The small size of the fluorescent azides used for detection results in a high degree of specimen penetration. It can be used to easily detect DNA replication in large tissue samples or organ explants with low proliferation and turnover of cells formerly believed to be in a “terminal” state of differentiation. Here we describe a protocol for the localization and identification of proliferating cells in quiescent or injured pulmonary vasculature, in a model of pulmonary veno-occlusive disease (PVOD). PVOD is an uncommon form of pulmonary hypertension characterized by progressive obstruction of small pulmonary veins. We previously reported that mitomycin-C (MMC) therapy is associated with PVOD in human. We demonstrated that MMC can induce PVOD in rats, which currently represents the sole animal model that recapitulates human PVOD lesions. Using the EdU assay, we demonstrated that MMC-exposed lungs displayed areas of exuberant microvascular endothelial cell proliferation which mimics pulmonary capillary hemangiomatosis, one of the pathologic hallmarks of human PVOD. In vivo pulmonary cell proliferation measurement represents an interesting methodology to investigate the potential efficacy of therapies aimed at normalizing pathologic angioproliferation.5-Ethynyl-2′-deoxyuridine (EdU) incorporation is becoming the gold standard method for  In 2008, Salic and Mitchison  develope in vivo labeling of DNA-duplicating proliferating cells with EdU over hours or even days allows the detection of rare events of proliferation. For this reason we used this technique to track EC proliferation in controls and in animals affected by pulmonary veno-occlusive disease (PVOD).The normal endothelium is usually considered to be a genetically stable, quiescent cell line . This meμm) with typical complex lesions, plexiform lesions, while PVOD preferentially affects the postcapillary venous pulmonary vessels [PVOD is a rare and devastating cause of pulmonary hypertension (PH). Idiopathic or familial pulmonary arterial hypertension (PAH) is characterized by major remodeling of precapillary small pulmonary arteries  [However, the pathophysiologic mechanisms of PVOD remain poorly understood, particularly due to the lack of PVOD animal models and few studies have addressed the pathobiology of this very rare disease in humans . PVOD man (GCN2) . Drug exn (GCN2) , 24. Somn (GCN2) .In rats, MMC exposure induced preferential remodeling of pulmonary venous and capillary compartments, representing currently the sole animal model of PVOD. MMC-induced PVOD in rats was also characterized by pronounced capillary proliferation within the alveolar septa mimicking human condition.Rats were housed at the Faculty of Pharmacy of Châtenay-Malabry . Experiments were conducted according to the European Union regulations (Directive 86/609 EEC) for animal experiments and complied with our institution's guidelines for animal care and handling. The animal facility is licensed by the French Ministry of Agriculture (agreement number B92-019-01). This study was approved by the Committee on the Ethics of Animal Experiments CEEA26 CAP Sud. Animal experiments were supervised by Dr. Frederic Perros . All efforts were made to minimize animal suffering.PVOD was induced in female Wistar rat (100 g) by MMC, as previously described .(3.1)Inject EdU intraperitoneally into the rats at 50 mg/kg, 24 h before sacrifice.(3.2) via the trachea with a cryoprotector medium  diluted in PBS (1 : 1). Ligate the trachea and flash-freeze the lungs in 2-methylbutane precooled over dry ice.After hemodynamic measurements, exsanguinate the rat by aortic puncture. Then, inflate the lungs(3.3)μm lung section using a cryotome and air dry.Cut 6 (3.4)4Cl solution.Fix dried lung sections with paraformaldehyde (PFA) 4% for 10 minutes at room temperature and quench free aldehyde groups from PFA fixation with 50 mM NH(3.5)Permeabilize with Triton 3% and saturate the tissue with human (10%) and rat (10%) serum in PBS for 1 hour at room temperature.(3.6)Detect EdU using the click-iT EdU imaging kit  according to the manufacturer's instructions.(3.7)α-SMA (clone 1A4) as previously described by Ranchoux and collaborators [Perform double immunostaining against endothelial cell markers, fibroblast, and smooth muscle cell markers. Pulmonary fibroblast cells can be identified using antibody against Vimentin-TRITC (Santa Cruz Biotechnology clone V9), pulmonary microvascular endothelial cells can be labelled using antibody against CD34 , and smooth muscle cells can be labelled using antibody against borators .(3.8)Analyse the lung sections under a fluorescence microscope by quantifying the number of EdU-positive cells.To evaluate cell proliferation in rats, we visualized the incorporation of exogenously supplied 5-ethynyl-2′-deoxyuridine (EdU), to identify cells undergoing DNA replication.μm sections of paraffin-embedded rat lung tissue were stained with Masson's trichrome as previously described [Five escribed .Recently, we described the first relevant animal model of PVOD induced by MMC exposure . We depiP < 0.001). These observations confirm an active proliferation of microvascular endothelial cells and fibroblast in the observed lesions. These lesions are similar to PVOD lesion observed in patient lungs and provide confirmation of MMC-exposed rats as a promising animal model of human PVOD.In order to examine cell proliferation in rats, we visualized the incorporation of exogenous EdU to identify cells undergoing DNA replication. As shown in  In vivo pulmonary cell proliferation measurement with EdU staining represents an important technique to investigate the potential efficacy of therapeutic interventions aimed at reversing pathologic angioproliferation.Cell proliferation analyses are crucial for the assessment of altered cell growth and cell differentiation, which is reminiscent of many diseases including PAH, PVOD, cancers, systemic hypertension, and arteriosclerosis. Cell proliferation analyses are also useful to evaluate the toxicity or/and the potential efficacy of therapeutic molecules. We recently demonstrated that two chemotherapeutic drugs (MMC and cyclophosphamide) could induce PVOD in rats . PVOD isα-SMA, CD34, and Vimentin, in addition to EdU staining.Compared with EdU incorporation, a major drawback of the BrdU staining method is the requirement for a DNA denaturation step, typically by hydrochloric acid treatment or by heating, in order to expose the incorporated BrdU to the anti-BrdU antibody. These harsh staining conditions can damage tissue structure and potentially destroy cellular epitopes , 2. With in vivo is also required. For instance, in organs with high cell turnover such as the intestine, a short duration of labeling (2 hours) was sufficient to designate a baseline position of proliferating crypt cells [In our study, EdU was injected intraperitoneally into the rats at 50 mg/kg, 24 h before sacrifice. However, depending on the cell type, organ of interest, or animal species of the disease model and other parameters, the protocol will need to be refined for optimal assessment of cell proliferation. Some authors have found that the number of EdU (+) cells was associated with EdU concentration, incubation time, and the volume of click reaction solution . For inspt cells . In somept cells  demonstrμm/h for the cell migration rates on the villi in the jejunum and ileum, respectively [EdU can also be used to track proliferating cell. Salic and Mitchison  used EdUectively . Hence, ectively  used thiectively  stated t


Heterogeneous base catalysts are generally more reactive than solid acid catalysts which require extreme operating condition for high conversion and biodiesel yield. In the present study, synthesis of biodiesel was studied by using edible  or non-edible (Jatropha) feedstock catalyzed by heterogeneous base catalysts such as supported alkali metal .The chemical characteristic, textural properties, basicity profile and leaching test of synthesized catalysts were studied by using X-ray diffraction, BET measurement, TPD-COAmong the solid base catalysts, calcium based mixed metal oxides catalysts with binary metal system (CaMgO and CaZnO) showed capability to maintain the transesterification activity for 3 continuous runs at ~ 80% yield. These catalysts render high durability characteristic in transesterification with low active metal leaching for several cycles. Industrialization processes continue to grow globally in par with human population which leads to the growing worldwide demand for energy as well as for petrochemical resources, coal and natural gases. This phenomenon has caused the depletion rate of fossil energy resources to increase exponentially and caused alarming environmental problems to the society. Recently, many fuel developers have showed interests in alternative renewable fuels to substitute or blend with petroleum-based fuels. An alternative fuel shall be easily available, environment friendly and techno-economically competitive ,2.Biodiesel plays a major role in energy sector due to its similar combustion properties with petroleum. Furthermore, biodiesel is sometimes more superior than petroleum diesel with improved physical and chemical properties, such as higher flash point, higher cetane number, ultralow sulfur content, better lubricity, improved biodegradability, and smaller carbon footprint -5. ChemiAccording to Meng et al. (2009), biological feedstock supply for biodiesel production covers more than 75% of the overall production cost .In general, biodiesel feedstock can be divided into 4 main categories which are: (a) edible vegetable oil, (b) non-edible vegetable oil, (c) waste or recycled oil and (d) animal fats . The mosInstead of edible palm oil, non-edible feedstock is getting interest as a biodiesel feedstock in biodiesel production. Amongst the varieties of non-edible plant oil, Jatropha is the most favorable for biodiesel production as they meet the major requirement of biodiesel standards of USA, Germany and European Standard Organization. Jatropha crops can be well adapted to arid and semiarid conditions like non-cropped marginal lands and waste lands with harsh environments. Hence, the cultivation cost is lower as these crops can still sustain reasonably high yield without intensive care. Besides, Jatropha oil possesses similar composition as other vegetable oil, which favors biodiesel production. In addition, Jatropha oil contains of toxic phorbol ester which is unsuitable for human consumption and thus reduces the competitive with food supply market ,16.A new type of heterogeneous catalysis technology has been developed to adapt the natural characteristics of biodiesel feedstock and existing transesterification technology. Utilization of heterogeneous catalyst for biodiesel production has offered some relief to biodiesel producers by improving their ability to process alternative and cheaper feedstock with simplified processes and cheaper manufacturing processes with prolonged catalyst lifetime. The three factors namely catalytic activity, catalyst life and oil flexibility have tremendous impact on the cost of biodiesel ,17. Acco2O3), (b) alkaline-earth metal oxides– magnesium oxide (MgO), calcium oxide (CaO) and strontium oxide (SrO) and (c) calcium-based mixed metal oxides– (CaMgO and CaZnO). This study was aimed to investigate the versatility of solid base catalysts with different chemical characteristic for transesterification of edible and non-edible biodiesel feedstock. The physicochemical properties  of synthesized solid base catalysts produced thru wet impregnation and co-precipitation techniques were investigated. Furthermore, effects of the catalyst loading and reaction time towards catalyst activity were investigated in order to optimize transesterification conditions. The reusability of the solid catalysts was evaluated by batch experiment and the reasons for the deactivation of the catalyst were also discussed by performed the catalyst leaching test.By drawing on this, an attempt has been made in the present work to produce biodiesel from edible  and non-edible (Jatropha) oils using heterogeneous base catalysts: (a) supported alkali metal catalysts– sodium hydroxide supported with alumina (NaOH/Al2O3), alkaline-earth metal oxide  and calcium-based mixed metal oxides (CaMgO and CaZnO) base solid catalysts was revealed by X-ray diffraction pattern  and sodium aluminate (NaAlO2) phases (JCPDS File No. 20-1073). The intensity of Al2O3 peaks were reduced when NaOH was introduced into Al2O3 support, this is due to well dispersion of NaOH on the Al2O3 support in the form of monolayer covering the surface of support. The results indicate that NaOH has reacted with Al2O3 thus giving rise to the formation of aluminates and is in good agreement with that reported by Arzamendi’s and Kim’s research groups [3 and SrO.2H2O phases (JCPDS File No. 1-084-1778 and 28-1222). Strontium compound is difficult to regenerate by thermal treatment, and their decomposition temperatures are higher than 1200°C [2 and hydrolyzed by moisture to form carbonate and hydroxide phase, respectively which compatible with the XRD result. The XRD patterns of calcium-based mixed metal oxides catalysts recorded the patterns of both samples (CaMgO and CaZnO) were corresponded to pure oxide, and no new crystalline phase attributable to the formation of mixed oxides could be detected. CaMgO catalyst showed the appearance of cubic CaO and hexagonal MgO phases where CaZnO catalyst also gave diffraction peaks of CaO and wurzite structure of ZnO phase at 2θ angles of 31.7°, 34.4° and 36.2° (JCPDS File No. 36-1451).The crystalline structure of alumina supported alkali metal  possess similar number of basic sites (~400 μ mol of CO2/g). This indicated that catalyst with multi-metal ion interaction showed synergy effect by enhancing the basicity on active site of catalyst [2 profile of CaO showed the desorption peak at 615°C, which slightly higher compared to the desorption temperature of calcium-based mixed metal oxides catalyst means (~570°C). This could be explained that higher temperature of heating is required to desorb the strong interaction of CO2 from the active basic sites of CaO. The presence of strong basic strength in single CaO or binary system of calcium-based catalyst formed strong bonding with acidic CO2, and thus higher temperature (minimum > 500°C) require for desorption process. From Table Several studies have reported that basicity of catalyst is the major key to improve the transesterification activity. Among basicity distribution strength , the number of strong basic sites that are considered to be the main parameter in yielding a high biodiesel yield . The tot- anions ,33. The catalyst . Howevercatalyst . In addicatalyst . The TPD2O3, MgO, CaO, SrO, CaMgO and CaZnO) were studied thoroughly by varying the transesterification conditions. The transesterification reaction was performed at different reaction time (2–6 h) and catalyst amount (1–5 wt.%) under constant reflux temperature (65°C) and methanol to oil ratio of 15:1.The transesterification of edible  and non-edible (Jatropha) oils in the presence of different solid base catalysts  were reutilized for another two runs for both edible palm and non-edible Jatropha oil . Extra washing step and thermal activation is required prior to each cycle to maintain high reusability. In order to evaluate the leaching of the catalyst, elemental analysis was performed to study the leaching of Ca, Mg and Zn phases for CaMgO and CaZnO catalysts. The result for the fresh and used catalyst was depicted in Table Under optimized conditions , the solid base catalysts (NaOH/Al2O3 powder with an aqueous solution of NaOH compounds. Al2O3 support was impregnated with 15 ml sodium hydroxide (50 wt.%) solution. The impregnate was dried in oven at 100°C overnight and undergone thermal treatment at 500°C.The catalyst was prepared by incipient wetness impregnation of γ-Al3), lime stone (CaCO3) and strontium carbonate (SrCO3).MgO, CaO and SrO were obtained after calcination of pulverized magnesium carbonate  in the air to produce mixed metal oxides.The mixed metal oxide catalysts were prepared by using co-precipitation method, and subsequent calcinations of the precursors. These catalysts were obtained by slowly adding a 2 M aqueous solution of the corresponding metal nitrates to an aqueous solution containing Naα radiation to generate diffraction patterns from powder crystalline samples at ambient temperature. Specific surface area of the catalysts was obtained by the BET (Brunauer-Emmer-Teller) method using Thermo Finnigan Sorptomatic 1900 series nitrogen adsorption/desorption analyzer. The total basicity and basic strength of the catalysts were measured by temperature programmed desorption of carbon dioxide (CO2-TPD) using a Thermo Finnigan TPDRO 1100 apparatus provided with a thermal conductivity detector. The elemental composition of fresh and used catalysts was determined by using inductively coupled plasma-atomic emission spectrometer (ICP-AES) analysis that conducted by using Perkin Elmer Emission Spectrometer Model Plasma 1000.The crystalline phases of synthesized catalysts were analyzed by powder X-ray diffraction analysis using a Shimadzu diffractometer model XRD 6000 with employing Cu-KThe biodiesel feedstock, palm oil and Jatropha oil were purchased from local market and Bionas Sdn. Bhd., respectively. Physicochemical properties of both palm and Jatropha oil were shown in Table 2O3, MgO, CaO, SrO, CaMgO and CaZnO) were used as catalysts for the production of biodiesel from edible palm and non-edible Jatropha oils. All the base catalysts showed the presence of strong basic strength on the active site except for MgO which contained dominant amount of medium basic sites. The transesterification activity of the heterogeneous base catalysts are correlated with basicity and basic strengths of the catalysts. The optimum conditions for solid base catalysts to achieve >90% of palm biodiesel is 3 wt.% of catalyst, 15:1 of methanol to oil molar ratio within 3 h. Jatropha biodiesel yield required higher amount of catalyst (4 wt.%) and longer reaction time (4 h) under reflux temperature to achieve yield > 80%. It is reasonable to conclude that the type of feedstock oil and chemical characteristic of solid base catalyst strongly affects the yield of biodiesel. Despite of good transesterification activity for solid base catalyst, a good candidate of basic catalyst should be able to tolerate free fatty acids and moisture content in feedstock oil. High catalyst’s reusability without leaching of active component is another desirable characteristic in good candidate catalyst. Calcium-based mixed metal oxides catalysts (CaMgO and CaZnO) render high durability characteristic in transesterification with low active metal leaching for several cycles. The strong interactions between active metals provide a superior synergism of high basicity and stability effect for transesterification reaction.Heterogeneous base catalysts (NaOH/AlThe authors declare that they have no competing interests.LHV conceived of the study, participated in its design and coordination and drafted the manuscript. JJC provided advice on the testing method, analyzed results and helped to draft the manuscript. NFA and RNM performed partial experiments and analyzed results. YHTY contributed to conception and catalyst design, reaction study and provided research advice in data interpretation. All authors read and approved the manuscript.First Author: Hwei Voon Lee.Co-Authors: Joon Ching Juan, Nurul Fitriyah binti Abdullah, Rabiah Nizah MF, Yun Hin Taufiq-Yap.


Nanochroloropsis salina cultures to multiple chemical treatments (antibiotics and quorum sensing compounds) and obtained dense time-series data on changes to the microbial community using 16S gene amplicon metagenomic sequencing  to measure microbial taxa-taxa abundance correlations. Short-term treatment with antibiotics resulted in substantially larger shifts in the microbiota structure compared to changes observed following treatment with signaling compounds and glucose. We also calculated operational taxonomic unit (OTU) associations and generated OTU correlation networks to provide an overview of possible bacterial OTU interactions. This analysis identified five major cohesive modules of microbiota with similar co-abundance profiles across different chemical treatments. The Eigengenes of OTU modules were examined for correlation with different external treatment factors. This correlation-based analysis revealed that culture age (time) and treatment types have primary effects on forming network modules and shaping the community structure. Additional network analysis detected Alteromonadeles and Alphaproteobacteria as having the highest centrality, suggesting these species are “keystone” OTUs in the microbial community. Furthermore, we illustrated that the chemical tropodithietic acid, which is secreted by several species in the Alphaproteobacteria taxon, is able to drastically change the structure of the microbiota within 3 h. Taken together, these results provide valuable insights into the structure of the microbiota associated with N. salina cultures and how these structures change in response to chemical perturbations.Open microalgae cultures host a myriad of bacteria, creating a complex system of interacting species that influence algal growth and health. Many algal microbiota studies have been conducted to determine the relative importance of bacterial taxa to algal culture health and physiological states, but these studies have not characterized the interspecies relationships in the microbial communities. We subjected  Open algal cultures are complex dynamic ecosystems inhabited by diverse microbial communities  that displayed community stability and resilience to environmental perturbations at a global level  and examined changes in the connectivities among taxa in the microbial community. We used longitudinal 16S gene amplicon sequencing of 23 samples descended from a single ancestral microbiota and built correlation networks to reflect the dynamics of the inter-taxa associations and to investigate variations in taxa organization in response to different chemical treatments. As a result, we identified five cohesive modules representing various chemical treatment responses. Subsequently, through network centrality analysis, our data showed that key nodes in the modeled network were primarily from the bacterial species belonging to Alteromonadeles and Alphaproteobacteria, suggesting species from these groups are of particular significance and serve as “keystone” OTUs in the microbial community associate with N. salina.Recently, we found a microbial community associated with Roseobacter clade of Alphaproteobacteria are important symbionts of microalgae  and the microbiota were generated by acclimating N. salina culture together with seawater microbiota from the coast of Santa Cruz, CA  were immediately spiked with two separate doses of sterile-filtered organic compounds  for all chemicals; Table −2s−1). An initial 4 ml was taken from each sample after 3 h and centrifuged 10,000 g for 5 min to pellet the bacterial community. The remaining 4 ml in each well were harvested after 24 h. Bacterial community pellets were stored at −80°C prior to DNA extraction.Artificial microcosms of na CCMP, 776 and tGenomic DNA was extracted from algal culture samples with associated microbiota using a ZR Fungal/Bacterial DNA MiniPrep  following the manufacturer's protocol. 16S gene PCR preparation with standard procedures with barcoded primer set 341F forward and 518R reverse primer as previously described . Forward and reverse sequences of paired-end sequences were assembled by aligning 3′ ends using SHE-RA software . The microbial community associated with N. salina from one untreated and four treated samples were collected at 3 and 24 h post inoculation, along with one starting microbiota (0 h), collectively generating 23 samples  using WGCNA, which has been shown to be robust for analyzing relative abundance data . Figure P < 0.05) and turquoise  modules were negatively correlated with treatment time. These data indicate treatment time was a primary factor in contributing to variations in species abundance in these particular studies, which is consistent with beta-diversity analysis using unweighted Unifrac metric measure . There were also no modules significantly associated with tetracycline under the studied concentrations. Figure P < 10−7). Taxonomy analysis showed the brown chemotype-specific module was enriched by the Rhodobacteraceae family , whereas the yellow module was enriched by the Alteromonadaceae family   and Alphaproteobacteria (14 ± 4%), comparted to 3 h  Flavobacteriia (39 ± 2%) and Alphaproteobacteria (32 ± 2%). Meanwhile, Gammaproteobacteria relative abundance at 24 h increased to 60 ± 9% compared to 3 h  Gammaproteobacteria (25 ± 9%). Of particular note, the microbiota  was composed of Flavobacteriia (31 ± 1%), Alphaproteobacteria (21 ± 0%), and Gammaproteobacteria (45 ± 2%), suggesting it exhibits some compositional similarities to 24 h microbiota  prevalent in F/2 medium  that do not significantly perturb bacterial abundance. Nevertheless, the integration of correlations into interconnected networks may be applied in bridging from bacterial species' activity to microbial community functionality.N. salina. The top Rhodobacteraceae OTU has low connectivity, suggesting it has few interaction with other OTUs in the network. Meanwhile, it has higher betweenness values compared to other OTUs. This pattern suggests modular organization of the network via a minimal number of connections required for information flow. These high-betweenness, low-connectivity OTUs may be conceptually thought of as “bridges” in the non-redundant shortest paths, the observation of which resembles previously reported properties of other biological networks  and microalgae may occur through physical attachment (Miller and Belas, Alteromonadaceae are dominant phylotypes among microbial communities in response to the flux of organic matter in phytoplankton blooms, demonstrating how species of Alteromonadaceae could participate in structuring microalgae bacterial communities (McCarren et al., Network centrality analysis has been successfully applied in multiple fields (Jeong et al., N. salina microbial communities to varying doses of TDA, which is secreted as a hallmark bioactive compound in many species of the Roseobacter clade from Rhodobacteraceae family (Berger et al., Phaeobacter, Silicibacter, Ruegeria, and Pseudovibrio (Berger et al., Rhodobacteraceae decreased, while bacteria in Alteromonadales order of unclassified family increased in composition (Figure Alteromonadales has not been documented. Meanwhile the large total number of bacterial populations and differential bacterial growth rates in microbiota might have compounded the observed relative abundance. In terms of community structure, we observed shifting of microalgae microbiota induced by TDA dosage, and a 3 h 500 nM TDA treatment sufficiently skewed the original microbiota structure toward the 24 h unperturbed microbiota. This suggests that, given the myriad complexity in microbiota, microbial community associated with N. salina potentially harbors a dynamic property that responds to the presence of infochemicals exemplified by TDA. While the concentration of TDA produced naturally in either sea water or within the phycosphere is not documented, the involvement of bioactive compounds such as TDA in modulating microbiota structure has profound implications for bacterial community assemblages. Indeed, multiple bioactive compounds with various activities and specificities, such as indole-3-acetic acid (Amin et al., Roseobacter strains from microalgae microbiota (Buchan et al., To explore the possibility of the key species having influential effects on the overall configuration of the microbiota, we looked for evidence of microbiota responsiveness to the proposed key species. We subjected the n Figure . These oN. salina if modeled from the species-species correlation network? Module formation in the microbiota network helps bridge the gap between deep knowledge of individual keystone OTUs and a systems-level view of the microbial community. But such application will have to tackle several issues. Given the connectivity of the correlation network, the correlation network model does not provide detailed mechanisms nor offer regulatory ramification of such variations. While accurate inter-OTU correlation analysis remains an area of active research, methods developed recently such as CoNet (Faust et al., N. salina may help us detect, investigate, and assess practical responsiveness of “key” species or probiotic species introduced into the microbiome to withstand environmental perturbations and improve microalgae production.Our finding from network analysis therefore offers a tangible experimental metagenomics framework to tackle the following question: what is the structure of microbial community associated with KS, TL, EY designed experiments. HG, EY, MT performed experiments. HG, KS analyzed and modeled data. HG, TD, EY, KS wrote manuscript.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.


Many of the pathways identified as affected by shock waves in the hair follicles have been previously reported to be TBI responsive in other organs such as brain and blood. The results suggest that the hair follicle has some common TBI responsive molecular signatures to other tissues. Moreover, various TBI-associated diseases were identified as preferentially affected using a gene network approach, indicating that the hair follicle may be capable of reflecting comprehensive responses to TBI conditions. Accordingly, the present study demonstrates that the hair follicle is a potentially viable system for rapid and non-invasive TBI diagnosis.With wide adoption of explosive-dependent weaponry during military activities, Blast-induced neurotrauma (BINT)-induced traumatic brain injury (TBI) has become a significant medical issue. Therefore, a robust and accessible biomarker system is in demand for effective and efficient TBI diagnosis. Such systems will also be beneficial to studies of TBI pathology. Here we propose the mammalian hair follicles as a potential candidate. An Advanced Blast Simulator (ABS) was developed to generate shock waves simulating traumatic conditions on brains of rat model. Microarray analysis was performed in hair follicles to identify the gene expression profiles that are associated with shock waves. Gene set enrichment analysis (GSEA) and sub-network enrichment analysis (SNEA) were used to identify cell processes and molecular signaling cascades affected by simulated bomb blasts. Enrichment analyses indicated that genes with altered expression levels were involved in central nervous system (CNS)/peripheral nervous system (PNS) responses as well as signal transduction including Ca BINT is considered a source of traumatic brain injury, or TBI Blast-Induced Neurotrauma (BINT) has become an issue for soldiers since high explosives were introduced to warfare in the mid-19Current TBI/mTBI research has focused on two interconnected streams: investigating molecular pathology and exploring efficient diagnostic targets. A good understanding of the molecular mechanisms behind mTBI symptoms would lead to the discovery of specific biomarkers that can be used for diagnostic purposes The mammalian hair follicle system represents a well-coordinated and highly sophisticated process that requires crosstalk between specialized cells, ranging from follicular stem cell populations to fully differentiated cells such as keratinocytes At its core, secondary damage of mTBI is a consequence of altered biological signal transduction and gene expression under traumatic conditions. Stress responsive signal pathways and gene expression subsequently lead to adjustment to metabolism and cellular processes. Studies have shown the involvement of pathways including G Protein-coupled Receptors (GPRs), Toll-Like Receptors (TLRs), Janus Kinase (JAK)/Signal Transducer and Activator of Transcription (STAT) as well as Mitogen-Activated Protein Kinase (MAPK) signaling cascades in TBI responses Rattus norvegicus) to characterize gene expression patterns in the hair follicle. An Advanced Blast Simulator (ABS) was developed and deployed to deliver a single pulse shock wave, and head-only exposures of rats were carried out in a manner that simulated primary blast, and included elements of concussive and whiplash forces. Studies have demonstrated that the amount and quality of hair follicle RNA extract is sufficient for microarray hybridization The current study utilizes hair follicles from a blast exposed rat model  located at DRDC (Defence Research and Development Canada), Suffield Research Centre  was used for producing simulated blast waves Male adult Sprague-Dawley (SD) rats  were used in the current study. The animals were acclimated to the experimental environment for at least one week before use. For exposure, rats were stabilized in plastic sleeves and 3% isoflurane (in oxygen) anaesthetic was conducted for minimum 8 min. The sleeves were then placed in the ABS with the rat head positioned in the test area for shock wave exposure . After exposure, rats were closely observed for signs of stress and general health either 1 or 7 days before being sacrificed. At the end of the observation period, rat whisker follicles (n = 4–8) were quickly pulled and placed in RNAlater . Samples were then stored at −80°C until further analysis. In conducting this research, the authors adhered to the “Guide to the Care and Use of Experimental Animals” and “The Ethics of Animal Experimentation” published by the Canadian Council on Animal Care. All experimental procedures were approved by the Animal Care Committee at Defence Research and Development Canada (DRDC).RNA was isolated from a pooled sample of 6–8 rat hair follicles using a Qiagen microkit . Briefly, follicle samples were homogenized with a sonicator  and a HiBind RNA Spin Column was then used to purify the RNA. Concentrations and integrity of total RNA were determined with a NanoDrop-2000 spectrophotometer and an Agilent Bioanalyzer 2000 A commercially available SurePrint G3 Rat GE 8×60K (G4853A) was purchased from Agilent . Microarray analysis was performed using hair follicles from three sham control rats and five rats that were exposed to simulated blast. Microarray hybridizations were performed according to the Agilent One-Color Microarray-Based Gene Expression Analysis protocol using Cyanine 3 (Cy3) and followed the protocol outlined in Langlois and Martyniuk (2013). Briefly, 200 ng total RNA/sample was used to produce cRNA (Agilent Low RNA Input Fluorescent Amplification Kit). Fragmentation of the cRNA  was followed by 17 h hybridization at 60°C. An ozone barrier slide was used to cover rat microarrays before being scanned using an Agilent High Resolution DNA Microarray Scanner. Expression data from .tif images were extracted by the Feature Extraction Software (v10.7.3.1). The quality of microarray data was evaluated by manual inspection of the quality control reports provided from the Agilent software and each microarray was deemed to be of high quality. Raw expression data was imported into JMP Genomics v5.1. Raw intensity data for each microarray was normalized using Quantile normalization and differentially regulated transcripts were identified, as determined using an one-way analysis of variance (ANOVA) followed by a False Discovery Rate (FDR) set at 5%. All raw microarray data for this experiment have been deposited into the NCBI Gene Expression Omnibus (GEO) database (GSE46367).A subset of genes was selected and subjected to qPCR analysis for validating microarray results. The genes were angiotensin I converting enzyme 2 (Ace2), Tlr2, phosphoprotein enriched in astrocytes 15a (Pea15a), AHNAK nucleoprotein (Ahnak), tp53 regulated inhibitor of apoptosis 1 (Triap1), actin related protein 2/3 complex subunit 4 (Arpc4), Stat5a, spindle and kinetochore associated complex subunit 2 (Ska2), serpin peptidase inhibitor (Sperini), serpin peptidase inhibitor clade A (Serpina1) and hemoglobin beta (Hbb). All the qPCR gene targets were involved in key cellular processes that were reported to be TBI responsive  and the dark intensity signal. Using these metrics, the limit of detection was estimated to be an intensity of 3.0 and all spots measured below this intensity were assigned a value of 3.0. Based upon the quality control report, Feature Extraction, no probe on the rat microarray was saturated. Functional enrichment for gene ontology was performed in JMP Genomics. A p-value (i.e. most significant) was used for gene set ranking and the background list for GSEA was the rat genome. Genes were permutated 1,000 times using the Kolmogorov-Smirnov classic approach as an enrichment algorithm. To broaden the analysis, all pathways were expanded to include cell processes and functional classes in target gene seeds. Gene set categories examined for enrichment within the microarray data included the curated Ariadne cell signaling and metabolic pathways as well as Gene Ontology (GO) terms. Sub-network enrichment analysis (SNEA) for proteins and chemicals regulating cell processes was also performed to identify gene networks regulated in hair follicles following blasting . Additional information on SNEA can be found in Martyniuk and Langlois and Chishti et al. Gene set enrichment analysis (GSEA) was conducted using Pathway Studio 9.0  and ResNet 9.0. The number of gene probes mapped in Pathway Studio was 39, 422. For duplicate probes, the probe that showed the lowest www.cabrillo.edu/~dbrown/tracker/)  also showed no visible injury in the animals experiencing shock wave or weight change comparing between test and sham control rats. Position and movement of the rat head upon shock wave challenge was analyzed using high-speed cameras and “Tracker” computer software (racker/) , S3. Thep-value<0.05. There were 646 transcripts that had a ±1.5-fold change (p<0.05). Transcripts that showed a 10-fold decrease in the follicles included tyrosine phosphatase-like A domain containing 2 (Ptplad2), arachidonate 12-lipoxygenase epidermal (Alox12e) and coiled-coil domain containing 68 (Ccdc68) while transcripts showing a 10-fold increase included basic helix-loop-helix family member e23 (Bhlhe23), hemoglobin alpha (Hba), hemoglobin adult chain 2 (Hba-a2) and hemoglobin beta (Hbb). The complete list of differentially expressed genes is found in 2 = 0.66, p = 0.004)  . Detailep≤0.01 upon blast exposure in rat hair follicles. As shown in p≤0.01 when comparing control rats with the animals after blast exposure (p≤0.01). Enriched genes were primarily associated with extracellular spaces , plasma membrane , reticulum  and nuclear chromosome. Visual representations of gene networks involved in the inflammatory responses and Ca2+ homeostasis can be found in Gene ontology functional analysis provided a first glimpse of the biological relevance of the potential TBI responses in mammalian hair follicle. Raw data were analyzed and enriched independently into three main domains: biological processes, molecular function and cellular component (the complete GO term list can be viewed in exposure . Enrichep<0.05) in hair follicles in rats upon blast simulation. Pathways were further grouped into three categories: receptor signaling (five pathways), cell signaling (seven pathways) and metabolism (nine pathways) (p≤0.01) compared to other differentially enriched pathways (p<0.05), reasoning the responses of these two pathway are more likely to happen. Genes connected to the TLR pathway  showed a decrease in steady state mRNA levels in the rat hair follicles   .Our resuollicles . In addiollicles . Beside ollicles . To be m(p<0.05) . Under tp<0.05) were located preferentially on a couple of rat chromosomes  on the chromosome 13 position 13p13 were preferentially down-regulated (∼40%) following blast . Six memGSEA was conducted on the genes that were differentially enriched on chromosome 1, 7 and 13, in order to explore the functional significance of those chromosomes in rat hair follicle upon blast exposure. Considering the number of pathways enriched on those chromosomes, it is beyond the scope of the current paper to discuss all the processes. Hence, we focus on the pathways that were also enriched in microarray GSEA analysis described in section 3.4. The complete GSEA results for all three chromosomes can be found in p≤0.01 , heart disease , blood circulation disorders , and inflammation , among others.SNEA analysis is also capable of mapping differentially expressed genes that are related to diseases. A total of 260 diseases were identified as enriched in the hair follicles from blast exposed rats  and Transient receptor potential cation channel subfamily v member 2 (TRPV2) 2+ into cells and compartments such as mitochondria and nucleus can initiate apoptotic signaling cascades as it leads a disruption in metabolic processes + transport were also enriched by GO term  and SNEA (potassium ion import/homeostasis) analyses. Such regulation was also proposed by Reinert et al. as another ion transmission related secondary injury following severe primary injuries of TBI Various types of signal transduction responses were also identified, including ion exchange – and GPR-dependent pathways. Firstly, our microarray and enrichment analyses suggest changes in expression profiles of genes encoding CaReceptor signaling pathway was another enriched signal transduction theme upon blast simulation by the enrichment analyses, as indicated by the GO term receptor clustering. Such pathways include GPR-dependent signaling (GO term enrichment) and its major regulator of the guanylate cyclase pathway (GSEA) TLR pathway is another signaling pathway affected at transcriptomic level in hair follicles following blast . It was previously demonstrated that TLR pathways were responsive to TBI in the brain of the mice and proposed as a biomarker for stroke in blood As mentioned above, some of the enriched signaling pathways upon blast exposure are directly linked to inflammatory responses, which is another well-known TBI response As a result of pro-inflammation signal transduction, multiple inflammatory responses were reported following TBI exposure, including elevation of intracranial pressure, accumulation of polymorphonuclear leukocytes as well as increased proliferation of Natural Killer (NK) cells Furthermore, genes involved in defense response, cellular defense response, response to mechanical stimulus and protein refolding upon were enriched in GO term analysis upon blast exposure. Biological processes related to cell survival and proliferation secondary injuries that are commonly associated with the initial trauma to the brain SNEA disease network analysis also suggests that hair follicle is able to reflect comprehensive TBI consequences beyond direct responses. The results suggest that the rat hair follicle system is able to reflect potential changes in blood pressure and related cardiovascular responses upon blast exposure. Cardiovascular homeostasis is a major theme enriched in rat hair follicle and essential to the health of both body and brain. Following moderate to severe TBI, this equilibrium is often disrupted and can result in the loss of blood pressure autoregulation and attenuation of the normal bradycardic response to injury Additionally, positional enrichment analysis suggested that chromosome 1, 7, 13 and corresponding regions on them were preferentially affected by blast simulation. Rat chromosome 1 (the largest rat chromosome) houses the highest number of genes that are responsive to neurological disorders Accordingly, the current study demonstrated that gene expression was responsive to blast exposure in rat hair follicles. Multiple gene set enrichment analyses suggested that several types of cell signal transduction cascades, cell survival and nervous system responses. The results suggested that, on a molecular level, rat hair follicle responds to traumatic conditions similarly to brain and blood of both human patients and lab rodent models . In addition, transcriptomics responses in the rat hair follicle corresponded to brain trauma related medical disorders as well as potential TBI-responsive chromosome positional enrichment. These data indicate that hair follicle may be a robust system with similar TBI molecular signatures that have been characterized in other systems. Furthermore, hair follicles can be easily obtained via simple plucking without professional skills. These features make hair follicle an optimal biomarker system for TBI in the context of military activities as well as other traumatic conditions that could lead to brain injury. In conclusion, the present study demonstrated for the first time that mammalian hair follicle is a potentially optimal system for both TBI pathology study and biomarker discovery.Figure S1Representative of a single shock wave. This shock wave is produced with a 25 psi target pressure.(TIFF)Click here for additional data file.Figure S2Displacement of rat nose in XY plane. Origin is initial resting position of nose. Origin is initial resting position of nose; the path of movement was tracked using high speed video and tracking the tip of the nose.(TIFF)Click here for additional data file.Figure S3Radial distance displacement of rat nose from origin. Origin is initial resting position of nose.(TIFF)Click here for additional data file.Figure S42+ homeostasis; red indicates that the gene is increased in transcript level.Factors involved in Ca(TIFF)Click here for additional data file.Figure S5(A) Linear regression analysis showing correlation between microarray and qPCR; fold change to control was plotted for both microarray and qPCR. (B) Comparison of transcript level change in rat hair follicle upon shock wave exposure between microarray and qPCR.(TIF)Click here for additional data file.Table S1Primers and their corresponding conditions for qPCR analysis.(DOCX)Click here for additional data file.Table S2(DOCX)Click here for additional data file.Appendix S1Gene expression data for the rat transcriptome.(XLS)Click here for additional data file.Appendix S2(XLSX)Click here for additional data file.Appendix S3(XLSX)Click here for additional data file.Appendix S4(XLSX)Click here for additional data file.Appendix S5Abbreviations.(XLSX)Click here for additional data file.


In this study, the validity of the magnetic resonance measures was supported bystrong correlations with clinical functional measures and the responsiveness to diseaseprogression over 1 year was shown to be better with MRI than with the clinicalfunctional tests. Notably, standardised response mean values were greater than 1 ininclusion body myositis and greater than 0·8 in Charcot-Marie-Tooth disease 1A,indicating that magnetic resonance measures are highly sensitive to disease progressionand more responsive than established clinical measures. Even though Charcot-Marie-Toothdisease 1A progresses slowly, magnetic resonance measures detected substantial increasesin disease pathological changes in 1 year. Thus, the encouraging results of Morrow andcolleagues’ study might have a profound effect on clinical trials, potentiallyleading to a need for fewer participants to show efficacy or futility, shorter trials,and ultimately more rapid approval of treatments. An additional advantage of magneticresonance measures compared with timed functional and strength measures, which arehighly relevant to paediatric neuromuscular diseases, is that they are not dependent onparticipant motivation, an issue that has been of concern in Duchenne muscular dystrophytrials.8 Alternatively,magnetic resonance spectroscopy (MRS) could be used to more directly measure1H2O T2  should be carefully explored, with the ultimate goal of using magneticresonance measures as surrogate endpoints in clinical trials.


Primary cortical neurons did not respond to direct stimulation by norepinephrine or amphetamine with increased neurite outgrowth. However, conditioned media from astrocytes exposed to norepinephrine or isoproterenol (a beta adrenergic agonist) significantly increased neurite outgrowth when applied to neuronal cultures. Adrenergic agonists also upregulated FGF-2 expression in astrocytes. Pharmacological analysis indicated that beta receptors and alpha1, but not alpha2, receptors were involved in both effects. Antibody neutralization studies demonstrated that FGF-2 was a critical contributor to neurite outgrowth induced by astrocyte-conditioned media. Taken together the present results suggest that noradrenergic activation, when combined with physical therapy, can improve motor recovery following ischemic damage by stimulating the formation of new neural pathways in an FGF-2-dependent manner.Previously we have shown that addition of amphetamine to physical therapy results in enhanced motor improvement following stroke in rats, which was associated with the formation of new motor pathways from cortical projection neurons of the contralesional cortex. It is unclear what mechanisms are involved, but amphetamine is known to induce the neuronal release of catecholamines as well as upregulate fibroblast growth factor-2 (FGF-2) expression in the brain. Since FGF-2 has been widely documented to stimulate neurite outgrowth, the present studies were undertaken to provide evidence for FGF-2 as a neurobiological mechanism underlying amphetamine-induced neuroplasticity. In the present study rats that received amphetamine plus physical therapy following permanent middle cerebral artery occlusion exhibited significantly greater motor improvement over animals receiving physical therapy alone. Amphetamine plus physical therapy also significantly increased the number of FGF-2 expressing pyramidal neurons of the contralesional cortex at 2 weeks post-stroke and resulted in significant axonal outgrowth from these neurons at 8 weeks post-stroke. Since amphetamine is a known releaser of norepinephrine,  Stroke remains a leading cause of death and disability worldwide In general, drugs that promote neural plasticity appear to facilitate physical therapy-aided motor improvement although there is great variability in outcomes depending on study design, drugs and rehabilitation methods employed. Drugs that increase the synaptic activity of the monoamines norepinephrine (NE), dopamine (DA) and serotonin(5-HT), alter gene transcription, protein synthesis and dendritic outgrowth in a manner similar to what is seen in animals exposed to an enriched environment and/or exercise and which appears to be associated with improved functional outcome following brain injury Preclinical studies indicate that fibroblast growth factor-2  FGF-2 is one of a number of neurotrophic factors that are upregulated in response to brain injury presumably to subserve protective/restorative roles and to restore homeostasis Guide for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee at Hines VA and the Animal Care Committee at University of Illinois at Chicago. Animals were maintained in a temperature and humidity controlled room under a 12∶12-h light/dark cycle. Food intake was moderately restricted throughout the study to maintain body weight at 95% of ad libitum weight. Water was available ad libitum.Adult male Long Evans, black-hooded rats (250–300 g) were used. All experimental procedures were in accordance with the Upon arrival to the vivarium animals were randomly assigned to groups that would ultimately comprise the experimental cohorts. This was done in order to reduce aggression caused by future shuffling of animals among established groups. Prior to stroke surgery animals were trained to criterion on the skilled forelimb reaching task and assessed for their baseline performance on the ladder rung walk test. The pre-operative criterion for skilled forelimb reaching was at least 16 successes in 20 attempts for 3 consecutive days. Animals then underwent middle cerebral artery occlusion essentially as previously described Immediately after surgery, groups of rats were randomly allocated to different experimental conditions as follows:vehicle administration/control housing (VEH+CON)vehicle administration/rehabilitation (VEH+REHAB)amphetamine administration/rehabilitation (AMPH+REHAB).Amphetamine administration in the absence of physical therapy was omitted as our previous study demonstrated that amphetamine alone does not improve motor performance or enhance neurite outgrowth from contralesional cortex On days of drug administration  activity sessions began after completion of daily behavioral testing and fifteen minutes following drug or vehicle administration. D-amphetamine sulfate  or vehicle  was administered subcutaneously.Skilled forelimb reaching was assessed by counting the number of times an animal grasped a sucrose pellet on the first attempt and placed it into the mouth (i.e. “first reach success”). Each testing session consisted of 20 reaching opportunities using the preferred forelimb. Attempts using the non-preferred forelimb were not included in analyses. Tests were performed daily. Ladder rung walk utilized a horizontal ladder runway (1 m×10 cm) with 2 Plexiglas walls on either side. Testing sessions consisted of 3 runway crossings and were performed weekly. Forelimb foot errors were defined as either a complete miss or complete slip. Details of behavioral testing can be found in our previous studies After 8 weeks of behavioral testing, animals were anesthetized with ketamine/xylazine . The sensorimotor cortex opposite to the stroke lesion site was exposed, and 2 injections of 1 µl each of a 10% biotinylated dextran amine (BDA) solution  were placed stereotaxically into the forelimb sensorimotor cortex . Two weeks after BDA injection, animals were overdosed with sodium pentobarbital  and perfused transcardially with 4% paraformaldehyde. Brains and spinal cords were removed, placed in 30% sucrose for 1–2 days, embedded in Tissue-Tek OCT compound , frozen, and stored at −80°C. Alternate coronal cryosections (30 µm thick) were reacted for BDA positive fibers or processed with Nissl stain and analyzed for lesion location and extent as previously described Anatomical structures were identified with the atlas of Paxinos and Watson. Quantification of sprouting corticorubral fibers from the contralesional side to the deafferented  red nucleus was performed by counting all BDA-positive fibers crossing the midline at the level of the red nucleus. The number of labeled cortico-efferent fibers in the cerebral peduncle ipsilateral to the BDA injection site was determined using NIH Image and used to correct for inter-animal variances in BDA tracing as described previously Following pentobarbital overdose animals were perfused with phosphate-buffered saline (PBS) followed by 4% paraformaldehyde in 0.1 M PBS. Animals were sacrificed at either 2 weeks post-op or 8 weeks post-op. Brains were post-fixed for 1 hr, cryoprotected then frozen. Free-floating cryostat sections (30 µm) were incubated in PBS containing 0.1% Triton X-100 (PBST) containing 5% normal goat serum and primary antibody  for 48 hr at 4°C. After PBST washes, sections were incubated in goat anti-mouse  for 1 hr at room temperature, washed and then incubated with mouse ClonoPAP  for 1 hr at room temperature. Visualization was performed using diaminobenzidine . Serially adjacent sections were Nissl-stained using toluidine blue for total cell counts and assessment of lesion size. For all analyses the slides were coded and investigators were blind to the treatment group.th section of the brain from approximately +2.0 mm to −1.0 mm from bregma. Counting was restricted to the region of the motor cortex extending from the primary motor cortex  to the primary somatosensory forelimb area . After outlining the region of interest cell counting was performed following the optical dissector protocol . Section thickness following histological processing averaged approximately 15 um. The guard zone and optical dissector height were set at 2 um and 10 um, respectively. Total cell population = total cells counted×1/ssf×1/asf×1/hsf. A Gunderson coefficient of error of estimation <0.1 was obtained.A systematic random sampling procedure was followed using every 10If the lesion did not impinge on the forelimb region of the sensorimotor cortex, and/or if subcortical damage was observed, then the animal was excluded from the study. Stroke volume was quantitatively analyzed on Nissl stained sections using NIH image as described previously by investigators blind to the treatment group Cell culture media, serum, and antibiotics were obtained from Life Technologies . Primary cultures of rat cortical astrocytes were prepared from postnatal day 2 rat pups essentially as described by McCarthy and de Vellis 3 neurons/cm2) initially in Neurobasal containing B27 and 0.5 mM glutamax supplemented with 10% FBS. The next day the media was exchanged for fresh Neurobasal containing B27/glutamax in which the FBS was omitted (hereafter referred to as N/B27). After another 24 hr, neurons were placed in fresh N/B27 or astrocyte-conditioned media prepared as described below. Following 24 hr incubation under experimental conditions coverslips were taken for immunocytochemical staining for microtubule associated protein 2 (MAP-2) as described below. Cells were maintained at all times at 37°C in a water-saturated environment with 5% CO2Primary cultures of rat cortical neurons were obtained from embryonic day 18 cortices obtained from BrainBits  prepared per the supplier's instructions. Cells were seeded on 12 mm poly-lysine-coated coverslips (approximately 5×106 cells). After 6 hr of incubation the media was collected and concentrated 10-fold using a 9000 MW cut-off centrifugal concentrator. This concentrate was re-constituted by 10 fold dilution with fresh N/B27 immediately prior to application to neurons.Conditioned media was prepared by exchanging normal growth media with N/B27 containing vehicle or the indicated drugs. Routinely, 20 ml of N/B27 was used for T75 flasks of astrocytes . Coverslips were then washed, incubated with secondary antibody  for 1 hr at room temperature, washed and incubated with DAPI and mounted on slides with Fluoromount-G .Following the indicated treatments astrocyte whole cell lysates were prepared in NuPage LDS sample buffer (Life Technologies) supplemented with protease inhibitor cocktail III , phosphatase inhibitor cocktails I and II  and 100 mM DTT with heating at 70°C for 10 min. Samples were fractionated on 10% Bis-Tris NuPage gels (Life Technologies) and proteins electrophoretically transferred to PVDF membrane. Following blocking for 90 min at room temperature with 5% BSA in TTBS  membranes were incubated in primary antibody  overnight at 4°C. Following washes and secondary antibody  incubation for 60 min at room temperature the immunoreactive bands were detected by chemiluminescence with ImmunStar AP substrate  and quantified on a Fluorchem SP imager (Alpha Innotech). Equal loading and uniformity of protein transfer were verified by stripping and reprobing for actin and GAPDH.2 each) were taken. After counting MAP-2 immunoreactive cells, the total neurite length was obtained by first occluding cell bodies and tracing over neuritic processes in Adobe Photoshop. The image was then inverted and converted to grayscale for quantification using ImageJ. For all analyses investigators were blind to the treatment group. Data are expressed and analyzed as percent of corresponding vehicle control.Assessment of neurite formation was carried out using NIH Image for PC (ImageJ) on fluorescence photomicrographs. For each experimental condition (i.e. coverslip) micrographs of 4 fields under a 20× objective  and ladder rung walking  assessed at 8 weeks after stroke.Anterograde tracing of contralesional corticomotor pathways revealed that amphetamine plus physical therapy also resulted in a significant increase in the number of labeled axons originating from the contralesional cortex and crossing the midline to innervate the deafferentated red nucleus . Fig. 2-Since amphetamine has been reported to upregulate growth factors in the brain, such as FGF-2, that could lead to axonal growth we assessed the effects of amphetamine on cellular FGF-2 expression Although upregulation of FGF-2 by amphetamine has previously been characterized as being localized to astrocytes the data from Noradrenergic stimulation has been shown to upregulate FGF-2 expression in astrocytes Inclusion of the beta adrenergic antagonist propranolol (PROP) during the 6 hr incubation of isoproterenol with astrocytes completely blocked the neurite-promoting effects of isoproterenol-conditioned medium when subsequently applied to cortical neurons . InteresTo establish that FGF-2 contributes to the neuritogenesis caused by conditioned media from adrenergically-stimulated astrocytes we first demonstrated that NE or isoproterenol upregulated FGF-2 expression in astrocytes. The present work extends our previous observations on pharmacological adjuncts in stroke rehabilitation. In this study we provide evidence to support FGF-2 as a mechanism underlying the neural plasticity and neurorestorative properties of amphetamine in stroke. We found that, following stroke in rats, short-term amphetamine paired with physical therapy induced an increase in the number of FGF-2 expressing pyramidal neurons in the unlesioned motor cortex as compared to physical therapy alone . This efIn the stereological analysis of FGF-2 expression pyramidal cells in the unlesioned cortex were identified by their distinct morphology and clear evidence of staining. Although no formal attempt was made to assess the intensity of cellular staining it appeared that FGF-2-like immunoreactivity in animals given amphetamine was more intense than in animals that had received vehicle. Previous studies have shown amphetamine to modulate FGF-2 expression. In these studies short-term amphetamine  induced a persistent increase in FGF-2 in the brain that lasted for at least 1 month To directly study a potential role for FGF-2 and the involvement of astrocytes in mediating neurite outgrowth we turned to a cell culture model. Since a major pharmacological action of amphetamine is to induce the release of norepinephrine we tested the effects of amphetamine and adrenergic agents on neurite outgrowth in rat cortical primary neurons in culture. Our results demonstrated that there was no direct effect of drugs on neurite outgrowth , but cleA pharmacological analysis of the adrenergic receptors involved in stimulating astrocyte production of neuritogenic factors revealed that both alpha and beta subtypes were involved. Conditioned media from astrocytes exposed to the non-selective beta agonist, isoproterenol, induced an equivalent amount of neurite outgrowth as conditioned media from norepinephrine-stimulated astrocyes . ConditiConsistent with the involvement of alpha1 receptors and the concept that FGF-2 may be a critical neuritogenic component of astrocyte conditioned media is the observation that alpha1-adrenergic stimulation of the FGF-2 promoter leads to an upregulation in FGF-2 synthesis The potential clinical utility of FGF-2 has been recognized for years. More recently, an analysis of gene expression associated with the learning of skilled motor tasks shows the involvement of FGF-2-related genes in this process In a larger context our findings are consistent with the wealth of preclinical studies that show that noradrenergic activation enhances motor rehabilitation after brain damage Stroke remains a leading cause of disability worldwide for which the development of more effective rehabilitative strategies is needed. Amphetamine, which induces the neuronal release of catecholamines , has been one of the most widely studied drugs for improving motor function following stroke


BRAF V600E mutation, which was negative.In 2006, a 58-year-old woman presented with thyrotoxicosis. She had undergone left hemithyroidectomy 14 years before for a benign follicular adenoma. Ultrasound imaging demonstrated bilateral cervical lymphadenopathy with enhanced tracer uptake in the left lateral neck on a Technetium-99m uptake scan. Fine-needle aspiration biopsy of a left lateral neck node was insufficient for a cytological diagnosis; however, thyroglobulin (Tg) washings were strongly positive. The clinical suspicion was of functionally active metastatic thyroid cancer in cervical lymph nodes. A completion thyroidectomy and bilateral cervical lymph node dissection were performed. Histology demonstrated benign multinodularity in the right hemithyroid, with bilateral reactive lymphadenopathy and 24 benign hyperplastic thyroid nodules in the left lateral neck that were classified as parasitic thyroid nodules. As there had been a clinical suspicion of thyroid cancer, and the hyperplastic/parasitic thyroid tissue in the neck was extensive, the patient was given ablative radioactive iodine (3.7 GBq). After 2 years, a diagnostic radioactive iodine scan was clear and the serum Tg was undetectable. The patient has now been followed for 7 years with no evidence of recurrence. Archived tissue from a left lateral neck thyroid nodule has recently been analysed for Thyrotoxicosis due to functional thyroid tissue in the lateral neck is very rare and may be due to metastatic thyroid cancer or benign parasitic thyroid tissue.Parasitic thyroid nodules should be considered as a differential diagnosis of lateral neck thyroid deposits, particularly where there is a history of prior thyroid surgery.Parasitic thyroid nodules may occur as a result of traumatic rupture or implantation from a follicular adenoma at the time of surgery.The use of ablative radioactive iodine may be appropriate, as resection of all parasitic thyroid tissue can prove difficult.BRAF mutational analysis of parasitic thyroid tissue may provide extra reassurance in the exclusion of papillary thyroid carcinoma. Parasitic thyroid nodules causing thyrotoxicosis are rare; the aetiology and natural history of this diagnosis is unclear. New techniques of mutational analysis of thyroid tissue may provide further prognostic information to guide patient management.3) toxicosis: thyroid-stimulating hormone (TSH), <0.01 mIU/l ; free T3, 8.0 pmol/l  and free thyroxine (T4), 17.4 pmol/l . Anti-thyroglobulin (Tg), anti-microsomal and TSH receptor antibodies were negative. Thyroid function tests one year before were normal.A 58-year-old woman presented in 2006 with a 3-month history of palpitations and sweating. Thyroid function tests showed tri-iodothyronine ; all of these lymph nodes had markedly increased vascular signal. One lymph node in the mid-right cervical area (22 mm diameter) had loss of hilar echoes.Technetium-99 thyroid scan  showed aFine-needle aspiration biopsy of bilateral lymph nodes provided insufficient material for cytological diagnosis. Tg washings were performed on the aspirate specimens. The left-sided nodule aspirate had markedly elevated Tg at 13239 μg/l; the right-sided aspirate was negative for Tg.The positive Tg level in the functioning thyroid tissue in the left side of neck, which appeared to be within lymph nodes, seemed to support a diagnosis of metastatic thyroid cancer. Therefore, the archived pathological slides and paraffin blocks from the left hemithyroidectomy specimen undertaken in 1992 were reviewed. The review confirmed the diagnosis of a benign follicular adenoma measuring 21 mm in diameter with no evidence of capsular penetration or vascular invasion, thus excluding the diagnosis of follicular carcinoma A and B. The provisional diagnosis was a functional metastatic thyroid cancer from a previously undiagnosed primary cancer. In preparation for surgery, she was rendered euthyroid with carbimazole 10 mg mane. A completion thyroidectomy with central and selective bilateral neck dissection  was then performed.Histopathology demonstrated a multinodular goitre within the right thyroid lobe with no features of malignancy. In the right neck dissection, all 27 lymph nodes were normal or had non-specific reactive changes. In the left neck dissection, there were 38 normal lymph nodes and 24 nodules  of hyperplastic thyroid tissue in soft tissue not associated with lymph nodes A and B. 131I 3.7 GBq dose after T4 withdrawal . The whole-body scan done 3 days after radioactive iodine ablation showed uptake in the thyroid bed only.At 2 months after surgery, she received ablative 123I scan done 32 months after the completion thyroidectomy, following Thyrogen stimulation , was clear with no uptake throughout the body and an undetectable Tg level (<0.2 μg/l). The patient has now been followed for 7 years with no evidence of recurrence on ultrasound and with serum Tg remaining undetectable (on thyroxine).A follow-up diagnostic whole-body BRAF V600E mutation. This was performed using immunohistochemistry and PCR ; all results were negative.Owing to the clinical suspicion of malignancy and the unknown natural history of parasitic thyroid nodules, archived thyroid tissue was tested for the Our diagnosis in this case, taking into account all the clinical and pathological features, is of functional parasitic thyroid nodules in the left lateral neck. Parasitic thyroid nodules are defined as accessory thyroid tissue with no association with lymph nodes, they may or may not be still attached to the thyroid gland proper by a thin fibrous band 4. Histology demonstrated hyperplasia of the thyroid gland with a follicular adenoma. The second reported case There are two similar cases reported in the recent literature both of which received radioactive iodine ablation post-operatively The differential diagnosis of our patient's presentation includes: seeding from prior thyroid surgery, thyroid rests of embryological origin and metastatic thyroid cancer despite bland histology. Seeding of thyroid tissue from surgery usually results in either multiple subcutaneous thyroid nodules or thyroid tissue surrounded by connective tissue in the field of a previous operation Thyroid rests from embryological origin occur in over 50% of patients Metastatic thyroid cancer despite bland histology was also considered a potential differential diagnosis. In this case, there was no association of the hyperplastic nodules with lymph node tissue. Re-examination of the left hemithyroidectomy specimen from 1992 confirmed the previous diagnosis of a follicular adenoma with no malignant features. A papillary thyroid microcarcinoma was also found; however, the parasitic thyroid nodules in our case had no nuclear features of papillary thyroid carcinoma. Given that the pre-surgical provisional diagnosis was of metastatic thyroid cancer, despite the lack of subsequent histological confirmation of malignancy, our patient was managed with radioactive iodine ablation due to the clinical suspicion of malignancy and the extensive and functionally active parasitic thyroid nodules.BRAF mutational analysis has been described as a promising diagnostic and prognostic indicator of papillary thyroid carcinomas BRAF V600E is an activating mutation that encodes B-Raf, a serine/threonine protein kinase that is involved in directing cell growth BRAF V600E mutation in papillary thyroid carcinoma is between 32 and 73% Molecular markers may be useful in this situation as a guide to the malignant potential of lateral thyroid tissue On balance, we feel that the most likely mechanism behind the development of the functional lateral thyroid nodules seen in this case is thyroidosis due to seeding from the previous thyroid surgery. Our case resembles the two previously reported cases where thyroid tissue was found at sites outside of the operative field (lateral neck See attached.L Baker was responsible for the case report and literature review. B Crawford edited and finalised the case report, and is the treating physician. A Gill, C Chan and B Lin assisted with pathology slides and discussion.


In this study, Lee and colleagues develop a genomic predictor that can identify patients at high risk for late recurrence of hepatocellular carcinoma (HCC) and provided new biomarkers for risk stratification. Typically observed at 2 y after surgical resection, late recurrence is a major challenge in the management of hepatocellular carcinoma (HCC). We aimed to develop a genomic predictor that can identify patients at high risk for late recurrence and assess its clinical implications.n = 396) who underwent surgery between 1990 and 2011 at four centers (210 recurrences during a median of 3.7 y of follow-up). In multivariate analysis, this signature was the strongest risk factor for late recurrence . In contrast, our previously developed tumor-derived 65-gene risk score was significantly associated with early recurrence (p = 0.005) but not with late recurrence (p = 0.7). In multivariate analysis, the 65-gene risk score was the strongest risk factor for very early recurrence  . The potential significance of STAT3 activation in late recurrence was predicted by gene network analysis and validated later. We also developed and validated 4- and 20-gene predictors from the full 233-gene predictor. The main limitation of the study is that most of the patients in our study were hepatitis B virus–positive. Further investigations are needed to test our prediction models in patients with different etiologies of HCC, such as hepatitis C virus.Systematic analysis of gene expression data from human liver undergoing hepatic injury and regeneration revealed a 233-gene signature that was significantly associated with late recurrence of HCC. Using this signature, we developed a prognostic predictor that can identify patients at high risk of late recurrence, and tested and validated the robustness of the predictor in patients  is diagnosed in its early stages, it can be treated by surgically removing part of the liver (resection), by liver transplantation, or by local ablation, which uses an electric current to destroy the cancer cells. Unfortunately, the symptoms of HCC, which include weight loss, tiredness, and jaundice (yellowing of the skin and eyes), are vague and rarely appear until the cancer has spread throughout the liver. Consequently, HCC is rarely diagnosed before the cancer is advanced and untreatable, and has a poor prognosis (likely outcome)—fewer than 5% of patients survive for five or more years after diagnosis. The exact cause of HCC is unclear, but chronic liver (hepatic) injury and inflammation  promote tumor development.Even when it is diagnosed early, HCC has a poor prognosis because it often recurs. Patients treated for HCC can experience two distinct types of tumor recurrence. Early recurrence, which usually happens within the first two years after surgery, arises from the spread of primary cancer cells into the surrounding liver that left behind during surgery. Late recurrence, which typically happens more than two years after surgery, involves the development of completely new tumors and seems to be the result of chronic liver damage. Because early and late recurrence have different clinical courses, it would be useful to be able to predict which patients are at high risk of which type of recurrence. Given that injury, inflammation, and regeneration seem to prime the liver for HCC development, might the gene expression patterns associated with these conditions serve as predictive markers for the identification of patients at risk of late recurrence of HCC? Here, the researchers develop a genomic predictor for the late recurrence of HCC by examining gene expression patterns in tissue samples from livers that were undergoing injury and regeneration.By comparing gene expression data obtained from liver biopsies taken before and after liver transplantation or resection and recorded in the US National Center for Biotechnology Information Gene Expression Omnibus database, the researchers identified 233 genes whose expression in liver differed before and after liver injury . Statistical analyses indicate that the expression of the HIR signature in archived tissue samples was significantly associated with late recurrence of HCC in three independent groups of patients, but not with early recurrence (a significant association between two variables is one that is unlikely to have arisen by chance). By contrast, a tumor-derived 65-gene signature previously developed by the researchers was significantly associated with early recurrence but not with late recurrence. Notably, as few as four genes from the HIR signature were sufficient to construct a reliable predictor for late recurrence of HCC. Finally, the researchers report that many of the genes in the HIR signature encode proteins involved in inflammation and cell death, but that others encode proteins involved in cellular growth and proliferation such as STAT3, a protein with a well-known role in liver regeneration.These findings identify a gene expression signature that was significantly associated with late recurrence of HCC in three independent groups of patients. Because most of these patients were infected with HBV, the ability of the HIR signature to predict late occurrence of HCC may be limited to HBV-related HCC and may not be generalizable to HCC related to other causes. Moreover, the predictive ability of the HIR signature needs to be tested in a prospective study in which samples are taken and analyzed at baseline and patients are followed to see whether their HCC recurs; the current retrospective study analyzed stored tissue samples. Importantly, however, the HIR signature associated with late recurrence and the 65-gene signature associated with early recurrence provide new insights into the biological differences between late and early recurrence of HCC at the molecular level. Knowing about these differences may lead to new treatments for HCC and may help clinicians choose the most appropriate treatments for their patients.http://dx.doi.org/10.1371/journal.pmed.1001770.Please access these websites via the online version of this summary at US National Cancer Institute provides information about all aspects of cancer, including detailed information for patients and professionals about primary liver cancer (in English and Spanish)The liver cancer The American Cancer Society also provides information about primary liver cancer (including a video about coping with cancer)The UK National Health Service Choices website provides information about primary liver cancer (including information about living with primary liver cancer)Cancer Research UK (a not-for-profit organization) also provides detailed information about primary liver cancerMD Anderson Cancer Center provides information about symptoms, diagnosis, treatment, and prevention of liver cancer (in English and Spanish)MedlinePlus provides links to further resources about  Liver cancer is the second-leading cause of cancer mortality worldwide, accounting for approximately 600,000 cancer-related deaths annually In HCC, two distinct types of recurrence are known. Early recurrence arises from primary cancer cells disseminating to the surrounding liver and is usually observed within the first 2 y after surgery. In contrast, late recurrence, which is typically observed >2 y after surgery, appears to be a result of chronic liver damage known as the “field effect,” and produces de novo tumors that are independent of resected primary tumors Chronic injury and inflammation are known to promote tumor development. HCC is one of the best known examples, as more than 90% of HCCs arise in the context of hepatic injury and inflammation We and other researchers have discovered several genomic predictors for recurrence of HCC n = 96) and Fudan University in China  were obtained from Gene Expression Omnibus  This is a retrospective multi-center cohort study aimed at investigating the association of a gene expression signature reflecting HIR in human liver with recurrence of HCC and developing a genomic predictor that can identify patients with a high risk of recurrence after surgical treatment. Archived tissue samples (tumors and matched surrounding non-tumor tissues) of 72 HCC patients (cohort 1) undergoing hepatectomy as primary treatment at the Dongsan Medical Center of Keimyung University, Daegu, Korea, and the Guro Hospital of Korea University College of Medicine, Seoul, Korea, between 26 December 2001 and 3 June 2011 were included in this study. Patients were consecutively enrolled and selected on the basis of the availability of both tumor tissues and non-tumor surrounding tissues. All liver tissues were frozen in liquid nitrogen and stored at −80°C until RNA extraction. The study protocols were approved by the Institutional Review Boards at Dongsan Medical Center, Guro Hospital, and the University of Texas MD Anderson Cancer Center, and all participants provided written informed consent. Patients in cohort 1 were followed up prospectively at least once every 3 mo after surgery. Recurrence-free survival, which was defined as the time from surgery to the first confirmed recurrence, was censored when a patient died or was alive without recurrence at last contact. To test the robustness of the genomic predictor, gene expression data from two independent cohorts were used. Gene expression and clinical data from Queen Mary Hospital of University of Hong Kong  and living donors (n = 8), respectively (GSE12720) n = 4, GSE15239).To identify a gene expression signature that faithfully reflects HIR in human liver, i.e., a HIR signature, we acquired gene expression data from liver biopsies of liver transplantation and hepatectomy patients from the National Center for Biotechnology Information's Gene Expression Omnibus database. The first and second sets of biopsies were taken from the livers of deceased donors . For each sample, 500 ng of total RNA was used for labeling and hybridization according to the manufacturer's protocols. After the bead chips were scanned with an Illumina BeadArray Reader, the microarray data were normalized using the quantile normalization method in the Linear Models for Microarray Data package in the R language environment p-value was less than 0.005. This stringent significance threshold was used to limit the number of false-positive findings. We also performed a global test of whether the expression profiles differed between two classes (two series of biopsies) by permuting the labels of which arrays corresponded to which classes. For each permutation, the p-values were recomputed, and the number of genes significant at the 0.005 level was noted. The proportion of the permutations that gave at least as many significant genes as the actual data was the significance level of the global test. A total of 325 gene features (representing 233 unique genes) were identified as the HIR signature.We identified genes that were differentially expressed between two series of biopsies in three different datasets . Genes were considered statistically significant if their t-test p-value was less than 0.005. To stratify HCC patients as being at low risk or high risk of de novo late recurrence of disease according to the HIR signature, we applied a classification algorithm based on a Bayesian compound covariate predictor , yielding 13 genes. Next, backward stepwise multivariable regression analysis was carried out to find genes as independent predictors of recurrence (p<0.1). Four genes  were identified from the analysis.Prediction analysis of microarrays (PAM) was carried out as described previously p<0.05 indicated statistical significance. All statistical analyses were conducted in the R language environment (http://www.r-project.org).Patient prognoses were estimated using Kaplan–Meier plots and the log-rank test. We used multivariate Cox proportional hazards regression analysis to evaluate independent prognostic factors associated with recurrence-free survival. Recurrence-free survival was defined as the time from surgery to the first confirmed recurrence; data were censored when a patient died or was alive without recurrence at last contact. As covariates we used sex, age, alpha-fetoprotein concentration, infection with HBV, liver cirrhosis, tumor size, tumor number, vascular invasion, Barcelona Clinic Liver Cancer (BCLC) tumor stage, 65-gene risk score, and the HIR signature z-score was used to estimate the activation state of the transcription factors. An absolute z-score of >2 was considered significant as suggested by Ingenuity Pathway Analysis. The overlap p-values generated by Fisher's exact test were used to estimate the statistical significance of overlap between the dataset genes and the genes regulated by a transcription factor.We used the gene network analysis built into Ingenuity Pathway Analysis to identify potential upstream transcription factors that regulate gene expression patterns enriched in the HIR subtype. The analysis is based on prior knowledge of expected effects of transcriptional factors on their target genes stored in the Ingenuity Knowledge Base Surrounding liver tissues from 20 patients with HCC (ten with early recurrence [≤2 y] and ten with late recurrence [>2 y]) were investigated for immunohistostaining validation. For comparison, normal liver tissues obtained from nine donors for liver transplantation were used as normal control. Representative sections of formalin-fixed, paraffin-embedded tissues were used for immunohistochemistry. Primary antibodies against STAT3  and p-STAT3  were used. Briefly, 4-µm-thick sections of the tissues were deparaffinized and rehydrated. After treatment with a 3% hydrogen peroxide solution for 10 min to block endogenous peroxidases, the sections were pretreated in 10 mM citrate buffer (pH 6.0) in a microwave oven for 15 min for antigen retrieval. After incubation with the primary antibodies, the sections were processed using the EnVision detection system (Dako) according to the manufacturer's instructions, and 3,3′-diaminobenzidine tetrahydrochloride was used as a chromogen. All sections were counterstained with Mayer hematoxylin. For interpretation of the immunohistochemical stain results, the staining intensities of STAT3 were graded on a scale of 0 to 2 , and the extent of distribution was rated on a scale of 0 to 3 . For p-STAT3, the staining intensities were graded on a scale of 0 to 3 , and the extent of distribution was also rated on a scale of 0 to 3 . The histoscore was determined by multiplying the intensity scores by the distribution scores .BIRC3, GADD45B, IL1RN, LDLR, MCL1, RALGDS, CDKN1A, CCL20, ADM, MYC, DUSP5, BCL3, SOD2, SERPINE1, PHLDA1, C13ORF15, IER3, CEBPD, and SLC2A3) were quantified using quantitative reverse transcription polymerase chain reaction (qRT-PCR) experiments. Total RNAs from the surrounding non-tumor liver tissues of 24 patients in cohort 1 were reverse-transcribed using a first-strand cDNA synthesis kit . The resulting cDNAs were assayed using OriGene primers specific to each gene and the Mastercycler ep realplex system (Eppendorf). Cycling conditions were 95°C for 30 s, followed by 40 cycles of 95°C for 5 s, 62°C for 30 s, and 72°C for 20 s. Relative amounts of mRNAs were calculated from the threshold cycle number using expression levels of GAPDH as an endogenous control. All experiments were performed in triplicate, and average values are presented.The mRNA expression levels of 19 genes (p<0.005) before and after hepatic injury with regeneration in the three data-sets. The expression of 325 probes representing 233 unique genes (the HIR signature) was significantly altered in all three datasets  were predicted to have a high probability (>0.5) of having the HIR signature (HIR subgroup), and 49 patients (68%), a high probability of having the QT signature (QT subgroup). Recurrence-free survival differed significantly by subgroup , but was significantly associated with late recurrence , the risk score was significantly associated with a high overall recurrence rate  and failed to identify patients at high risk for late recurrence (p = 0.7), suggesting that this tumor-derived signature is more directly correlated with recurrence mediated by intrahepatic metastasis of primary tumors, which usually occurs within 2 y after surgery Using gene expression data from tumors, we had previously developed a 65-gene risk score that can identify patients at high risk for HCC recurrence after surgery p = 0.7) or intermediate recurrence (p = 0.3) but remained significantly associated with late recurrence (p = 0.001) (p<0.001) but not with the intermediate recurrence group (p = 0.4) or the late recurrence group (p = 0.7) (Because recurrence is the leading cause of death among HCC patients during the first year after surgery = 0.001) . The tump = 0.7) . The facp = 0.003) and BCLC stage (p = 0.001), which is the most frequently used clinical staging system and already a well-known risk factor for HCC recurrence (p = 0.008). The HIR signature was not a significant predictor of very early recurrence.We next assessed the prognostic association between the two predictors (the HIR signature and the 65-gene risk score) and known demographic and clinical risk factors. For the very early recurrence subgroup , significant predictors of recurrence-free survival were the 65-gene risk score (currence  [5]. Whep = 0.002) and BCLC stage (p = 0.08) were important predictors of recurrence-free survival (p = 0.002). The 65-gene risk score was not a significant predictor of late recurrence.In the late recurrence subgroup , the HIR signature . The significant association between the HIR signature and late recurrence remained the same regardless of the cutoff time for late recurrence , we then limited our analysis to patients whose tumors were BCLC stage 0 or A at the time of surgery   . It is ap<0.001) . Furtherp<0.001) .To gain better insight into the molecular characteristics of the HIR signature, we categorized genes according to their known functions based on the Ingenuity Knowledge Base repository p<0.001 by two-sample t-test) and identified 177 genes (p<0.001) and NOTCH1 (p<0.001) were again significantly activated in the HIR subgroup  in surrounding tissues using qRT-PCR methods. Consistent with our prediction, expression of downstream targets was significantly higher in HIR subtype than QT subtype samples  (Because the number of genes in the HIR signature (233 genes) is too large for easy translation of current findings into clinical practice, we estimated the minimum number of genes that can faithfully predict the HIR and QT subgroups. When the PAM algorithm that can find subsets of genes that best characterize each class p<0.001) . Furtherp<0.001) .RALGDS, IER3, CEBPD, and SLC2A3) as independent predictors of recurrence. We next tested whether the expression data of these four genes would be sufficient to construct a reliable prognostic model. Similar to the HIR20 model, the four-gene prognostic model (HIR4) showed significant concordance with original model   . The prop<0.001) . Taken tBIRC3, GADD45B, IL1RN, LDLR, CDKN1A, CCL20, DUSP5, BCL3, SERPINE1, PHLDA1, and C13ORF15) were extremely high , suggesting that the expression data of most genes would be highly reproducible. In addition, our data demonstrated high concordance of four genes selected from multivariate logistic regression analysis  after resection of the primary tumor. The robustness of this signature was validated using three independent cohorts with a total of 396 patients. Our results also suggest that signaling pathways activated during HIR may play important roles in de novo late recurrence of HCC, and are potential explorable therapeutic targets for prevention of de novo recurrence of HCC.We further demonstrated that our previously developed tumor-derived 65-gene risk score was strongest for predicting very early recurrence (<1 y). Thus, genetic information from tumors may be limited for predicting very early recurrence, but surrounding non-tumor liver tissues may contain genetic information sufficient for predicting late recurrence. These findings support a previously developed notion that early recurrence might be largely due to metastatic tumor regrowth of primary tumor while late recurrence might be due to de novo tumor development from non-cancerous lesions with predisposing risk A significant association of STAT3 activation with late recurrence was suggested by the gene network analysis with the HIR signature and later validated by immunohistostaining of the surrounding liver tissues. Our findings are in good agreement with previous studies demonstrating that STAT3 is necessary for both liver regeneration following hepatic injury and hepatocarcinogenesis in mouse models A previous study identified a prognostic 186-gene expression signature (the Broad signature) from surrounding non-tumor liver tissues of HCC patients that was significantly associated with late recurrence of HCC We also assessed the minimum number of genes required for a reliable prediction model by several independent approaches. First, PAM showed that ten to 20 genes would be sufficient to construct a reliable predictor with an approximately 10% miscalculation rate. Second, we found that a 20-gene predictor (HIR20 model) worked as well as the full 233-gene HIR predictor in identifying patients at high risk for late recurrence. Third, multivariate logistic regression analysis revealed that as few as four genes (HIR4 model) were sufficient to construct a reliable predictor. Lastly, the robustness of microarray data was validated by qRT-PCR experiments with the same RNAs used for microarray experiments. Thus, our study also clearly demonstrated the feasibility of developing reliable predictors with a small number of genes with the use of relatively simple technology such as qRT-PCR.Our study has some limitations. Tissues and clinical data were retrospectively collected. Thus, our models need to be validated in a prospective study, although we obtained the same results for three independent patient cohorts. It is also important to point out that prognostication of the HIR signature may be limited to HBV-related HCC because the majority of the patients in our study were HBV-positive. This could limit the generalizability of our results. While the validation of the HIR signature in three independent cohorts of patients with different ethnic and environmental backgrounds enrolled in Korea and China strongly supports the potential generalizability of our models, it will be necessary to test our models in patients with different etiological backgrounds such as HCV or obesity in a future study.In summary, we showed that two different genomic predictors can identify patients at high risk for early and late HCC recurrence. Because these recurrences are clinically different entities with distinctive biological characteristics, separate rational management or treatment recommendations can be developed. For example, patients at high risk of late recurrence may benefit from the use of JAK/STAT and NOTCH1 pathway inhibitors after surgical resection Checklist S1STARD checklist.(DOC)Click here for additional data file.Figure S1Kaplan–Meier survival plots of recurrence-free survival of patients with late recurrence.(PDF)Click here for additional data file.Figure S2Kaplan–Meier survival plots of recurrence-free survival of patients in three recurrence groups.(PDF)Click here for additional data file.Figure S3Kaplan–Meier survival plots of recurrence-free survival of patients with early HCC recurrence stratified by the HIR signature.(PDF)Click here for additional data file.Figure S4Kaplan–Meier survival plots of recurrence-free survival of patients with early HCC recurrence stratified by 65-gene risk score.(PDF)Click here for additional data file.Figure S5Differentially expressed genes between HIR and QT subgroups of HCC in three cohorts.(PDF)Click here for additional data file.Figure S6STAT3 and NOTCH1 networks in HIR subgroup of surrounding non-tumor tissues from HCC patients.(PDF)Click here for additional data file.Figure S7Expression of genes regulated by NOTCH1 in surrounding non-tumor tissues from HCC patients.(PDF)Click here for additional data file.Figure S8Miscalculation rate of prediction models with a given number of genes.(PDF)Click here for additional data file.Figure S9Significance concordance between expression data from microarray experiments and qRT-PCR experiments in 15 genes selected for validation of microarray data.(PDF)Click here for additional data file.Figure S10Significance concordance between expression data from microarray experiments and qRT-PCR experiments in four genes selected from multivariate logistic regression analysis.(PDF)Click here for additional data file.Table S1Genes in the HIR signature.(DOCX)Click here for additional data file.Table S2Overlap of predicted outcomes by two genomic predictors.(DOCX)Click here for additional data file.Table S3Functional categories of HIR signature(DOCX)Click here for additional data file.Table S4Transcription factors activated in HIR gene expression signature.(DOCX)Click here for additional data file.Table S5Expression and phosphorylation of STAT3 in liver tissues.(DOCX)Click here for additional data file.Table S6Functional categories of genes in the 65-gene risk score.(DOCX)Click here for additional data file.Table S7Selected 20 genes from top ten functional categories.(DOCX)Click here for additional data file.Table S8Concordance between the HIR and HIR20 models.(DOCX)Click here for additional data file.Table S9Concordance between the HIR and HIR4 models.(DOCX)Click here for additional data file.Text S1Supporting text.(DOCX)Click here for additional data file.