graft survival in two different models of skin allografts. First, when the allogeneic B16F10 melanoma cells (H-2b/I-Ab) are subcutaneously injected in BALB/c hosts (H-2d/I-Ad), they are rejected due to MHC disparity. However, when those cells were injected in the presence of DnaK, they could form tumors in the hosts  was also found to promote the immunosuppressive M2-like macrophage phenotype and favor tumor growth in a murine melanoma model  from he hosts . In situma model . Togetheon Tregs , 62.In addition to Hsp60 and Hsp70 family members, Hsp90 proteins have also been shown to have protective properties. For example, subcutaneous treatment with mouse gp96 was shown to delay skin allograft rejection in minor and major mismatch models . In anotThere is now substantial evidence to demonstrate the immunosuppressive potential of Hsps. These studies indicate that the anti-inflammatory properties of Hsps warrant further investigation into Hsp-based treatments for contexts in which repression of immune responses is desirable. As discussed here, Hsp treatments have been effective agents to inhibit alloimmunity and extend solid organ transplant survival in mice. One could hypothesize Hsp treatments that promote a tolerogenic environment to also have therapeutic applications for various autoimmune and inflammatory diseases.It remains to be seen how universal the application of immune-regulatory properties of Hsps can be applied to transplants of different cells and tissues. It is also important to note that Hsps were reported to amplify inflammatory  and immuIn solid organ transplantation contexts, however, most studies have indicated higher levels of intracellular and extracellular Hsps extend graft survival. This is likely due to a combination of the cytoprotective properties of Hsps enabling better survival following IRI and subsequent reduced DAMP release and inflammation as well as the immunomodulatory effect extracellular Hsps have upon multiple cell types including macrophages, T cells, and DCs. The resulting Hsp-induced immuno-biology described has included modulation of APCs to induce tolerogenic responses and regulatory T cells and decreased alloreactive T cell generation Figure .ex vivo manipulation of organs before the transplant in order to increase Hsps levels could constitute a promising approach in decreasing initial ischemic damage and inflammation, improving organ survival. Administration of Hsp-derived peptides or ex vivo expansion of Treg with Hsp-derived peptides could be an alternative strategy to improve solid organ outcome. Elucidation of the innate cell subsets and the receptors by which Hsps can specifically interact with will be extremely important to optimize Hsp-based therapy design.Thus, TB, BL, RL, and CB wrote the paper. TB did the cartoon.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Klippel-Feil syndrome (KFS) is characterized by the failurein segmentation of two or more vertebrae due to an abnormaldivision of the mesodermal somites and has been reported tobe associated with cardiac and central nervous systemanomalies. We report a rare occurrence of concurrentKlippel-Feil anomaly, tethering and dermoid cyst of dorsalspine masquerading Potts’ disease in an eighteen years oldfemale. In rare instances the KFS can be associated withintracranial or spinal tumors, most frequently dermoid orepidermoid cysts.Congenital spinal malformation, Klippel-Feil syndrome,dermal sinus, dermoid cysts In rare instances the KFS can be associated withintracranial or spinal tumors, most frequently dermoid orepidermoid cysts1, 4, 5. We report the rare occurrence ofconcurrent Klippel-Feil anomaly associated with intraspinallesion, tethering and dermoid cyst misinterpreted as Potts’ disease.Klippel-Feil syndrome (KFS) is characterized by the failurein segmentation of two or more vertebrae due to an abnormaldivision of the mesodermal somites An 18- year old female presented with a history ofprogressive weakness of both lower limbs with difficulty inwalking. She had been bedridden for one month. She hadhesitancy of micturition, but no urinary retention. There wasno history of upper limb weakness. There was no history ofcontact with tuberculosis. General and systemicexaminations were unremarkable. Higher mental functionsand cranial nerves were normal. Motor and sensory functionsin upper limbs were normal. There was increased tone in both the lower limbs. Sensations were decreased to allmodalities below D8 level. Power was grade II/V in thelower limbs. Bilateral knee and ankle jerks were exaggeratedand the plantar response was extensor. AP and lateralradiographs of the dorsal spine showed reduced disc spacebetween D8 and 9 vertebral bodies and scalloping of theposterior margins 2, 5. Congenital fusion of thevertebrae in Klippel-Feil syndrome is due to failure ofnormal segmentation of the cervical somites during the thirdto eighth week of gestation4. It has been suggested that adisturbance in the mesoderm before the fourth week ofgestation might play an important part in the causation ofthese anomalies1. A shortening of the spine because of areduction or fusion in the number of somites may result inaltered tissue tension, which could lead to entrapment ofdermal elements4. The classic clinical triad of a short neck,low hairline, and limitation of movement of the neck which is seen in approximately 52% of patients with KFS4 can beabsent in patients where there is no involvement of thecervical spine. Although MRI is the best investigation toshow the extent of intra-spinal lesions but supplementingwith plain radiograph and CT scan provide details of thebony pathology. A total surgical resection is the mainstay oftreatment of these lesions5. In the current case, initialradiographs showed decreased disc space, MRI showedapparently smaller vertebrae, the findings consistent with anintraspinal cystic lesion, leading to misdiagnosis as spinaltuberculosis; however a definitive diagnosis was made afterhistopatholigcal examination.An array of congenital central nervous system abnormalitieshave been described in patients with Kilppel-Feil syndrome whichdevelop - during the same intra-uterine period as thedevelopment of the somites


To overcome the limitations of independent component analysis (ICA), today’s most popular analysis tool for investigating whole-brain spatial activation in resting state functional magnetic resonance imaging (fMRI), we present a new class of local dimension-reduced dynamical spatio-temporal model which dispenses the independence assumptions that severely limit deeper connectivity descriptions between spatial components. The new method combines novel concepts of group sparsity with contiguity-constrained clusterization to produce physiologically consistent regions of interest in illustrative fMRI data whose causal interactions may then be easily estimated, something impossible under the usual ICA assumptions. There is an ever-growing and pressing need for accurately describing how brain regions are dynamically interrelated in resting state fMRI . Thanks To circumvent limitations of modelling high-dimensionality systems, Wikle and Cressie  proposedModel robustness has alternatively been sought by indirect means as, for example, thru LASSO regression  and basiThe present paper extends the results in  about loDSTM problems may be formulated as state-space models  mentclass2pt{minimussed in  on \documentclass2pt{minimGiven By considering that ov space , approximentclasspt{minimaFor each value of  is obtainctional  but in tIn practice, we still need to estimate nt}λm in  for signIf The next step consists of determining which time series of wavelet coefficients Clusterization begins with each Even though the dissimilarity measure  already The next step consists of estimating the observation matrix mentclass2pt{minimAccording to the approximation model,In fact, each spatial component The remainder of the algorithm consists of applying the traditional EM algorithm for timation  using thUsing simulated data to examine algorithm performance under different conditions, we created a vector time series corresponding to points on a discretized one-dimensional space consisting of entclass1pt{minimaentclass1pt{minimaWe used Daubechies (D2) functions to transform the data and gauged performance by executing 100 Monte Carlo simulations leading to the mean and deviation results as shown in Fig. The mean absolute values of the correlation coefficient between the simulated and estimated sources versus SNR in Fig. For further illustration purposes, we used fMRI images from seven healthy volunteers under a resting state protocol .Whole-brain fMRI images (ument}4) . To aid Motion and slice time correction and temporal high-pass filtering . Preprocessing included slice time correction, motion correction, skull stripping, spatial smoothing  and temporal high-pass filtering , subcortical (2) and cerebellar (19) regions. Cortical and subcortical spatial components  as presented here addresses an approach to source estimation and localization in resting state fMRI data analysis that dispenses with artificial stochastic model assumptions, such as those used in classical blind source separation , independent component analysis (ICA) and non-negative matrix factorization (NMF) , 19, 21)The cortical components identified by LDSTM Fig.  reflect  line of ’s observPDC analysis shows a network where the information flows from regions in the superior parietal cortex (SPC) to regions in the cerebellum (CER) and anterior cingulate. As expected, the right SPC sends information to the left CER, and left SPC sends information to the right CER. Although the relationship between these structures is known, this stresses two main systems engaging in the mentioned network. The connectivity between SPC and CER is in line with recent studies showing evidence of a cerebellar-parietal network involved in phonological storage . In addiIn our model, LDSTM identified approximately 50 % of components in the cerebellum. This result is surprising as the rate of cerebellar components identified in resting state using ICA is below 20 % in general . Some ofSince spatial wavelet analysis efficiently encodes the data neighbourhood information via a orthogonal transformation, the present method properly addresses a number of issues involving whole-brain connectivity estimation. The first one is associated to the lack of knowledge about the spatial localization of the sources. The method provides a data-driven approach to locate the main sources of data variability, thus avoiding the effects and uncertainties due to a priori region of interest delineation. The second aspect is that the new method naturally employs multi-scale transformations to create a compact model of the images, a feature of growing importance as higher-resolution images are sure to become available in the near future and whose computational processing load may be thereby substantially mitigated. Finally and most importantly, unlike ICA, the method permits deeper connectivity analysis between the identified spatial components as no independence assumption is made ’a priori’.Various method extensions are possible, especially when it comes to estimate appropriate regularization parameter choice as a function of the amount of noise present in the data. In the present implementation, spatial noise is assumed homogeneous and normally distributed which implies a chi-squared distribution for wavelet coefficient variance. Examination of wavelet coefficients variance for real fMRI data, however, points to the need to consider heavy-tailed distributions, so that a more general approach is currently being developed to estimate wavelet domain noise variance from a finite mixture of exponential distributions that could then be used to quantify the level of data sparsity.Here, an EM-based algorithm was presented for LDSTM identification. By projecting high-dimensional datasets into smoothness spaces, one can describe the system’s spatial components via a reduced number of parameters. Further dimension reduction and denoising is obtained by soft-vector thresholding under contiguity-constrained hierarchical clustering. Finally, simulated results corroborate that the new algorithm can outperform the traditional EM approach even under mild conditions. Even with very large datasets as in the fMRI example, LDSTM shows promise in its ability to parcelate the human brain into well-localized physiologically plausible regions of spatio-temporal brain activation patterns.