This article reviews the synthesis, structural and optical characterizations of some novel luminescent two dimensional organic-inorganic perovskite (2DOIP) semiconductors. These 2DOIP semiconductors show a self-assembled nano-layered structure, having the electronic structure of multi-quantum wells. 2DOIP thin layers and nanoparticles have been prepared through different methods. The structures of the 2DOIP semiconductors are characterized by atomic force microscopy and X-ray diffraction. The optical properties of the 2DOIP semiconductors are characterized from absorption and photoluminescence spectra measured at room and low temperatures. Influences of different components, in particular the organic parts, on the structural and optical properties of the 2DOIP semiconductors are discussed. Two dimensional organic-inorganic perovskites 2DOIPs) are a wide class of materials, with general chemical structures (R-NHDOIPs are (OILED) ,6. Anoth (OILED) ,8,9, and (OILED) ,11. More (OILED) .mensions ,15,16,17Energy gap in an organic material is defined as the energy difference between the highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) bands. Since the energy gap of the organic layers is usually much larger than that of the inorganic layers, the electronic structure of 2DOIPs can thus be regarded as a multi-quantum-well, consisting in inorganic wells and organic barriers. The optical properties of the 2DOIPs are generally decided by the inorganic layers. Due to the large variety of MXThe 2DOIP crystals exist as bulks, thin films, and nanoparticles ,19,20,21Amines ) and PbIet al. have developed a single source thermal ablation method [2DOIP films prepared through thermal ablation have also been reported ,29,30. Tn in ref.. These tWe have realized luminescent 2DOIP nanoparticles by a spray-drying (or nebulization/lyophilization) method . Similaro 500 nm .Atomic force microscopy (AFM) is a powerful tool for determining the surface topography with nanometer scale resolution ,32,33. Din films .X-ray diffraction (XRD) is a frequently used method to characterize the ordering within the self-organized 2DOIPs ,35. Figu 6.325 Å , thus th 6.325 Å . The XRDmparable . Howevermparable .I are the intensities of a monochromatic light of wavelength , but so4 in ref ). PhotolI is the integrated intensity of the PL spectrum, P is the power of excitation light, and a is the absorption factor at the excitation wavelength (325 nm here). As an example, we were able to demonstrate that the PL efficiencies of the PhE-PbBrIn , we haveectively .The 2DOIP nanoparticles show also good PL efficiency. r 2DOIPs .e 30 meV . Thus, te 30 meV . In addiThe absorption wavelength range of the 2DOIPs is related to the nature of the inorganic components. In the 2DOIPs with general chemical structures , 2-phen, 10, 12 . The orge factor . The lator X = I . The radIn the 2D (R-NHl chains ,58 or cyl chains  usually X4 films . BesidesWe reviewed different methods of synthesis, structural and optical characterizations of the luminescent two-dimensional organic-inorganic hybrid perovskite semiconductors (R-NH


Increased plasma homocysteinemia is considered a risk factor of dementia, including Alzheimer’s disease (AD) and vascular dementia. However, the reason elevated plasma homocysteinemia increases the risk of dementia remains unknown. A pathological hallmark of AD is neurofibrillary tangles (NFTs) that consist of pathologically phosphorylated tau proteins. The effect of homocysteine (Hcy) on tau aggregation was explored using human neuroblastoma M1C cells that constitutively express human wild-type tau (4R0N) under the control of a tetracycline off system, primary mouse cultured neurons, and by inducing hyperhomocysteinemia in a mouse model of tauopathy (HHCy mice). A wide range of Hcy concentrations (10–1000 µM) increased total tau and phosphorylated tau protein levels. Hcy activated glycogen synthase kinase 3, and cyclin dependent kinase 5, major tau phosphokinases, and inactivated protein phosphatase 2A, a main tau phosphatase. Hcy exhibited cytotoxic effects associated with enhanced activation of caspase. Truncation of tau in the C-terminus, the cleavage site of caspase 3  was also increased. Total tau, phosphorylated tau, as well as C-terminal cleaved tau were increased in the sarkosyl insoluble tau fraction. Hcy also increased the level of tau oligomers, as indicated by the tau oligomer complex 1 (TOC1) antibody that specifically identifies oligomeric tau species, in the tris insoluble, sarkosyl soluble fraction. The levels of TOC1-positive oligomeric tau were increased in brain lysates from HHCy mice, and treating HHCy mice with S-adenosylmethionine, an intermediate of Hcy, reduced the levels of oligomeric tau to control levels. These observations suggest that Hcy increases the levels of phosphorylated tau as well as truncated tau species via caspase 3 activation, and enhanced tau oligomerization and aggregation. Alzheimer’s disease (AD), progressive supranuclear palsy, corticobasal degeneration, argyrophilic grain disease, Pick’s disease, frontotemporal dementia with parkinsonism linked to chromosome 17, Niemann–Pick disease type C, and chronic traumatic encephalopathy are among a group of disorders collectively known as tauopathies because they are characterized by the accumulation of pathologically modified tau proteins ,3. A numHyperhomocysteinemia (HHCy) also is considered an important risk factor for dementias, including AD  and vasc12 and folate are cofactors in one-carbon metabolism where they promote the remethylation of Hcy, while vitamin B6 converts Hcy to cystathionine. Hcy accumulates when there are deficiencies in folate ethanesulfonic Acid (HEPES) (pH 7.5), 5 mM MgCl2, 150 mM NaCl, 20% glycerol, 5 mM sodium pyrophosphate, 30 mM β-glycerophosphate, 30 mM sodium fluoride, and a protease inhibitor cocktail) containing 100 µM Suc-LLVY-AMC fluorogenic substrate  in 96 well plates for measuring chymotrypsin-like activity ,66. Fluot-test  or by a two-tailed one-way analysis of variance (ANOVA) with the Student–Newman–Keuls post-hoc test. Differences were considered significant when p < 0.05 . Data are shown as the mean ± standard deviation (SD) from the minimum of three experiments. Statistical comparisons were assessed by two-tailed Student’s 6, or B12 deficiency, causes misregulation of kinases and phosphatases that affect tau, caspase activation, and impaired proteasomal function. Accordingly, tau accumulation occurs, phosphorylation of tau increases, caspase-cleaved tau appears, and tau oligomers form, all of which are associated with cell toxicity.HHCy, which can be induced by folate, vitamin B


Rheumatoid arthritis is an inflammatory condition characterized by overzealous inflammation that leads to joint damage and is associated with an increased incidence of cardiovascular disease. Statins are frontline therapeutics for patients with cardiovascular disease and exert beneficial actions in rheumatoid arthritis. The mechanism that mediates the beneficial actions of statins in rheumatoid arthritis remains of interest. In the present study, we found that the administration of 2 clinically relevant statins—atorvastatin (0.2 mg/kg) or pravastatin (0.2 mg/kg)—to mice during inflammatory arthritis up-regulated systemic and tissue amounts of a novel family of proresolving mediators, termed 13-series resolvins (RvTs), and significantly reduced joint disease. Of note, administration of simvastatin (0.2 mg/kg) did not significantly up-regulate RvTs or reduce joint inflammation. We also found that atorvastatin and pravastatin each reduced systemic leukocyte activation, including platelet-monocyte aggregates (∼25–60%). These statins decreased neutrophil trafficking to the joint as well as joint monocyte and macrophage numbers. Atorvastatin and pravastatin produced significant reductions (∼30–50%) in expression of CD11b and major histocompatibility complex class II on both monocytes and monocyte-derived macrophages in joints. Administration of an inhibitor to cyclooxygenase-2, the initiating enzyme in the RvT pathway, reversed the protective actions of these statins on both joint and systemic inflammation. Together, these findings provide evidence for the role of RvTs in mediating the protective actions of atorvastatin and pravastatin in reducing local and vascular inflammation, and suggest that RvTs may be useful in measuring the anti-inflammatory actions of statins.—Walker, M. E., Souza, P. R., Colas, R. A., Dalli, J. 13-Series resolvins mediate the leukocyte-platelet actions of atorvastatin and pravastatin in inflammatory arthritis. In arthritides, cardiovascular complications are thought to arise from the chronic inflammatory state that characterizes these conditions and causes >50% of premature deaths in patients with arthritis (Cardiovascular disease (CVD) is the primary cause of global mortality, representing ∼30% of all deaths  up-regulates the formation of 13R-HDPA , a key intermediate in the biosynthesis of RvT that is then donated to neutrophils where it is converted to bioactive molecules . This family is composed of 4 molecules, namely RvT1, RvT2, RvT3, and RvT4 , d4-prostaglandin (PG) E2 and d4-leukotriene (LT) B4), which were purchased from Cambridge Bioscience  or provided by Charles N. Serhan ; atorvastatin, pravastatin sodium salt, simvastatin sodium salt, and celecoxib (Cambridge Bioscience); Dulbecco’s PBS , RPMI-1640, collagenase D, DNAseI, paraformaldehyde, Histopaque-1077, trisodium citrate, HBSS, penicillin-streptomycin, and dextran from Leuconostoc spp. , IL-1β, and TNF-α ; fetal bovine serum, CD11b-PE-Texas Red, CD62P-Brilliant Violet 650, and CountBright Absolute Counting Beads (Thermo Fisher Scientific); whole blood lysing reagents kit ; collagenase type II ; and anti-mouse IgG, CD64-PE, Ly6G-Alexa Fluor 700, Ly6C-Brilliant Violet 785, CD45-APC/Cy7, IA/IE-Brilliant Violet 650, CD11b-Alexa Fluor 488, Siglec F-APC, CD115-Brilliant Violet 711, CD43-Brilliant Violet 510, Ly6C-Alexa Fluor 488, CD11c-Brilliant Violet 785, CD115-Brilliant Violet 711, CD43-Alexa Fluor 647, and CD41-Brilliant Violet 510 .Materials included liquid chromatography–grade water, methanol and acetic acid , Eclipse C18 Poroshell column , and C18 solid-phase extraction columns ; synthetic and authentic standards for liquid chromatography–tandem mass spectrometry quantitation and deuterium (d) internal standards  were procured from Charles River Laboratories . Experiments strictly adhered to United Kingdom Home Office regulations  and Laboratory Animal Science Association Guidelines . All animals were provided with standard laboratory diet and water −/− that contained 0.05% ethanol) via i.v. injection on d 3, 5, and 7. Clinical scores were monitored daily by using a 26-point arthritic scoring system. Swelling and redness of mouse ankles/wrists, pads, and digits were inspected daily as previously described  were administered K/BxN serum  on d 0 and 2 to initiate inflammatory arthritis . Mice weescribed . Blood aIn select experiments, mice were given 10 mg/kg celecoxib 1 h before statin injections. Blood and paws were collected either on d 8 after arthritis onset or 2 h after statin injection on d 7.via cardiac puncture. Cells were incubated with Fc-blocking IgG and fluorescent-labeled Abs  for 45 min on ice. Cells were washed and incubated with 0.1% live/dead stain for 30 min on ice. Red blood cells were lysed and fixed using the whole blood lysing reagent Kit. Staining was then evaluated by using LSRFortessa cell analyzer  and analyzed using FlowJo software .Whole blood was collected by using heparin-lined syringes g for 10 min. Isolated cells were suspended in DPBS−/− that contained 0.02% bovine serum albumin and 1% Fc-blocking IgG (v/v) and were incubated with 0.1% live/dead stain for 20 min on ice. Cells were washed using DPBS−/− and incubated with fluorescent-labeled Abs  for 45 min on ice. These were then washed and fixed using 1% paraformaldehyde. CountBright Absolute Counting Beads were used for leukocyte enumeration. Staining was evaluated using LSRFortessa cell analyzer and analyzed using FlowJo software.Paws were harvested and leukocytes were isolated as previously described . In brie2).Umbilical cords were collected by the midwifery staff of the Maternity Unit, Royal London Hospital , and HUVECs were isolated as previously described . Cells wg for 20 min). Platelet-rich plasma was removed as previously described . Red blood cells in the resulting pellet were lysed by using ice-cold water and transferring to HBSS. Cells were obtained via centrifugation at 250 g for 10 min.Neutrophils were isolated from the blood of healthy consenting donors in accordance with the Declaration of Helsinki and Queen Mary Research Ethics Committee (QMREC)–approved protocol (QMREC 2014:61). Blood was collected by using 3.2% sodium citrate as an anticoagulant and centrifuged (100 escribed . Red blo5 cells/cm2) were incubated with IL-1β (10 ng/ml) and TNF-α (10 ng/ml) for 16 h, then with atorvastatin, pravastatin, or vehicle (DPBS that contained 0.05% ethanol) for 30 min at 37°C. Four million neutrophils were added to each well and cells were incubated for 60 min (37°C). Incubations were quenched with ice-cold methanol that contained deuterated internal standards and samples were taken for lipid mediator profiling.Incubations were conducted as previously described . In brie8-5S- hydroxyeicosatetraenoic, d4-LT B4, d5-LXA4, d4-PGE2, and d5-RvD2—was added to samples. These represent the various chromatographic regions of identified lipid mediators to allow quantification and assessment of sample recovery. Lipid mediators were extracted and profiling conducted as previously described (Ice-cold methanol that contained 500 pg of each deuterated (d) internal standard—described , 21, 22.2HPO4 and 0.4% NaH2PO4) for 48 h. These were then placed in 10% EDTA in DPBS for 2 wk. When decalcified, paws were embedded in wax as previously described  and 1-way ANOVA with post hoc Dunnett’s, Sidak’s, or Tukey’s multiple comparisons test. Where appropriate, 1-sample Student’s t test compared with normalized vehicle or 2-way ANOVA were used. The criterion for statistical significance was P < 0.05.Results are presented as means ± d-series resolvins and RvTs . Of note, the concentrations of these mediators were within their described bioactive ranges .We first investigated whether atorvastatin regulated RvT formation during inflammatory arthritis and whether this action was unique to this statin or shared with other clinically relevant statins, namely pravastatin and simvastatin –11. To tand RvTs B, C. Theand RvTs B and at and RvTs C 21). U. Ud-serie ranges . Pravast4, PGD2, PGE2, PGF2α, and thromboxane B2 in mice that were administered either pravastatin or atorvastatin . In these mice, we also found an increase in plasma levels of RvT concentrations, with RvT1 being increased by both atorvastatin and pravastatin, whereas RvT4 was only increased by atorvastatin .Assessment of plasma mediator levels demonstrated decreases in LT BSupplemental Fig. 1 and Supplemental Table 3). In these mice, we also found decreased circulating amounts of inflammation-initiating eicosanoids, including PGD2 and PGE2. Assessment of plasma lipid mediator profiles from mice that were administered pravastatin also demonstrated a marked increase (>100%) in peripheral blood RvTs, with RvT1 demonstrating the greatest increase compared with vehicle-treated mice . In these mice, we also observed decreases in circulating PGD2 (∼46%) and PGE2 (∼29%). Together, these results demonstrate that atorvastatin and pravastatin increase both joint and plasma RvTs and decrease systemic inflammation during inflammatory arthritis.Given that statins are rapidly cleared from circulation with a half-life of ∼14 h  and ∼3 hSupplemental Fig. 2).Given that in the vasculature, RvT is produced during neutrophil endothelial interactions , we nextWe next investigated whether atorvastatin and pravastatin at doses that increased RvTs also reduced joint inflammation. Arthritis was initiated and mice were treated and disease progression monitored as described above. In mice that were administered vehicle, signs of disease were observed as early as d 2, and disease severity reached a maximum at d 6 with a score of 11.9 ± 0.9, after which disease activity plateaued to d 7 A. SimilaWe next assessed whether atorvastatin, pravastatin, and simvastatin displayed joint protective actions. Hematoxylin and eosin–stained sections of knee joints from mice that were administered atorvastatin and pravastatin demonstrated reduced leukocyte infiltration, pannus formation, and joint damage compared with vehicle-treated mice, whereas these parameters were unaltered in mice that were administered simvastatin E. Togethn = 9 mice) on these monocyte subsets. Atorvastatin administration also regulated neutrophil and platelet responses, significantly reducing neutrophil CD11b expression, platelet-neutrophil aggregates . Similarly, leukocyte trafficking and activation in joints was increased to levels that were similar to those found in vehicle-treated mice . Together, these findings demonstrate that inhibition of COX-2 reduced RvT production and abolished the joint and systemic protective actions of pravastatin and atorvastatin in inflammatory arthritis.Inhibition of COX-2 reversed the actions of atorvastatin on leukocyte trafficking and activation in the joint, increasing the numbers of nonclassic monocytes D, neutroFindings presented in this manuscript demonstrate that both atorvastatin and pravastatin increase RvT production in inflammatory arthritis. Up-regulation in tissue and blood concentrations of these specialized proresolving mediators was associated with a reduction in joint disease activity as well as joint leukocyte trafficking and activation. In addition, both atorvastatin and pravastatin decreased systemic inflammation, reducing platelet, monocyte, and neutrophil activation. The protective actions of these statins were reversed by COX-2 inhibition. Of note, simvastatin did not increase RvTs and displayed blunted actions in the regulation of joint disease and leukocyte responses. Together, these findings establish the rank order potency of atorvastatin, pravastatin, and simvastatin in regulating RvTs as well as the role of these molecules in mediating the protective actions of these statins.4, which mediates their actions in cardiovascular and mucosal protection, respectively. In cardiac myocytes atrovastatin promotes 15-epi-LXA4 formation by increasing the phosphorylation of 5-lipoxygenase . We also found that the RvT pathway, at least in part, was responsible for the protective actions of this statin in controlling various aspects of inflammatory arthritis, including joint and vascular inflammation , although this was to a much lesser extent than atorvastatin and pravastatin. These results suggest that simvastatin is not as effective at promoting COX-2 S-nitrosylation as the other 2 statins. Furthermore, joint and plasma 15-epi-LXA4 were not significantly regulated in these mice, which underscores the role of RvTs in mediating the actions of these statins .We recently demonstrated that, in the vasculature, atorvastatin up-regulates the formation of RvTs and that this pathway was responsible for mediating the protective actions of statins in bacterial infections . By usinammation . This reammation  and 6. O4 and LTD4 . These findings support the role of RvTs in mediating the protective actions of these 2 statins in regulating systemic inflammation and processes that are associated with the development of CVD.Leukocytes, including monocytes, are implicated in the propagation of various aspects of CVD , 27. Stuand LTD4 . In the via release of proteinases and production of extracellular traps (15In rheumatoid arthritis, unabated leukocyte recruitment and activation leads to a perpetuation of joint inflammation and joint destruction. Among the leukocytes that are recruited to the joint, nonclassic monocytes were recently found to be essential in initiating joint inflammation in murine arthritis , whereasar traps . The infar traps . Resultsar traps  and 6. Rar traps . This is traps 15, 39, 40.Taken together, these results establish the rank order potencies of atorvastatin, pravastatin, and simvastatin at regulating RvTs. Joint and systemic increases in these proresolving mediators also correlated with the ability of each of these statins to dampen various aspects of local and systemic inflammation, including edema, leukocyte, and platelet activation. Thus, these results establish a novel mechanism of action for atorvastatin and pravastatin in regulating inflammation in arthritis. In addition, they also provide potential novel functional biomarkers for measuring the efficacy of statins in controlling local and vascular inflammation in patients with rheumatoid arthritis.


Triportheus genus. While in Erythrinidae distinct XY and/or multiple XY-derived sex chromosome systems have independently evolved at least four different times, representatives of Triportheus show an opposite scenario, i.e., highly conserved ZZ/ZW system with a monophyletic origin. In both cases, recent molecular approaches, such as mapping of repetitive DNA classes, comparative genomic hybridization (CGH), and whole chromosome painting (WCP), allowed us to unmask several new features linked to the molecular composition and differentiation processes of sex chromosomes in fishes.Fishes exhibit the greatest diversity of species among vertebrates, offering a number of relevant models for genetic and evolutionary studies. The investigation of sex chromosome differentiation is a very active and striking research area of fish cytogenetics, as fishes represent one of the most vital model groups. Neotropical fish species show an amazing variety of sex chromosome systems, where different stages of differentiation can be found, ranging from homomorphic to highly differentiated sex chromosomes. Here, we draw attention on the impact of recent developments in molecular cytogenetic analyses that helped to elucidate many unknown questions about fish sex chromosome evolution, using excellent characiform models occurring in the Neotropical region, namely the Erythrinidae family and the  For cytologically distinguishable sex chromosomes to emerge, one of the homologues needs to acquire at least two linked alleles that are advantageous to one sex only and possibly detrimental to the other ,2. The nUnlike well-established sex chromosome systems in mammals, snakes, and birds ,7, fish 1X1X2X2/♂X1X2Y, ♀XX/♂XY1Y2, ♂ZZ/♀ZW1W2 multiple ones [1Z1Z2Z2/♀Z1Z2W1W2 and ♀X1X1X2X2/♂X1Y1X2Y2 [Remarkably, even considering the small number of known cases, several different sex chromosome systems have been described for diverse fish taxa ,15,16, dple ones ,17,18, aX1Y1X2Y2 ,20. The X1Y1X2Y2 .Triportheus [Leporinus [Characidium species [Sex chromosomes often emerged independently and at various times in many fish lineages, following distinct patterns of differentiation even in closely related species ,23,24,25portheus ,27 and seporinus ,29, and  species ,31. Simi species , sex chr species ,15,32,33 species ,35,36. I species  or even  species . Finally species .The above-mentioned features qualify some fish species as excellent models to examine sex chromosome evolution, calling for more in-depth cytogenetic and genomic studies. While the identity, structure, and expression of particular genes are still rather poorly explored in fish sex determination and differentiation ,37,38,39Triportheus genus from the Triportheidae family. These fish groups have been our investigation systems for years, for which a considerable amount of conventional and molecular cytogenetic data show differential pathways on the differentiation process of the sex chromosomes.In this sense, our present review emphasizes two alternative models to investigate the evolution of sex chromosomes among Neotropical fishes, exemplified by species from the Erythrinidae family and the Hoplias , Hoplerythrinus , and Erythrinus , providing a rare opportunity to gain insights into the evolutionary forces that drive the origin of nascent sex chromosomes, the evolution of the sex pair and speciation processes. In fact, the lack of heteromorphic sex chromosomes, as well as the occurrence of sex chromosome systems with distinct evolutionary stages of differentiation, can be found among different populations of the wolf fish Hoplias malabaricus and/or the red wolf fish Erythrinus erythrinus [Erythrinidae is a small characiform family, widely distributed in the Neotropical region, with only three genera: ythrinus ,43. Notes Gill, 103, HopleH. malabaricus and E. erythrinus [H. malabaricus and four (A–D) in E. erythrinus have already been identified, respectively, based on major differences in diploid chromosome numbers (2n), chromosome morphology, and sex chromosomes [Based on a multitude of chromosomal and molecular data, especially within the last 20 years, it has become increasingly apparent that we are dealing with a group of species instead of a single taxonomic unit, both for ythrinus ,44. Fromomosomes ,45,46,47H. malabaricus, karyomorph B  exhibits the most differentiated ♀XX/♂XY sex chromosome system, where the subtelocentric X chromosome is clearly distinguished from the small-sized metacentric Y by a conspicuous heterochromatic block on its long (q) arms [Within (q) arms ,49. Seve(q) arms ,51. Thes(q) arms ,52. Note(q) arms . Indeed,(q) arms .1X1X2X2/X1X2Y type [Karyomorph C , is characterized by morphologically undifferentiated XX/XY sex chromosome system evidenced by a small, but considerable, heterochromatin accumulation on the exclusively proximal region of the X chromosome . CGH resX2Y type . StabiliX2Y type , thus poX2Y type  reportedX2Y type . TherefoX2Y type .1Y2 neo-sex chromosomes, where the unusual acrocentric Y1 chromosome carries the male-specific region that is also enriched by several types of repetitive DNAs [Karyomorph F  also exhibits a nascent sex chromosome system of XX/XY type . The malive DNAs .H. malabaricus, the species E. erythrinus exhibits extensive karyotype diversity among populations, with four karyomorphs (A–D) currently recognized [1X1X2X2/X1X2Y sex chromosome system, though they still differ in 2n and karyotype composition. Chromosomal mapping of repetitive DNA by means of FISH (fluorescence in situ hybridization) demonstrated the nature of chromosomal rearrangements and genomic modifications leading to establishment of this X1X2Y sex system. More specifically, the neo-Y owns its origin to a centric fusion event. In support of this, FISH with telomeric probe highlighted the presence of interstitial telomeric sequences (ITSs) in the centromeric region of the neo-Y chromosome, the only metacentric element in the karyotype [Similarly to cognized . While karyotype .H. malabaricus karyomorph B (HMB-X), (ii) the X1 chromosome of H. malabaricus karyomorph D (HMD-X1), (iii) the Y chromosome of H. malabaricus karyomorph F  and (iv) the Y chromosome of E. erythrinus karyomorph D (ERY-Y), were used in WCP experiments, providing the pathways of the sex chromosomes differentiation and important hints about interspecific and inter-karyomorph relationships  comprises 19 extant species popularly known as freshwater sardines [Lignobrycon, Engraulisoma, Clupeacharax, and Agoniates [Triportheus, its origin dates back to 26.2 ± 6.5 Myr, where Triportheus auritus phylogenetically represents the oldest lineage , with evident signs of monophyletic origin  [T. auritus [Triportheus species reflects the probable role of these sequences in the evolution of the ZZ/ZW sex chromosome system of this genus [Besides the reduction in size, the W chromosome of  regions ,67,68. Irms (Wq) ,73,74. Irms (Wq) ,75. Simirms (Wq)  have alr auritus  and in s auritus ,79, whil auritus . The maiis genus .Triportheus has been also associated with other repetitive DNA sequences. The W chromosome of Triportheus albus also possess another multigene family, the U2 small nuclear RNA (snRNA). Until now, only a few cases are known to occur in fish sex chromosomes . Micros regions ,74,81,82Triportheus genus. In fact, the Z-probe painted the whole length of the Z chromosome equally in all species, showing its conserved nature in both size and genetic content [T. auritus, demonstrating that the ZW system in Triportheus had a common origin within the genus [Lignobrycon myersi (Triportheidae) [Triportheus as a unique model providing a particular scenario of the evolution of sex chromosomes among the Neotropical fishes.The WCP using both W- and Z-derived probes confirmed the hypothesis about the origin of the ZW sex chromosome system within the  content ,71  , as welltheidae) , open neTriportheus highlight a case of monophyletic and stable sex chromosome system, but with different stages of degeneration the sex-specific chromosome. Nonetheless, both models share some common properties, such as repetitive DNA sequences playing a substantial role in the differentiation processes of heteromorphic sex chromosomes. In contrast, the multiple neo-sex chromosomes, arising through the structural rearrangements, do not undergo progressive heterochromatinization [Triportheus species represent only a fraction of possible evolutionary scenarios within the spectacular fish sex chromosome diversity among lower vertebrates.In the light of recent cytogenetic progresses, we aimed to illustrate here two contrasting scenarios of sex chromosome evolution in Neotropical fishes: (i) representatives of the Erythrinidae family show high variability and multiple origins of their sex chromosome systems, and (ii) representatives of the genus nization —a featurnization ,20,85,86nization . In thisAs illustrated above, the recruitment of WCP and its cross-species application together with CGH-based experiments represent powerful toolbox for deciphering homologies between distinct sex chromosomes on the interspecific or interpopulational level. However, what accounts for such rapid evolution of sex chromosomes among fishes is yet not fully understood. To obtain a more informed and comprehensive picture, next generation sequencing (NGS) data could be harnessed as a valuable option for studying the genetic basis of sex determination, providing opportunities to identify homologies between sex chromosomes across populations and species in a high throughput manner. Besides, searching for genome-specific satellite DNAs and their subsequent FISH mapping might add another layer of investigation and bring a more complex view about the process underlying fish sex chromosomes evolution.


Next-generation sequencing (NGS) in thyroid cancer allows for simultaneous high-throughput sequencing analysis of variable genetic alterations and provides a comprehensive understanding of tumor biology. In thyroid cancer, NGS offers diagnostic improvements for fine needle aspiration (FNA) cytology of thyroid with indeterminate features. It also contributes to patient management, providing risk stratification of patients based on the risk of malignancy. Furthermore, NGS has been adopted in cancer research. It is used in molecular tumor classification, and molecular prediction of recurrence and metastasis in papillary thyroid carcinoma. This review covers previous NGS analyses in variable types of thyroid cancer, where samples including FNA cytology, fresh frozen tissue, and formalin-fixed, paraffin-embedded tissues were used. This review also focuses on the clinical and research implications of using NGS to study and treat thyroid cancer. An understanding of the molecular mechanisms of tumor formation is mandatory for accurate diagnoses and personalized treatments. Previously, single gene assays were commonly used for finding molecular alterations in tumors. Presently, NGS technology provides the simultaneous analysis of hundreds of genes of interest, using targeted sequencing panels . Thus, NBRAF, NRAS, HRAS, KRAS) and chromosomal rearrangements , which vary with histologic subtype [Thyroid cancer is the most common malignancy of the endocrine organs; its prevalence is increasing, more than tripling during the last three decades . PapillaA majority of previous studies using NGS in thyroid cancer analyzed variable specimen sample types and histologic subtypes Table . In clinBRAF (59%) was the most frequent, followed by PIK3A (11%), TP53 (7%), and NRAS (4%). In contrast, 83% of FVPTCs had mutated genes: RAS (73%) was the most frequent, followed by BRAF (7%) and TSHR (3%) [BRAF V600E-mutated PTCs and 8 BRAF-wild type PTCs [BRAF V600E-mutated PTCs and BRAF-wild type PTCs [BRAF V600E-mutated PTCs, and HLAG, CXCL14, TIMP1, and IL1RAP were over-expressed. In BRAF-wild type PTCs, 4 immune function genes  were most significantly differentially expressed, and exhibited a high degree of correlation with lymphocytic infiltration [ETV6-NTRK3 rearrangement in 14.5% of radiation-associated PTCs and 2% of sporadic PTCs [ETV6-NTRK3 rearrangement may be a key mechanism of radiation-induced carcinogenesis [Several studies have applied NGS to variable subgroups of PTCs. Nikiforova et al. analyzed FFPE or frozen tissue from 27 classic PTCs and 30 FVPTCs, using the ThyroSeq NGS panel targeting 12 genes with 34 amplicons on the Ion Torrent PGM sequencer . The resSHR (3%) . Smallriby PIK3A %, TP53 .The Cancer Genome Atlas (TCGA) Network described the genomic characterization of 496 PTCs, and generated data using whole genome sequencing. This was done on the NGS platform and by a multiplatform analysis of SNP arrays, DNA methylation, and reverse phase protein arrays . In PTCsETV6-NTRK3 fusion was identified in 18% of samples, and was associated with aggressive histologic features such as non-encapsulation, solid/insular/trabecular patterns, extensive glandular involvement, and thick tumor fibrosis [BRAF V600E mutation, RET/PTC1/3 fusion, and TERT promoter mutation [Regarding pediatric thyroid carcinoma, Picarsic et al. analyzed 17 pediatric PTCs  from FNA, fresh frozen tissue, and FFPE samples. Mutation analysis with a 7-gene mutation panel using real-time PCR and ThyroSeq v2 on the Ion Torrent PGM sequencer showed that: (1) The detection rate of molecular alterations was increased by up to 87% by the ThyroSeq v2 NGS assay compared to an increase of 60% by the 7-gene mutation panel. (2) In pediatric thyroid carcinoma, chromosomal rearrangement (53%) was more common than point mutation (33%). (3) fibrosis . Ballestfibrosis . No addimutation .Follicular carcinoma (FC) is a well-differentiated thyroid carcinoma and the second-most common thyroid cancer after PTC. It accounts for 10% of the total thyroid cancers . AlthougNRAS (N = 9), KRAS (N = 2), HRAS (N = 1), TSHR (N = 4), TP53 (N = 4), and PTEN (N = 1) [N = 18) and oncocytic type (N = 18) samples showed distinguished genetic alterations. In the conventional type FCs, NRAS was the most frequently affected gene, followed by TSHR and KRAS. TP53 was the most commonly mutated gene in the oncocytic type FCs, followed by HRAS, KRAS, and PTEN [A previous study retrospectively collected FFPE or fresh frozen tissue samples from 36 FCs to study 12 cancer genes and 34 amplicons using the ThyroSeq panel on the Ion Torrent PGM sequencer. The analysis detected mutations of  (N = 1) . Interesand PTEN .MDM2, FLI1), transcription factors and repressors , epigenetic enzymes , and protein kinases . (2) Single nucleotide variants were the most common types of mutations, and large structural variants were the least frequent. (3) A novel translocation in DERL/COX6C was detected. (4) Somatic alteration affected non-coding gene regions and exhibited high penetrance. These results suggest that FC has significant molecular heterogeneity, since FC reveals far more complex somatic alterations than PTC, and each tumor harbors distinct somatic alterations.Swierniak et al. analyzed 26 follicular adenomas, 22 FCs, and 34 paired normal thyroid tissue samples. Targeted NGS of 372 genes using the TruSeq kit on the Illumina HiSeq 1500 platform yielded the following results : (1) SomPoorly differentiated carcinoma (PDC) and anaplastic carcinoma (AC) are rare types of thyroid carcinoma, each with a prevalence of 10% , 22, andNRAS, PIK3CA, GNAS, and BRAF in PDCs, and TP53, BRAF, RAS, PIK3CA, PTEN, and CTNNB1 in ACs.In a study using the ThyroSeq panel on the Ion Torrent PGM sequencer, 12 genes with 34 amplicons were analyzed from FFPE or fresh frozen tissue from 10 PDCs and 27 ACs. The study showed that 30% of PDCs had mutations, whereas 74% of ACs had mutations [TP53 mutations were identified in all but 2 cases [CDH1, FANCD2, CHECK2, ADH1B, GPC3, TP53, and PTEN genes were altered in PDCs, and ATM, HNF1A, MET, NF1, TP53, PTEN, MSH2, RB1, NBN, NF1, MUTYH, TSC2, HRAS, and EGFR genes were altered in ACs [Sykorova et al. analyzed fresh frozen tissue samples from 3 PDCs and 5 ACs using the TruSight Cancer Panel, targeting 94 cancer-related genes on the Illumina MiSeq sequencer . All PDC 2 cases . CDH1, Fd in ACs . HoweverN = 80) or fresh-frozen tissue (N = 37) samples from 34 PDCs and 33 ACs [TP35, TERT promoter, PI3 K/AKT/mTOR pathway effector, SWI/SNF subunit, and histone methyltransferase. (2) In PDCs, clinicopathologic features were different based on the genetic alterations: 92% of RAS mutations were found in PDCs met the Turin criteria, whereas 81% of BRAF mutations were found in PDCs met the Memorial Sloan-Kettering Cancer Center (MSKCC) criteria. PDCs harboring BRAF mutations were smaller and had frequent nodal metastasis, whereas RAS-mutant PDCs showed larger tumor sizes and a higher rate of distant metastasis. (3) The association of mutation between EIF1AX and RAS was notable in both PDCs and ACs. EIF1AX mutations have been reported in approximately 1% of PTCs, and known to be mutually exclusive for BRAF and RAS mutations. However, EIF1AX mutations were found in 11% of PDCs and 9% of ACs, and 93% were associated with RAS mutations. (4) Chromosomal rearrangements  were found in 14% of PDCs, but were absent in ACs.Landa et al. performed NGS using the MSK-IMPACT cancer exome panel, and analyzed 341 genes from FFPE (d 33 ACs . The anaRET proto-oncogene mutation [Medullary carcinoma (MC) is a neuroendocrine tumor originating from C-cells, and accounts for approximately 5% of total thyroid cancers . MC is cmutation , 31.RET mutations, 3 (20%) were HRAS mutations, and 1 (7%) was a KRAS mutation [RET mutations (60%), 3 HRAS mutations (15%), 1 KRAS mutation (5%), 1 STK11 mutation (5%), and 3 samples where mutations were undetected (15%) [RET status was evaluated with both the NGS and Sanger sequencing methods, and NGS showed higher sensitivity than the Sanger method. NGS identified an additional 3 RET mutations, which were undetected by the Sanger method. Although multiple mutations were found in MCs by the NGS assay, a relevant therapeutic target has not been identified, and further investigation is required to improve the treatment of MCs.Nikiforova et al. analyzed 12 genes and 34 amplicons using the ThyroSeq panel on the Ion Torrent PGM sequencer with FFPE or fresh frozen tissue samples from 15 sporadic MCs. Mutations were identified in 11 (73%) MCs, of which 7 (47%) were mutation . Simboloed (15%) . RET staOne of the most profound limitations of applying NGS for thyroid cancer is a lack of sufficient evidence-based framework applicable to the clinical practice. As discussed in this review, the number of existing studies using NGS to analyze thyroid cancer stands at less than 15. Also, most of the previous studies were carried out at single institutes using specific subtypes of thyroid cancer in small sample sizes, rather than all types of thyroid cancers; therefore, resulting data would still be insufficient for making decisions on either patient diagnosis or treatment in clinical practice. To overcome such limitations, a large-scaled, global, and multicentered NGS study for thyroid cancer is required.BRAF V600E detected by NGS, surgical resection would be the most appropriate treatment option, since BRAF V600E is a highly specific mutation for PTC [RAS mutation can be detected in FVPTC and follicular neoplasm that require surgical excision, while also being present in benign adenomatous nodules [In a thyroid nodule with indeterminate cytology and  for PTC . In cont nodules  that do BRAF, RAS, and RET mutations, NGS technology facilitated detection of new somatic alterations in thyroid cancer such as MITF, MDM2, JAK3, FLI1, IDH1 etc. [In addition to well-known DH1 etc. , all in Most of NGS panels applied in thyroid cancer studies cover hotspot mutations, and are highly sensitive for evaluation of limited regions of selected genes; however, relevant mutation could be missing if not appropriately mapped. As described in previous studies, targeted NGS with specific gene panel showed high PPV. However, NGS analysis using 7-gene panel in previous study showed that 30–35% of thyroid cancer patients were still negative for mutation, and low NPV would require diagnostic surgery in benign nodules to prevent missing cancers . High seBRAF V600E is the most common and the earliest genetic event in PTC [The fast-evolving NGS technology offers a cost-effective approach for cancer genomics, as well as in thyroid cancer. In future prospects of thyroid cancer, NGS can be used to detect circulating tumor cells or cell-free plasma DNA to identify early relapse and/or residual disease. Previous studies reported presence of circulating tumor cells or cell-free plasma DNA in thyroid cancer patients , 41, whit in PTC , 43, andThe emergence of NGS technology has provided in-depth analysis of multiple, diverse cancers by a number of devices and gene panels, and has led to more effective options for cancer screening, prevention, diagnosis, prognosis, and targeted therapy. The use of NGS to study thyroid cancer has improved our understanding of the molecular genetics of thyroid cancer. In thyroid nodules of indeterminate cytology, such as FN/SFN and AUS/FLUS, the NGS test detected multiple genetic alterations and identified patients with a high risk of malignancy. Risk stratification using molecular signatures offers many more precise treatment options during patient management. The application of NGS for PTC, FC, MC, PDC, and AC revealed novel genetic alterations which were not detected by past sequencing methods. Newly discovered genetic alterations include genes associated with tumor recurrence and distant metastasis, which are candidates for molecular prognostic markers. However, limitations are also present with NGS, arising from variable sample types, multiple platforms and gene panels, and variable analysis programs, each of which can confound results. Standardization for quality control and the data-analytic process is needed to minimize the discrepancies between analyses. For poor-prognostic histologic types of thyroid cancer—MC, PDC, and AC—NGS studies identified several novel genetic alterations, but drug-actionable target genes have not been identified yet, and further investigation is required. Nevertheless, development of new sequencing technologies, such as NGS, enhances the cancer genome body of knowledge, and allows for more effective cancer screening, prevention, diagnosis, and monitoring. This in turn provides for better precision medicine and more curative cancer treatments.


Two hundred and thirty five patients with osteosarcoma, aged less than 40 years, and treated by amputation or radiotherapy, were entered in a randomised trial of two forms of adjuvant chemotherapy. A two drug regimen, namely vincristine 2 mg m-2 (maximum 2mg) followed by methotrexate 200 mgm-2, given every three weeks, was compared with a three drug regimen, comprising the same vincristine-methotrexate treatment, alternating every three weeks with doxorubicin 60 mg m-2. Both regimens were continued for 54 weeks. Forty-one patients were excluded, most because of inadequate histology, leaving 194 patients for analysis. One hundred and seventy of these had immediate amputation, 14 were treated by a policy of radiotherapy, with surgery delayed for 9 months, provided no distant metastases had appeared, and 10 by a policy of radiotherapy only. Patients have been followed-up for between 26 and 94 months after entry to the trial. The 2- and 5-year survival rates were 48% and 27%. No significant difference in survival was observed between the two regimens, but toxicity was less with the two drug regimen.


The ability to respond to changes in the extra-intracellular environment is prerequisite for cell survival. Cellular responses to the environment include elevating defense systems, such as the antioxidant defense system. Hypoxia-evoked reactive oxygen species (ROS)-driven oxidative stress is an underlying mechanism of retinal ganglion cell (RGC) death that leads to blinding disorders. The protein peroxiredoxin 6 (PRDX6) plays a pleiotropic role in negatively regulating death signaling in response to stressors, and thereby stabilizes cellular homeostasis.We have shown that RGCs exposed to hypoxia (1%) or hypoxia mimetic cobalt chloride display reduced expression of PRDX6 with higher ROS expression and activation of NF-κB. These cells undergo apoptosis, while cells with over-expression of PRDX6 demonstrate resistance against hypoxia-driven RGC death. The RGCs exposed to hypoxia either with 1% oxygen or cobalt chloride (0-400 μM), revealed ~30%-70% apoptotic cell death after 48 and 72 h of exposure. Western analysis and real-time PCR showed elevated expression of PRDX6 during hypoxia at 24 h, while PRDX6 protein and mRNA expression declined from 48 h onwards following hypoxia exposure. Concomitant with this, RGCs showed increased ROS expression and activation of NF-κB with IkB phosphorylation/degradation, as examined with H2DCF-DA and transactivation assays. These hypoxia-induced adverse reactions could be reversed by over-expression of PRDX6.Because an abundance of PRDX6 in cells was able to attenuate hypoxia-induced RGC death, the protein could possibly be developed as a novel therapeutic agent acting to postpone RGC injury and delay the progression of glaucoma and other disorders caused by the increased-ROS-generated death signaling related to hypoxia. Such changes often include hypoxia, the scarcity of oxygen can lead to cell injury and death by apoptosis. Recent evidence has shown an increase of intracellular ROS expression in cells during hypoxia, with the source of the increase being the mitochondria ,2. Mamma hypoxia , but the hypoxia , followi hypoxia . Hypoxia hypoxia ,23,24. O hypoxia ,25, caus hypoxia ,22,26,27 hypoxia . NF-κB i hypoxia ,30. A we hypoxia . In glau hypoxia ,33. Init hypoxia ,34.2+-independent phospholipase A2 activities [2 and/or CoCl2, a hypoxia mimetic, as a model system to explore the underlying event(s) of hypoxia-induced RGC death. We have shown that RGCs facing hypoxia for longer periods display elevated expression of ROS and reduced levels of PRDX6, and that RGCs over-expressing PRDX6 gain resistance against hypoxia-evoked generation of ROS and ROS-induced cellular insults, by negatively regulating death signaling.Moreover, the over-expression of intracellular ROS caused by extracellular stressors is controlled by antioxidant defenses such as catalase, SOD, glutathione peroxidase, and, most importantly, the newly discovered peroxiredoxins -39. The tivities ,39-44. Rtivities ,43,45-48tivities ,40,48-50tivities ,48,51. Rtivities ,53 and itivities , psoriastivities ,56, athetivities . Thus, ativities ,59. Howetivities ,37, we tIn this study, we investigated whether chronic hypoxia evoked the production of ROS in RGCs, and whether over-expression of ROS initiated NF-κB-mediated death signaling by phosphorylation/degradation of IkB, resulting in RGC death. In addition, by using transfection and transactivation assays, we showed that ROS-mediated suppression of Prdx6 mRNA and protein expression in RGCs bearing higher levels of ROS could be attenuated by the over-expression of PRDX6. Collectively, these findings provide a foundation for rational use of antioxidant-based therapeutics for treating or preventing/delaying RGC death from ROS driven oxidative stress under hypoxic conditions.2. The cells reaching confluency were trypsinized and subcultured using 1:20 split. RGCs of 5 to 7 passages were used to carry out the experiments.RGC-5  were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 μl/ml streptomycin at 37°C with 5% COCells were either exposed to 1% oxygen using hypoxic chamber or they were treated with cobalt chloride, a hypoxia mimic ,61 at va4) were grown in 48 well plates and treated with 50, 100, 150, 200 or 400 μM of CoCl2 for 24, 48 or 72 h. After treatment period, a colorimetric MTS assay (Promega) was performed as described earlier [490 nm value was measured after cobalt chloride treatment at specified duration with an ELISA reader. The values are represented as a percent change to matched controls within cell types.Cells , following the company's protocol. Briefly, cells were grown in 4 chambered slide, washed with PBS and fixed in freshly prepared 4% paraformaldehyde in PBS (pH 7.4), followed by incubation in permeabilization solution  for 2 min on ice. Cells were rinsed twice with PBS, and incubated in a TUNEL reaction mixture for 60 min at 37°C in the dark. Cells were rinsed three times with PBS. After mounting, samples were microphotographed using a micro-scope , and analyzed. To determine the total dead cells, other than only the apoptotic cells, RGCs were stained with trypan blue solution (0.4%), where non viable cells are stained with trypan blue which is normally excluded by the live cells.-1139 to +109 bp was isolated from mouse genomic DNA and sequenced [for, 5'-CTGAGAGCTCCTGCCATGTTC-3'; Construct Bfor, 5' CTTCCTCTGGAGCTCAGAATTTAC-3'; Construct Cfor, 5'-CACAG-AGCTCGTTCTTGCCACATC-3'; Constructs A, B, and Crev, 5'-CAGGAACTCGAGGAAGCGGAT-3'. We used construct B in the present study.The 5'-flanking region from Intracellular redox state levels were measured using the fluorescent dye, H2-DCFH-DA as described earlier ,43. BrieNuclear, cytoplasmic extracts and total Cell lysates were prepared as described previously . Equal a® LC480 Sequence detector system (Roche Diagnostic Corporation). We used primers specific for Prdx6 , NF-κB (forward 5'-TTTCCCCTCATCTTTCCCTC-3' and reverse 5'-TGTGCTTCTCTCCCCAGG-3') and β-actin (forward 5'-CGTGGGCCGCCCTAGGCACCA-3' and reverse 5'-TTGGCCTTAGGGTTCAGGGGGG-3'). The primers were synthesized at University of Nebraska Medical Center, DNA Facility, Omaha, NE, USA. The comparative Ct method was used to calculate relative fold expression levels using the e-Roche software. The Cts of target genes were normalized to the levels of β-actin as an endogenous control in each group.To monitor the levels of Prdx6, NF- κB (p65), and β-actin mRNA in RGCs, total RNA was isolated using the single-step guanidine thiocyanate/phenol/chloroform extraction method  and converted to cDNA using Superscript II RNAase H-Reverse Transcriptase. Quantitative real-time PCR was performed with SYBR Green Master Mix  in a RocheA full length cDNA of Prdx6 was isolated from human lens epithelial cell cDNA library using sense (5'-ATGCCCGGAGGTCTGCTTCTCGGGG-3') and antisense (3'-AATTGGCAG CTGACATCCTCTGGCTC-3') primers, and the resultant amplified product was cloned in pcDNA3.1/NT-GFP-TOPO vector procured from Invitrogen . This coData are presented as Mean ± S.D. of the indicated number of experiments. Data were analyzed by Student's t-test when appropriate. A p value of < 0.05 was defined as indicating a statistically significant difference.2) to expose RGCs to hypoxic stress. We examined whether RGCs exposed to such stress showed reduced survival and died with apoptosis and displayed higher ROS levels. Results from cells exposed to cobalt chloride were compared with those from RGCs exposed to 1% O2.Recently reports have documented that hypoxia-induced elevation in intracellular ROS is a cause of pathophysiology in cells and tissues . However2 .Cells exposed to cobalt chloride showed decrease in cell survival, with levels depending on concentration and exposure time  or to 1% O2) for 24 h. CAT activity assessed with CAT-ELISA as described in Methods revealed activation of NF-κB  nuclear translocation Figure  and IkB PRDX6 exerts its protective function by regulating ROS expression and blocking cell death signaling. To determine the efficacy of PRDX6 in abolishing hypoxia-evoked ROS-driven damage to and death of RGCs, we transfected RGCs with pGFP-PRDX6  as described in Experimental Procedures, and transfection efficiency was equalized with OD obtained from GFP fluorescence at Ex485/Em530. We also confirmed over-expression of pGFP-PRDX6 using Western analysis Figure  As expec2 (hypoxia) in retinal tissues may cause severe damage, subsequently leading to blinding disease.Changes in oxidant and antioxidant balance may alter cellular homeostasis; cells may go through survival or death pathways, depending upon expression of ROS and antioxidant defense capacity. Eye tissues are constantly exposed to external and internal environmental stresses such as sunlight, chemical irritations, and hypoxia. These stressors may lead to blinding disorders of the eye by inducing abnormalities in the homeostatic system of cells. Interruption of normal blood flow to an organ causes ischemia-hypoxia, which can result in tissue injury in many organs, including the heart, liver, lungs, and retina, all physiologically active tissues. Stresses or deficiency of OThe manner in which chronic hypoxia causes cell or tissue damage or cell death has been described in several recent reports documenting that the hypoxia evokes overproduction of ROS, which are a major culprit in cell damage . Moreov2 treatment and found that RGC death following hypoxia was predominantly apoptotic, although both apoptotic and necrotic cell death have been observed [Recently, several reports have shown that hypoxic stress induces ROS production, which, if not quenched, leads to cellular pathophysiology ,65,69,70observed ,72. Furtobserved , and theobserved  as a resobserved .Interestingly, our present study found increased expression of PRDX6 for the first 24 h of hypoxia exposure Figures , but wheThe identification of genes or their products involved in etiology of oxidant-mediated pathology has already led to important insights into the cellular response to stress and mechanisms of oxidant damage. In previous reports, we described the regulation of PRDX6 gene expression by NF-κB ,10, and We have found that hypoxia induces ROS-driven RGC death caused by down regulation of PRDX6 in cells under prolonged hypoxia, and ROS expression is causally associated with PRDX6 expression level. Results further revealed that ROS are differentially regulated during hypoxia; however, increased expression of ROS, due to deficiency of PRDX6, indeed reflects pathophysiology of RGCs. Because delivery of PRDX6 may attenuate RGC death by optimizing intracellular ROS and NF-κB activation, PRDX6 should be considered as therapeutic agent for hypoxia-induced disorders. Further detailed research will be needed to elucidate the mechanisms involved in the PRDX6-mediated protection of RGCs.DPS and NF conception and designing of experiments; RT, LSK, NF, performing experiments and acquisition of experimental data; DPS, NF, RT, LSK, BC, EK, NF, AK, analysis and interpretation of data and writing and revising of manuscripts.


To the Editor: Since 2003, linezolid has typically been used to treat infections caused by multidrug-resistant gram-positive cocci such as vancomycin-resistant Enterococcus faecium and methicillin-resistant Staphylococcus aureus  in Greece.From July through October 2007, nonrepetitive coagulase-negative staphylococci that exhibited resistance to linezolid were isolated from blood cultures from 4 separate patients hospitalized in the ICU at Sismanoglion General Hospital of Athens, a 450-bed tertiary care hospital. The ICU is a 10-bed, level II unit, comprising 2 rooms with 1 bed each and 2 rooms with 4 beds each. Each isolate was recovered in 2 of 2 blood culture sets per patient, indicating true bacteremia. The demographic and clinical information for the patients is described in the tuf gene followed by sequencing analysis  according to manufacturer’s instructions. Resistance genes mecA,vat,vga, erm, aac(6´)-Ie+aph(2″), ant(4′)-Ia, and aph(3′)-IIIa, as markers for resistance to β-lactams, dalfopristin, macrolides, and aminoglycosides, were identified by PCR as previously reported  after digestion of chromosomal DNA with SmaI  and a molecular method based on the S. cohnii subsp. ureolyticus. The API Staph system has correctly identified 2 of them; the remaining 2 isolates were falsely characterized as S. xylosus. According to disc diffusion test results, the isolates were resistant to cefoxitin, oxacillin, penicillin, rifampin, quinupristin-dalfopristin, erythromycin, clindamycin, fusidic acid, tobramycin, gentamicin, and linezolid. MICs were linezolid 32, oxacillin 256, quinupristin-dalfopristin 8, vancomycin 2, teicoplanin 2, tigecycline 0.125, and daptomycin 0.5 mg/L. Molecular methods detected the following resistance genes: mecA, ermA,aac(6´)-Ie+aph(2″), and aph(3´)-IIIa. The isolates, despite their resistance to streptogramins, were negative for vat and vgaA genes. In addition, all isolates carried the G2576T mutation and had 4 of 5 mutated alleles. No isolate carried the cfr gene or any mutation on ribosomal protein L4 and L22 genes. PFGE results indicated that all isolates were clonally related, belonging to the same clone.The molecular method identified the isolates as Outbreaks caused by linezolid-resistant staphylococci are rare worldwide , and the outbreak strain was not recovered from other patients in the ICU or in other departments of the hospital after the initial outbreak. In conclusion, to avoid spread of staphylococcal resistance in ICUs, measures such as hand hygiene and adequate central venous catheter handling should be taken, and policies regarding antimicrobial drug use must be applied.The 4 patients were treated in the same room by the same personnel; thus, a potential explanation for this outbreak is patient-to-patient transmission of linezolid-resistant strains on the hands of healthcare personnel. However, cultures of ICU personnel  grew only methicillin-resistant


The processes that drive fibrotic diseases are complex and include an influx of peripheral blood monocytes that can differentiate into fibroblast-like cells called fibrocytes. Monocytes can also differentiate into other cell types, such as tissue macrophages. The ability to discriminate between monocytes, macrophages, fibrocytes, and fibroblasts in fibrotic lesions could be beneficial in identifying therapies that target either stromal fibroblasts or fibrocytes.in vitro and in sections from human lung. We found that markers such as CD34, CD68, and collagen do not effectively discriminate between the four cell types. In addition, IL-4, IL-12, IL-13, IFN-γ, and SAP differentially regulate the expression of CD32, CD163, CD172a, and CD206 on both macrophages and fibrocytes. Finally, CD49c (α3 integrin) expression identifies a subset of fibrocytes, and this subset increases with time in culture.We have identified markers that discriminate between human peripheral blood monocytes, tissue macrophages, fibrocytes, and fibroblasts. Amongst these four cell types, only peripheral blood monocytes express the combination of CD45RO, CD93, and S100A8/A9; only macrophages express the combination of CD45RO, 25F9, S100A8/A9, and PM-2K; only fibrocytes express the combination of CD45RO, 25F9, and S100A8/A9, but not PM-2K; and only fibroblasts express the combination of CD90, cellular fibronectin, hyaluronan, and TE-7. These markers are effective both These results suggest that discrimination of monocytes, macrophages, fibrocytes, and fibroblasts in fibrotic lesions is possible, and this may allow for an assessment of fibrocytes in fibrotic diseases. There are multiple sources of fibroblast-like cells present in fibrotic lesions and healing wounds in vitro are able to develop further into myofibrocytes, a myofibroblast-like population of cells that express α-SMA and are able to contract collagen gels, in an in vitro model of wound contraction Fibrocytes appear to differentiate from CD14+ peripheral blood monocytes in vitro and in vivoAt present, there is no single specific marker for fibrocytes. The combination of intracellular collagen staining and the expression of CD45 or LSP-1, plus either CD34 or CXCR4, has been considered as a sufficiently accurate criterion to discriminate fibrocytes from leukocytes, dendritic cells, endothelial cells and tissue-resident fibroblasts Another major difficulty in analyzing the role of fibrocytes in fibrotic diseases is the inability to identify markers specific for fibroblasts in vitro-cultured macrophages , in vitro cultured fibrocytes , and in vitro cultured fibroblasts  with commercially available reagents to identify markers that can accurately discriminate between these populations.In this report, we have screened freshly isolated human peripheral blood monocytes (10–15 µm cells that express CD14 with a bilobed nucleus), 5 cells per ml in a humidified incubator containing 5% CO2 at 37°C. Immediately following Ficoll separation PBMC incubated in 8-well glass slides for 1 hour at 37°C were used to analyze, by immunohistochemistry, the expression of markers on monocytes ex vivo. PBMC were also cultured for 7 days in the presence of 10% fetal calf serum and 10 ng/ml M-CSF , which are standard conditions for the culture of human macrophages Human peripheral blood was collected into heparin vacutainer tubes  from healthy adult volunteers with specific approval of Rice University's Institutional Review Board. Written consent was received and all samples were de-identified before analysis. Peripheral blood mononuclear cells (PBMC) were isolated by Ficoll-Paque Plus  as described previously Normal human dermal fibroblasts  and MRC-5 fetal fibroblasts  were cultured in RPMI-1640 (Sigma-Aldrich) supplemented with 10% FCS, 100U/ml penicillin, 100 µg/ml streptomycin, and 2 mM glutamine, as described previously Serial 5 µm thick sections from patients with Chronic Obstructive Pulmonary Disease (COPD) or Interstitial Lung Disease (ILD) were obtained from the National Heart Lung and Blood Institute-sponsored Lung Tissue Research Consortium (LTRC), with specific approval of Rice University's Institutional Review Board. Written consent was received and all samples were de-identified before analysis. Sections were either standard formalin fixed paraffin-embedded sections or “HOPE fixed” before paraffin-embedding 2 anti-mouse IgG, biotinylated goat F(ab')2 anti-rabbit IgG , biotinylated mouse F(ab')2 anti-rat IgG, or donkey F(ab')2 anti-goat IgG . All secondary antibodies were used at 2.5 µg/ml in PBS-BSA for 30 minutes. Biotinylated antibodies were detected by a 1/200 dilution of ExtrAvidin alkaline phosphatase (Sigma-Aldrich or Vector Laboratories) in PBS-BSA. Staining was developed with the Vector Red Alkaline Phosphatase Kit (Vector Laboratories) for 7 minutes. Sections were then counterstained for 10 seconds with Gill's hematoxylin #3 (Sigma-Aldrich) diluted 1∶5 with water, rinsed in water, and then treated with Scott's tap water substitute  for 30 seconds. Slides were then dehydrated through 70%, 95%, and 100% ethanol, cleared with xylene, and mounted with VectaMount (Vector Laboratories). All procedures were performed at room temperature. Non-specific esterase (α-Naphthyl Acetate) staining was performed according to the manufacturer's protocol .For all slides, non-specific binding was blocked by incubation in PBS containing 4% BSA (PBS-BSA) for 60 minutes. Endogenous biotin was blocked by the addition of streptavidin and biotin solutions in PBS-BSA, following the manufacturers' instructions . Slides were then incubated with 5 µg/ml primary antibodies in PBS-BSA for 60 minutes (see 2 anti-mouse IgG (Southern Biotechnology), Rhodamine Red-X donkey F(ab')2 anti-rabbit IgG (Jackson ImmunoResearch), or NL637 donkey anti-goat IgG (R&D Systems). After washing, the biotinylated antibodies were detected with Streptavidin-Alexa 488 (Invitrogen) in PBS-BSA. Sections were then fixed for 30 minutes in 70% ethanol containing 0.1% Sudan Black B  to quench tissue autofluorescence For immunofluorescence staining, following incubation with primary antibodies as described above, slides were labeled with biotinylated goat F(ab')2 anti-rabbit IgG. After washing, the slides were incubated for at least 60 minutes with PBS containing 50% normal mouse serum  and 2% BSA. After washing, the slides were then incubated with mouse IgG1 monoclonal antibodies, and then incubated with isotype-specific biotinylated goat F(ab')2 anti-mouse IgG1 (Southern Biotechnology). After washing, the biotinylated antibodies were detected with Streptavidin-Alexa 647 (Invitrogen). Sections were then fixed for 30 minutes in 70% ethanol containing 0.1% Sudan Black B, washed in PBS, and mounted with VectaShield containing DAPI. All procedures were at room temperature.For staining using both mouse IgG1 and IgG2a monoclonal antibodies, slides were first incubated with mouse IgG2a antibodies  and rabbit anti-collagen antibodies. After washing, the slides were incubated with Alexa-488 goat anti-mouse IgG (Invitrogen) and Rhodamine Red-X donkey F(ab')2 anti-mouse IgG  in PBS-BSA. For 3 color flow cytometry, PBMC were then incubated in PBS-BSA containing 20% normal mouse serum  for 30 minutes on ice, to block any remaining anti-mouse IgG binding sites. PBMC were then washed twice in ice-cold PBS and then stained on ice for 30 minutes with CD16-PE (BD-Biosciences) and CD14-PE/Cy7  in PBS-BSA containing 20% normal mouse serum. PBMC were analyzed by flow cytometry using an Accuri C6 cytometer . Peripheral blood monocytes were identified by their size, granularity, and expression of CD14. At least 5,000 CD14 positive monocytes were examined for each marker.PBMC were analyzed by flow cytometry as described previously Immunohistochemistry images were captured on an Axioskop microscope  using an AxioCam MRc5 digital camera (Zeiss) and AxioVision software (Zeiss). Immunofluorescence images were captured on an Axioplan2 microscope (Zeiss) with a CoolSNAP HQ digital camera  and Metamorph software . Images were analyzed with ImageJ .Statistical analysis was performed using GraphPad Prism 4 software . Differences between multiple groups were assessed by ANOVA using Tukey's post-test. Significance was defined as p<0.05. In the figures, *** indicates p<0.001.in vitro, or isolated from tissues in vivo, have been identified with a variety of markers, including LSP-1, α-SMA, CD13, CD34, CD45, CCR2, CXCR4, and collagen-I Fibrocytes, either cultured under a variety of conditions in vitro differentiated macrophages, we were able to compare directly the expression of markers on these cell types in the same cultures. As reported previously, we found that lymphocytes , ex vivo monocytes , in vitro cultured macrophages , and fibrocytes, all express CD43, CD44, CD45, LSP-1, CD29 (β1 integrin), and CD18 (β2 integrin)   and 3 , . Wend CXCR4  and 12. ex vivo monocytes (in vitro for 24 hours had low to undetectable levels of CD14 and CD93 (data not shown) We next assessed whether we could identify markers that could discriminate cells that had the potential to differentiate into fibrocytes. We screened PBMC at time points before fibrocytes appeared in the cultures. As described previously, compared to onocytes  and 5, min vitro and in vivo activated macrophages express different markers when exposed to cytokines, immune complexes, or bacterial products Both in vitro and in vivo, and that this process appears to be dependent on the FcRγ chain, a common membrane-signaling component of CD64, CD32a, and CD16 in vitro  and interstitial lung disease (ILD) with antibodies against collagen-I, CD32, CD45RO, CD68, CD93, CD164, LSP-1, PM-2K, and TE-7  appears to be a marker of fibrocytes that appear after an extended period in culture.In this report, we stained human monocytes, macrophages, fibrocytes, and fibroblasts with a large panel of antibodies to find markers that can distinguish these cells. We found that we can exploit the expression of PM-2K on macrophages and the lack of PM-2K expression on fibrocytes to distinguish fibrocytes from macrophages in human The PM-2K antibody binds to a 150 kD cytoplasmic protein that appears to be specific for tissue macrophages, but the gene has yet to be identified Several groups have shown that fibrocytes have a progressive loss of hematopoietic markers as they differentiate from monocytes It is difficult to discriminate fibrocytes with low levels of the hematopoietic markers CD34 and CD45 from mesenchymal fibroblasts in tissue sections. We observed that, as reported previously, fibroblasts express CD68, which precludes it as a marker to discriminate fibroblasts from macrophages and fibrocytes We have also been able to show that the loss of CD33, CD35, CD93, and PNA expression on monocytes and the gain of 25F9 expression on macrophages can be used to differentiate these two cell types. The loss of CD33, CD35, CD93, PNA, and CD14, and the gain of 25F9, CD34 and CD49c will distinguish monocytes from fibrocytes. Finally, the expression of CD11a, CD11b, CD11c, CD18, CD43, CD45, and LSP-1 on monocytes, macrophages, and fibrocytes, and the expression of CD90, hyaluronan, and fibronectin on fibroblasts distinguish these two sets of cells.For our analysis of fibrocyte markers, we used commercially available reagents to remove the issue of limited availability of antibodies from non-commercial sources. This has the added advantage that differences in the expression patterns or numbers of cells between laboratories is less likely to be due to variations in antibody preparation, and more likely to represent variation due to tissue processing or an actual variation in the biological process.These studies indicate that the discrimination of monocytes, macrophages, fibrocytes, and fibroblasts is possible. In particular, the accurate discrimination of macrophages, fibrocytes, and fibroblasts will be beneficial in quantifying these cells in fibrotic lesions to determine if an increased number of one or more of these cell types is associated with a fibrotic disease.


To the Editor: Q fever is a worldwide zoonosis caused by the obligate intracellular pathogen Coxiella burnetii (C. burnetii infection (C. burnetii infection.C. burnetii DNA was detected on the lymph nodes with a C. burnetii–specific pair of primers that amplified an htpAB-associated repetitive element (C. burnetii with immunoglobulin (Ig) G antibody titer to phase 1 and phase 2 antigen of 800 and 1,600, respectively, and IgM antibody titer to phase 2 antigen of 50.A 70-year-old man was admitted to the hospital because of weight loss, night sweats, and a continuous high-grade fever of 2 months’ duration. His past medical history was unremarkable, except for pulmonary tuberculosis treated 55 years earlier and chronic glaucoma. He lived in a rural area and had rare contact with cattle. On admission, his body temperature was 39.5°C; his right laterocervical lymph nodes were enlarged (3 cm x 4 cm) and inflamed. Blood values were unremarkable except for an elevated C-reactive protein level of 150 mg/L . A computed tomography scan of the chest showed hilar calcifications and enlarged mediastinal lymph nodes. A biopsy of cervical lymph nodes indicated granulomatous lymphadenitis with foci of necrosis. C. burnetii with an IgG antibody titer to phase 1 antigen of 320.A 44-year-old man was admitted to the hospital because of a continuous low-grade fever of 3 months’ duration. He had worked as a farmer for 15 years and assisted in the birth of sheep and cattle. On admission, his body temperature was 38°C, and right inguinal lymph nodes were inflamed, measuring 4 x 4 cm. A lymph node biopsy showed granulomatous lymphadenitis with stellate abscesses surrounded by palisading epithelioid cells. Serologic testing by indirect IF was positive for For both patients, results of Ziehl staining and Lowenstein  cultures of gastric aspirates (x 3) and lymph node specimens were negative for mycobaceria, as were the results of tuberculin skin tests. Other diseases were ruled out, including brucellosis, yersiniosis, bartonellosis, and chlamydial infections (by serologic testing) and fungal infections (parasitologic studies on lymph node tissue). Antinuclear antibodies were absent, and angiotensin-converting-enzyme values were normal. Both patients received doxycycline, 200 mg once a day, and rifampin, 600 mg twice a day, for 1 year, and the symptoms resolved . For patient 1, serologic testing after 1 year of treatment showed an IgG antibody titer to phase 1 antigen of 320.C. burnetii was the likely etiologic agent, given the results of polymerase chain reaction and serologic studies (patient 1) or the patient’s occupation and results of the serologic testing (patient 2). Moreover, for both, no other potential cause could be identified, and the response to doxycycline-rifampin regimen was favorable. We suggest that granulomatous lymphadenitis be added to the list of atypical presentations of Q fever.Granulomatous lymphadenitis has been described during mycobacterial infections, tularemia, cat scratch disease, yersiniosis, lymphogranuloma venereum, histoplasmosis, coccidioidomycosis, and chronic granulomatous diseases (


DA926692 in the bone marrow of a childhood T-ALL case showing a t translocation as the sole chromosome abnormality. The breakpoints, defined at the sequence level, mapped within HPS5 (Hermansky Pudlak syndrome 5) intron 1 at 11p15.1, and DA926692 exon 2 at 2q11.2. The translocation was accompanied by a submicroscopic inversion that brought the two genes into the same transcriptional orientation. No chimeric trancript was detected. Interestingly, Real-Time Quantitative (RQ)-PCR detected, in the patient's bone marrow, expression of a 173 bp product corresponding to the 3' portion of DA926692. Samples from four T-ALL cases with a normal karyotype and normal bone marrow used as controls were negative. It might be speculated that the juxtaposition of this genomic segment to the CpG island located upstream HPS5 activated DA92669 expression. RQ-PCR analysis showed expression positivity in 6 of 23 human tissues examined. Bioinformatic analysis excluded that this small non-coding RNA is a precursor of micro-RNA, although it is conceivable that it has a different, yet unknown, functional role. To the best of our knowledge, this is the first report, in cancer, of the activation of a small non-coding RNA as a result of a chromosomal translocation.Chromosomal translocations play a crucial role in tumorigenesis, often resulting in the formation of chimeric genes or in gene deregulation through position effects. T-cell acute lymphoblastic leukemia (T-ALL) is associated with a large number of such rearrangements. We report the ectopic expression of the 3' portion of EST  Acquired balanced chromosomal translocations provide crucial diagnostic and prognostic data in cancer patients. They are probably pathogenetically significant as initiating events if present as the only cytogenetic changes . These rT-ALL, as other hematological malignancies, is associated with a number of chromosomal abnormalities, resulting either in a position effect or in a gene fusion .DA926692.Here we report the cloning of a novel balanced t translocation, found as the sole cytogenetic abnormality in the BM of a childhood T-ALL case, accompanied by the ectopic expression of the 3' portion of EST A 14-year-old male patient was referred to our hospital for rhinolalia. Total body CT disclosed a diffuse enlargement of the rhinopharynx vault, lymphoid hyperplasia of the Waldeyer's tonsillar ring, and a mediastinal enlargement. BM aspiration showed 90% of blast cells. Flow cytometry revealed that blast cell population was positive for CD 45, 2, 5, 7, and cytoplasmic CD3, while negative for CD1a and surface CD3. These findings were consistent with a diagnosis of early T-ALL, thus the patient started treatment based on AIEOP-BFM ALL 2000 protocol   Fig. 1A1A. FISH ion Fig. .The break on chromosome 2 was mapped within the interval chr2:96,884,868–97,812,993, delimited by the BACs RP11-1141J14  and RP11-716G6  at 2q11.2, yielding FISH signals on der(2) and der(11), respectively Figs. . It was HPS5 and GTF2H1, produced a fragment of approximately 800 bp. The sequence (Accession No. EU617360) showed that chromosome 11 at nt 18,300,006 (HPS5 intron 1) was fused with chromosome 2 at nt 97,384,504 (EST DA926692 exon 2)  Fig. . UnexpecDA926692/HPS5. Vectorette PCR with a forward primer  revealed that chromosome 2, at nt 97,384,507 (DA926692 exon 2), was fused with chromosome 11 at nt 18,299,998 (HPS5 intron 1)  Fig. . Again, HPS5/DA926692 chimeric transcripts, failed.5'- and 3'-RACE-PCR as well as RT-PCR experiments, aimed at cloning possible HPS5  on chromosome 11, and ANKRD36, FAHD2B, KIAA1641, ZAP70 on chromosome 2, revealed no misexpression in the present case compared to other T-ALL cases without the t translocation (data not shown).Further RQ-PCR analyses aiming at quantifying the expression levels of genes flanking the breakpoints, i.e. PS5 Fig.  and GTF2DA926692. A primer set specific for exon 1, upstream to the breakpoint, revealed no statistically significant difference in expression level compared to controls . Similarly, miRNA predictions by ProMiR II  and miR-abela  did not retrieve any positive results (data not shown).We hypothesized that this ncRNA could represent a precursor of an miRNA, but the miRNA prediction table produced by miRRim DA926692 might be part of a more complex predicted gene. Notably, the N-SCAN PASA-EST track showed that DA926692 and the EST CD110063, located in a double position both proximally  and distally  to DA926692  translocation in T-ALL not resulting in any DA926692 in the patient's BM  could have activated its expression in the BM cells of the patient.Here, the only observed gene expression change consisted in the ectopic expression of the 3' portion of  BM Fig. . One hypDA926692 was isolated from a small intestine cDNA library, described as similar to Ig kappa chain V-I region HK103 precursor , but no known function has yet been reported. Its expression in some human adult tissues , commonly found as transcriptional and translational regulators ,10. A vaDA926692 in chilhood T-ALL could have a pathogenetic role, even if of still unclear significance. It is presently unknown, however, if the described rearrangement represents a primary or a secondary genetic aberration at disease onset. Further studies on similar cases may be extremely important to confirm this new impact of chromosomal translocation, i.e. activation of ncRNA in tumors.In this context, we could speculate that the ectopic expression of the 3' portion of HPS5): Hermansky-Pudlak syndrome 5 isoform b; (GTF2H1): general transcription factor IIH, polypeptide 1; (nt): nucleotide; (RQ-PCR): Real-Time Quantitative PCR; (ANKRD36): ankyrin repeat domain 36; (FAHD2B): fumarylacetoacetate hydrolase domain containing; (KIAA1641): hypothetical protein LOC57730; (ZAP70): zeta-chain associated protein kinase 70 KDa; (ORFs): Open Reading Frames; (miRNAs): microRNAs; (ncRNA): non-coding RNA.(T-ALL): T-cell Acute Lymphoblastic Leukemia; (TCR): T-Cell Receptor; (CT): Computed Tomography; (BM): Bone Marrow; (PB): Peripheral Blood; (FISH): Fluorescence In Situ Hybridization; (BAC): Bacterial Artificial Chromosome; (The authors declare that they have no competing interests.MCG and AL designed research and performed molecular analyses. FA and IV contributed in clinical data collection and analysis. SC and AL performed classical cytogenetic analysis and contributed in the development of the study. LI performed molecular cytogenetic analysis. PD performed bioinformatic analysis. IP contributed in molecular data analysis and interpretation of the results. MR and GB gave final approval to the manuscript. CTS supervised the study and drafted the manuscript.


Ionizing radiation (IR) therapy is a primary treatment for glioblastoma multiforme (GBM), a common and devastating brain tumor in humans. IR has been shown to induce PI3K-Akt activation in many cell types, and activation of the PI3K-Akt signaling pathway has been correlated with radioresistance.Initially, the effects of IR on Akt activation were assessed in multiple human GBM cell lines. Next, to evaluate a potential causative role of IR-induced Akt activation on radiosensitivity, Akt activation was inhibited during IR with several complementary genetic and pharmacological approaches, and radiosensitivity measured using clonogenic survival assays.Three of the eight cell lines tested demonstrated IR-induced Akt activation. Further studies revealed that IR-induced Akt activation was dependent upon the presence of a serum factor, and could be inhibited by the EGFR inhibitor AG1478. Inhibition of PI3K activation with LY294002, or with inducible wild-type PTEN, inhibition of EGFR, as well as direct inhibition of Akt with two Akt inhibitors during irradiation increased the radiosensitivity of U87MG cells.These results suggest that Akt may be a central player in a feedback loop whereby activation of Akt induced by IR increases radioresistance of GBM cells. Targeting the Akt signaling pathway may have important therapeutic implications when used in combination with IR in the treatment of a subset of brain tumor patients. Glioblastoma multiforme (GBM), or grade IV astrocytoma, is the most common and lethal primary malignant brain tumor in humans -3. Despi2 (PIP3). PIP3 is converted to inactive phosphatidylinositol  P2 (PIP2) by the PTEN lipid phosphatase, which is commonly deleted or mutated in GBM [The PI3K-Akt signaling pathway is a ubiquitous and evolutionarily conserved signaling cascade that is involved in numerous cellular functions, including apoptosis, cell proliferation, differentiation, migration, and metabolism ,5. Activd in GBM ,11,12.3 through pleckstrin-homology (PH) domains, and translocate to the plasma membrane where they are activated via phosphorylation at residues Ser473 and Thr308. Once activated, Akt promotes cellular proliferation and inhibits apoptosis through phosphorylation of multiple substrates, including caspase-9, Bad, GSK3, and forkhead transcription factors, such as FKHR (FOX1), FKHRL (FOXO3), and AFX (FOXO4) [The most important downstream effector of PI3K signaling is the serine/threonine kinase Akt . There are three closely related Akt isoforms in mammalian cells, including Akt1 (PKBα), Akt2 (PKBβ), Akt3 (PKBγ) . All Akt (FOXO4) ,13.Activation of PI3K-Akt signaling is important in most human malignancies, including hematopoietic, melanoma, non-small cell lung, pancreatic, endometrial and ovarian, breast, prostate, hepatocellular, and brain cancers ,7,11. PTin vitro [PTEN is among the most frequently mutated or deleted tumor suppressor genes in GBM, as genetic and epigenetic alterations have been identified in at least 60% of patients . Importain vitro ,19, and in vitro ,16,20.Activation of the PI3K-Akt signaling pathway is associated with radioresistance in many cancers, including those of the colon, bladder, prostate, head and neck, cervix, and brain ,22. InhiImportantly, IR has been shown to induce Akt activation in multiple cell types, including some human GBM cells -31. In t2. LY294002 and doxycycline were purchased from Sigma, AG1478 from Biosource, SH-5 from Calbiochem, and MK-2206 from Selleck Chemicals.U87MG, MO59J, LN18, H4, A172, DBTRG-05MG, LN229, and HS683 cells were obtained from the American Type Culture Collection, and were cultured in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% FBS and 1% penicillin/streptomycin. U87MG cells containing transgenes for inducible wild-type PTEN, or the phosphatase-inactive mutant form of PTEN, PTEN-C124S, were gifts from Dr. Georgescu , and wer137Cs irradiator at ~2 Gy/min.Sub-confluent cell monolayers were irradiated using a J.L. Shepard Mark I 3VO4, 1 μg/ml leupeptin supplemented with proteinase inhibitor cocktails (Roche) and phosphatase inhibitor cocktails (Sigma). Cell lysates were separated by SDS-PAGE and transferred to PVDF membranes. After probing with primary antibodies, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody, and visualized by ECL (Pierce). Antibodies specific for total Akt and phospho-Akt (Ser473) were obtained from Cell Signaling Technologies. Antibodies specific for PTEN (clone 6H.1) was from Cascade Bioscience, and that for α-tubulin was from Neomarkers.Cells were lysed in lysis buffer  containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na2 tissue culture flasks in normal medium. Colonies were allowed to grow for 14 days before staining with a 0.2% crystal violet/formalin solution, and counted under stereomicroscopy. Colonies were defined as clusters of >50 cells. Colony-forming efficiency is reported as the survival fraction, which is defined as the total number of clones in irradiated cells divided by total number of clones in otherwise identical unirradiated cells. Each point on the survival curve represents the mean surviving fraction from at least three replicates. Cell survival measurements were fitted to a linear quadratic mathematical model using the GraphPad Prism 4 program [Cells in exponential growth phase were irradiated as described above. Prior to irradiation, cells were treated with LY294002, AG1478, SH-5, or doxycycline as described in the Figure legends. At 4 - 24 hr post-radiation, the cells were detached from the culture dish with trypsin, and were seeded at various dilutions into 25 cm program .We began our studies by testing the effect of IR on Akt phosphorylation in eight GBM cell lines. Akt activation was assessed by comparing the levels of basal Akt phosphorylation to that present 1 hr after a single dose of 6 Gy radiation. IR led to increased phosphorylation of Akt in three of the cell lines , which reached maximal levels within 1 hr of IR treatment, and maintained an elevated level for several hours Fig. . From thU87MG cells, which harbor a mutationally inactivated PTEN gene by virtue of homozygous splice site mutations , were chWe next investigated the mechanism of IR-induced Akt phosphorylation, and began by testing for a serum requirement for this effect. As shown in Fig. As EGFR  is commonly activated by genomic amplification in GBM and has previously been implicated in radiation resistance ,10,20,35We next tested if IR-induced Akt signaling modulated the radiosensitivity of GBM cells. First, a PI3K inhibitor was used to inhibit IR induced Akt activation, as PI3K is the upstream signaling molecule for Akt. Cells were pretreated for 1 hr with LY294002, which is a potent inhibitor of PI3K, followed by irradiation at 0 - 9 Gy. The cells were incubated overnight subsequent to removing the drug 4 hr after IR, and their reproductive growth ability was measured using clonogenic survival assay as described in the Methods.As shown in Fig. As we had previously shown that EGFR activation was required for IR-induced Akt activation Fig. , we nextIn addition to abolishing PI3K activity, LY294002 has been reported to inhibit other PI3K-like kinases (PIKK), such as mTOR , DNK (DNA-dependent protein kinase), and ATM (ataxia telagiectasia mutated protein) . These kU87MG cells have mutant PTEN genes , leadingIn the absence of doxycycline treatment, both cell lines expressed little PTEN protein and high levels of phospho-Akt Fig. . TreatmeNext, these U87MG clones were treated with or without doxycycline for 24 hr, followed by radiation treatment. 4 hr after IR, the cells were trypsinized and subjected to clonogenic survival assays. As shown in Fig. Next, we used Akt inhibitors to directly inhibit IR induced Akt activation, and assessed the effect on radiosensitivity. Two different Akt inhibitors, SH-5 and MK-2206 were tested.50~4 μM [SH-5 is a structurally modified phosphatidylinositol ether lipid analogue (PIA) that binds to the PH domain of Akt . SH-5 ha50~4 μM ,45. We f50 at 8 nm, 2 mM, 65 mM for Akt1, Akt2 and Akt3 respectively [Another tested Akt inhibitor MK-2206 showed similar effect. MK-2206 is a potent allosteric Akt inhibitor with ICectively . 1 hr trTaken together, these results indicate that Akt is an important downstream effector of PI3K signaling in modifying the response of human GBMs to IR treatment.Our results demonstrate that irradiation leads to activation of the Akt signaling pathway in a subset of GBM cell lines. IR-induced Akt activation was dependent upon the presence of serum factors, and could be inhibited by the EGFR inhibitor. Inhibiting PI3K, EGFR and Akt activation during irradiation increased the radiosensitivity of U87MG cells.The U87MG cell line is frequently used as a GBM model, and contains wild-type p53 and mutant PTEN. Our results show that IR induces Akt activation without changing levels of total Akt. However, this effect is substantially less robust in serum-free medium. The fact that radiation-induced Akt activation depends on the presence of serum factors suggests that activation of growth factor receptors is involved in this process.Overexpression of EGFR is one of the most prominent abnormalities associated with GBMs. Approximately 50% of GBMs contain over-active EGFR, typically through EGFR gene amplification or the expression of an active EGFR mutant. The expression of EGFRvIII, a common constitutively active EGFR mutant, increases radioresistance in immortalized normal human astrocytes . ClinicaIncreased Akt activation is associated with radiation resistance in various tumor types. However, most experiments have compared the radiosensitivity of cells with different levels of basal Akt activation ,47,48. SAmong the eight GBM cell lines tested for Akt activation, both LN18 and LN229 contain wild-type PTEN, and irradiation induced Akt activation in LN18 cells, but not in LN229 cells. All of the other six GBM cell lines contain mutant PTEN, but the effects of radiation on Akt activation were not consistent. Further experiments are needed to determine if activation of Akt by irradiation is related to the genetic status of PTEN or other factors critical to this signaling pathway.In conclusion, our findings indicate that Akt activation may have a critical role in radiosensitivity in a subset of GBM cells. Selective inhibitors that specifically target Akt signaling may have important therapeutic implications when used in combination with radiation in the treatment of GBM patients.The authors declare that they have no competing interests.HL performed experiments, HL, JSK, and TW designed experiments, and HL and TW wrote the manuscript. All authors have reviewed and approved the manuscript.