Reactive oxygen and nitrogen species have cell signaling properties and are involved in a multitude of processes beyond redox homeostasis. The peroxiredoxin (Prdx) proteins are highly sensitive intracellular peroxidases that can coordinate cell signaling via direct reactive species scavenging or by acting as a redox sensor that enables control of binding partner activity. Oxidation of the peroxidatic cysteine residue of Prdx proteins are the classical post-translational modification that has been recognized to modulate downstream signaling cascades, but increasing evidence supports that dynamic changes to phosphorylation of Prdx proteins is also an important determinant in redox signaling. Phosphorylation of Prdx proteins affects three-dimensional structure and function to coordinate cell proliferation, wound healing, cell fate and lipid signaling. The advent of large proteomic datasets has shown that there are many opportunities to understand further how phosphorylation of Prdx proteins fit into intracellular signaling cascades in normal or malignant cells and that more research is necessary. This review summarizes the Prdx family of proteins and details how post-translational modification by kinases and phosphatases controls intracellular signaling. The relative reactivity of these species spans several log orders of magnitude to affect the half-life and distance traveled within the intracellular environment. The hydroxyl radical is the most reactive of these species with a half-life on the order of 10−9 s, while H2O2 is much less reactive in comparison with a half-life of 10−3 s [In biological systems, redox reactions can regulate intracellular levels of reactive oxygen (ROS) and nitrogen species (RNS) to control a multitude of intracellular processes. These reactive species include both radical and non-radical forms of ROS and RNS such as superoxide anion  [2O2 load as well as the extracellular secretion of H2O2, which can reach 2 µM in stimulated neutrophils [There are diverse sources of ROS that emerge from several organelles within the cell. The intracellular concentration of Hto 10 nM . Mitochopiration , but thepiration  and the  in vivo . Complex in vivo ,8 and II in vivo  produce by MnSOD . The stevely low  based onM−1·s−1)  and mitoM−1·s−1) . Other oM−1·s−1) , the endM−1·s−1) ,15 and lM−1·s−1) . The plaM−1·s−1) , prostagM−1·s−1) , lipoxygM−1·s−1)  and xantM−1·s−1)  and tranM−1·s−1) , respecttrophils . Reactive species have a complex relationship in cancer. An overabundance of reactive species can cause cancer initiation and drive progression and accumulation of additional DNA mutations to support further growth . Ant2O2 is a particularly important reactive molecule with second messenger function. Spatiotemporal features of the molecule enable it to possess reactivity, yet move through biological macromolecular microenvironments intracellularly within different organelle compartments and intercellularly to neighboring cells. Organ and cellular systems coordinate the balance of the H2O2 through enzymatic-catalyzed reductant proteins in conjunction with the pro-oxidant sources mentioned above to form an interconnected network that maintains homeostasis or drives oxidative signaling. The enzymatic metabolism of H2O2 is primarily catalyzed through the action of catalase, glutathione peroxidases (GPx) and peroxiredoxins (Prdx). While all three enzymatically metabolize H2O2, important biochemical and biological differences exist. Insight into the basal function of the three enzymes in vivo can be drawn from the phenotypic effects observed in gene knockout studies. Deletion of catalase displays no overt pathological changes under basal conditions in mice, while GPx1 knockout mice were found to be smaller sized [Her sized  and harber sized ,29,30. Ter sized . Deletioer sized ,33. Prdxer sized .2O2 via an iron heme porphyrin complex [2O2 is enzymatically scavenged through oxidation followed by reduction of the active site heme iron. The active site heme is initially oxidized to an oxyferryl species, which is subsequently reduced by a second H2O2 molecule to regenerate enzymatic function. Sequestration of catalase to a single organelle enables control of peroxisomal H2O2 levels but also requires H2O2 derived from other intra- and extracellular sources to diffuse to the peroxisome in order for catalase-dependent catalysis to occur. Catalase exhibits a high H2O2 turnover rate [m close to 100 mM in human erythrocytes [2O2 concentrations are low. Contrasting catalase, GPx family members are found within several organelles. There are eight family members in the glutathione peroxidases (GPx) family (GPx1-8). GPx1-4 and 6 utilize a selenocysteine active site and glutathione (GSH) as a co-factor to reduce H2O2 [. GPx1 and 4 are present in most tissues, with GPx1 expression found in the cytoplasm and mitochondria, while the phospholipid hydroperoxide reducing GPx4 is found in the plasma membrane and cytoplasm [5 M−1 s−1 [Catalase is localized within peroxisomes and catalyzes the decomposition of H complex . A two-sver rate , but seqhrocytes , yield auce H2O2 ,38. GPx1 M−1 s−1 . These p2O2 to water. The family is subdivided into groups classified as 2-Cys (Prdx1-4), atypical 2-Cys (Prdx5) and 1-Cys (Prdx6) isoforms based on their structure and mechanism of action [p) is conserved among all family members at roughly 50 amino acids from the N-terminus. The Cysp is highly reactive to H2O2 due to its surrounding amino acids, which yield rate constants on the order of 106 to 108 M−1 s−1 [p under conditions of heightened H2O2 concentrations, resulting in a cysteine sulfinic acid, which can be further oxidized to a Cys sulfonic acid to cause irreversible inactivation of the enzyme [p [2O2 induced sulfinylation reactions that are characteristically observed in typical 2-Cys Prdxs [The Prdx family has six members (Prdx1-6) that are present in many cellular compartments. Prdx1, 2 and 6 are located in the cytoplasm, and nucleus; Prdx3 is localized to the mitochondria; Prdx4 is found in the endoplasmic reticulum; and Prdx5 is located in the peroxisomes, cytoplasm and mitochondria . The Prdf action . The act M−1 s−1 . Typicale enzyme ,44. Typinzyme [p ,44,45. Tys Prdxs ,45,46.p to the mechanistically important resolving Cys located on the opposing Prdx homodimerization partner near the C-terminus. Prdx1 and 2 homodimers can associate non-covalently to form larger decameric complexes that are ordered as a pentamer of dimers to form a doughnut-like structure [p is oxidized by H2O2 to a sulfenic acid moiety, which then forms a disulfide bond with the resolving Cys on the homodimerization partner [p can become overwhelmed in the presence of high levels of H2O2 and become overoxidized to form Cys sulfinic or further sulfonic moieties that lack peroxidase activity. The rate constant of the sulfenic acid form of Prdx2 with H2O2 to form the sulfinic Prdx is on the order of 104 M−1 s−1 [p can be adducted with GSH (rate constant 500 M−1 s−1) under physiological concentrations to protect from overoxidation and recycle with Grx1 [Homodimerization of Prdx proteins in a an N-terminus head to C-terminus tail fashion enables 2-Cys family members to align the Cystructure ,46. In 2 partner   enable protein tyrosine kinases (PTKs) to phosphorylate downstream targets without PTP-dependent removal of the phosphate moiety. PTPs are more readily targeted by H2O2 due to the enhanced reactivity of the low pKa active site cysteine residue, which causes increased susceptibility to oxidation compared to PTKs. Cell signaling cascades, therefore, utilize local H2O2 to coordinate signal transduction pathways to promote growth [2O2 within different intracellular compartments, cells require not only the production of H2O2 but the inactivation of peroxidases to drive phenotypic responses. As Prdx proteins are highly sensitive and enzymatically responsive to low levels of H2O2, the cell has developed pathways to inhibit peroxidase activity within the cell during cell proliferation. Suppression of Prdx1 activity by phosphorylation has been shown to play a role during the cell cycle in two different intracellular compartments, namely at the plasma membrane during growth factor signaling and within the nucleus during the transition to mitosis.Outside of oxidation of the Cysch as pH , ionic sch as pH  and tempch as pH . Expressch as pH ), but toe growth ,68. To d2O2 through the action of Nox [2O2 production by NOX1 is not sufficient alone to promote and sustain signaling [2O2, thereby allowing target proteins such as the Src family kinases to become oxidized [2O2 near lipid rafts is necessary to both simultaneously activate Src family kinases and inactivate PTPs. H2O2-mediated inhibition of PTPs is therefore crucial for growth factor signaling as activation of PTKs alone is not adequate to promote sufficient levels of protein tyrosine phosphorylation [A classic counter-example to the redox relay cell signaling coordination by Prdx described above is provided by phosphorylation of Tyr194 on Prdx1 by Src . Local in of Nox  at the pn of Nox ,72. Of iignaling ,69. Rathp was found to be protected from overoxidation in phosphorylated protein during co-treatment of cells with growth factor and H2O2. In vitro studies of the phosphorylated protein found the dimeric form of the Prdx1 was present without decamers. In vivo wound healing experiments exhibited a peak in Tyr194 phosphorylation after 1 day, which remained for 1 week.Phosphorylation on Tyr194 in response to treatment of cells with EGF or PDGF displayed isoform selectivity for Prdx1 in comparison to Prdx2 . Studies2O2 accumulation through inactivation of Prdx1 by phosphorylation is also important in the nucleus during mitosis [2O2 [2O2 enables active Cdk1-cyclin B to transition cells to late mitosis where Prdx1 can be dephosphorylated to promote deactivation of Cdk1 via dephosphorylation by the now activated Cdc14B. The importance of the centrosomal H2O2 during the transition to mitosis was evaluated by expressing catalase fused to a centrosomal targeting sequence, which inhibited entry into mitosis. Local H mitosis . During sis [2O2 . Inactiv2O2 in vitro and ex vivo. In the absence of TOPK via siRNA decrease, cells were more sensitive to UVB-induced apoptosis. Melanoma cells expressing wild-type but not Ser32Ala Prdx protein was more resistant to UVB irradiation. Whether Prdx2 can also be phosphorylated by TOPK is unknown but may not be seen in experiments with RPMI7951 cells, which only express high levels of Prdx1. While the studies above indicate post-translational modifications that serve to reduce peroxidase activity, not all Prdx phosphorylation is repressive to enzymatic activity. The T-cell-originated protein kinase (TOPK) can phosphorylate Prdx1 on Ser32 to enhance peroxidase activity . Investi2 (PLA2) activity, which is unique to the Prdx family. As Prdx6 phosphorylation increases PLA2 activity, phosphorylation of the protein may mirror protein overexpression seen in bladder [2 activity [2 (aiPLA2) functions to degrade surfactant phospholipids and aide DPPC  synthesis through the deacylation-reacylation pathway, by providing the required substrate, lyso-PC. Phosphorylation of Prdx6 provides an important functional role as a PLA2; phosphorylated Prdx6 maintains activity at both acidic and cytosolic pH [2 activity through the MAPK pathway [2 activity compared to Thr177Ala protein. Phosphorylation of Prdx6 has been found to attenuate phospholipase A bladder , liver [ bladder  and lung bladder ,79,80 caactivity . Acidic solic pH . Treatme pathway . ERK1, E−/−Prdx6 PMVEC displayed dramatically decreased plasma membrane translocation of the NOX2 components Rac1 or p47phox following Ang II stimulation. Upon treatment with the known MAPK inhibitor, U0126, Prdx6 phosphorylation, PLA2 activity and ROS were significantly decreased. This result suggested that MAPK activation mediates phosphorylation of Prdx6 resulting in its translocation to the cellular membrane, whereby, PLA2 activity can function to promote NOX2 activation [2 activity when compared to wild-type protein [Phosphorylation of Prdx6 on Thr177 was found to be critical for activation of NOX2 in pulmonary microvascular endothelial cells (PMVEC) by angiotensin II (Ang II) or PMA . Phosphotivation . Whethertivation ,83. Chhu2O2 in the cell, phosphorylation of Prdx proteins has been tied to cell death. Interestingly, Prdx1 phosphorylation at Thr90, which aids in driving the cell cycle through mitosis in the nucleus as described above, can also be targeted by tumor suppressor proteins. The difference between activation of survival or death pathways based on phosphorylation of the same protein at the same amino acid highlights the importance of location and timing of Prdx phosphorylation and the possible involvement of additional phosphorylation of Prdx1 at Thr183 to control activity. The following sections will detail the mechanistic studies that implicate Prdx phosphorylation with death. While some examples below highlight neurodegenerative pathways as examples of inactivation by phosphorylation, further cancer-focused work needs to be undertaken to extend these mechanisms as both Cdk5 and LRRK2 activity have been implicated in cancer. Cdk5 activity has been noted to be involved in proliferation, migration, invasion, metastasis, the epithelial to mesenchymal transition, the DNA damage response and angiogenesis in many forms of human cancer . The LReference ,86. 2O2 cause Prdx1 to bind Mst1, which was found to promote Mst1 activation and enhance apoptosis [2O2. In contrast, phosphorylation of the Thr90 residue of Prdx1 by Mst1 and possibly Mst2 has been described to inactivate peroxidase activity [−/−Prdx1 mouse embryonic fibroblasts (MEFs) showed heightened levels of the DNA damage biomarker phosphorylated Ser139 H2AX following treatment with H2O2. This finding suggests that the inactivation of Prdx1 by Mst1 could potentially cause a positive feedback loop whereby excess H2O2 further activates Mst1. That inactivation of Prdx1 by phosphorylation can promote Mst1 activation, while knockdown of Prdx1 can inhibit Mst1 activity, suggests that a more complex pattern of regulation is present that may involve both oxidation and phosphorylation.Mammalian sterile 20–like kinase-1 (Mst1) and Mst2 have been shown to suppress tumor formation in the liver and intestines in vivo ,88,89,90poptosis . Loss ofactivity . Full-leactivity . Whetheractivity . Express+ treated cells in vitro and MPTP treated mice in vivo. Furthermore, Cdk5 activation was shown to mediate dopaminergic (DAergic) neurodegeneration by downregulation of Prdx2 peroxidase activity which ultimately leads to significant elevations of intracellular ROS and DAergic neuron death [Prdx2 phosphorylation has been primarily investigated in pathological brain diseases and injury. Mitochondrial dysfunction and excessive oxidative stress are believed to be critical facilitators of the pathogenesis of Parkinson’s disease (PD). Neurons have oxidative metabolism systems in place, such as Prdxs, to manage and prevent the accumulation of deleterious levels of oxidative stress but can be overwhelmed when ROS scavengers are decreased or inactivated. In the context of PD, Prdx2 associates with a Cdk5 kinase complex via p35 or the proteolytically cleaved products of p35, p10 and the hyper-activated p25 . Cdk5/p3on death . This obon death . Anotheron death . Expresson death , p35 seeon death . Cdk5-de−/−p35 neurons. Glutamate-induced neuronal cell death was rescued by viral expression of wild-type or Thr89Ala Prdx2 but not Thr89Glu Prdx2. Ischemia-dependent neuronal cell death generated by 4-vessel occlusion in hippocampal CA1 neurons could also be rescued by expression of wild-type Prdx2 but not Thr89Glu in vivo [The Cdk5-Prdx2 pathway has also been shown to cause neuronal death in ischemia models . Glutama in vivo . PhosphoDrosophila model that expressed activated kinase mutant LRRK2, where overexpression of Prdx3 rescued flies from decreased lifespan, depletion of dopaminergic neurons and dysfunctional mitochondria in flight muscles of flies [Drosophila with the Prdx mimetic ebselen, which diminishes H2O2 and peroxynitrite. Prdx3 is expressed in mitochondria and has been shown to be important for maintaining mitochondria mass and membrane potential in Myc-transformed fibroblasts and breast cancer cells . The debof flies . The impde novo expression of unmodified protein during signaling events. While information regarding the dephosphorylation of Prdx family members is relatively scarce, some information is available. The protein phosphatase PP2A has been identified to dephosphorylate Prdx1 when phosphorylated at Thr90, which could be blocked with okadaic acid [−/−Pin1 MEFs. The association between Pin1 and Prdx1 was enhanced in the presence of 50 μM H2O2 and could be inhibited by pharmacologically blocking Cdk1 activity or mutation of Thr90 Prdx1 to Ala. Further experiments found that Pin1 could also bind Prdx2, 3 and 4 but not in cell lysates containing Thr-Pro motif mutants Thr89Ala Prdx2, Thr146Ala Prdx3 and Thr162Ala Prdx4. Reports detailing the importance of interactions between kinases and Prdx family members suggest dephosphorylation may represent an equally significant process. The absence of a Prdx phosphate removal mechanism would require phosphorylated Prdx protein to be degraded or sequestered for future coordination of various signaling pathways. Protein recycling through proteasomal degradation may occur, but this would represent an energy-intensive process requiring aic acid . Dephosp−/−Ptp1b MEFs exhibited significantly higher levels of Prdx4 phosphorylation in the presence or absence of G-Csf stimulation when compared to Ptp1b-proficient MEFs. It is unclear which Tyr was phosphorylated in −/−Ptp1b MEFs or the functional consequences, but the authors suggested that the Tyr phosphorylation may decrease Prdx4 peroxidase activity. Overall, the existence of phosphatases that dephosphorylate Prdx family members is an area that requires further exploration. Dephosphorylation of Prdx4 has also been demonstrated by Ptp1b . GranuloThe current state of knowledge of phosphorylation as a means to modulate Prdx function has been relatively understudied. While biochemical details exist for modification at Ser32, Thr90, Thr183 and Tyr194 and attempts have been made to evaluate Thr18, the consequences of phosphorylation of Prdx1 at multiple sites are unclear. Phosphorylation of Prdx1 Thr90 and Thr183 would be expected to perform similar functions as both sites are targeted by Mst1, decrease peroxidase activity and promote the formation of decameric complexes, but not all combinations are as straightforward. There are questions regarding what occurs when contrasting sites are phosphorylated that cause distinct changes to the oligomeric tertiary structure or peroxidase activity. Phosphorylation of Ser32 has been shown to enhance peroxidase activity, but whether this can exert a dominant effect to rescue the decreased peroxidase functionality of Prdx phosphorylated at Thr90, Thr183 or Tyr194 is unknown. The general structure of a phosphorylated Thr90 or Thr183 Prdx1 decamer may also collapse following Tyr194 phosphorylation, which has been shown to lead to homodimers of Prdx1 primarily. Multi-site phosphorylation may be precluded from occurring in some cases due to changes in the three-dimensional protein structure that prevent sequential kinase binding from occurring, but many other proteins have been shown to have multi-layered signal transduction cascades of regulation. The existence of positive and negative phosphorylation feedback loops to control post-translationally modified proteins temporally has been well documented in transcription factors, kinases and other enzymes. In our current era where large resource experimental datasets have become more readily available due to technical advancements, large proteomic datasets can be mined to determine if protein modifications are present under a myriad of conditions. Published datasets have emerged that support existence of novel phosphorylated forms of Prdx family members. As modifications of plants have been well described in a previous review by Liebthal et al. , we willPhosphorylation of other members of the Prdx family has been less well described. There is evidence that the cdc2-cyclin B complex is capable of phosphorylating Prdx1, 2, 3 and 4 in vitro but cell compartmentalization concerns were suggested by the authors to preclude Prdx3 and 4 from the likelihood of interacting with Cdk proteins . While C2 activity but peroxidase specific phosphorylation sites have not yet been detailed. Six phosphorylation sites of Prdx6 have been described in large proteomic datasets (+/+bi-1) and −/−bi-1 mice found that Tyr phosphorylation was increased in −/−bi-1 liver, brain, heart, lung and kidney tissues [In comparison to Prdx1 and 2, the number of phosphorylation sites present in large datasets is less abundant for the rest of the protein family. Delineation of the effects of phosphorylation of Prdx3 for Thr146 in LRRK2 kinase mutant cells and flies has been described and suggests phosphorylation is a mechanism utilized to alter peroxidase activity, but other sites have yet to be studied ,100. Thrdatasets . Tyr89 i tissues . The ide2O2, ROOH and ONOO− concentration within multiple cellular organelles. Research efforts have therefore primarily focused on how oxidation of the peroxidatic Cys perturbs cell signaling. While there are many important unrealized details and properties of Prdx oxidation that remain, the effects of other forms of post-translational modifications to Prdx proteins represent another exciting area of exploration. Dynamic changes to oxidation, phosphorylation and other forms of post-translation modifications can change three-dimensional structure and function of Prdx proteins to coordinate the various processes outlined above. In addition, control of Prdx through phosphorylation further integrates redox and signaling cascades. Inactivating phosphorylations of Prdx proteins likely prevent Prdx redox relay reactions as the peroxidatic Cys loses peroxidase activity. Prdx phosphorylation cascades would thus enable attenuation of Prdx-dependent oxidative equivalent transfer to partner proteins and inhibit redox signaling. The role of peroxidase activating phosphorylation sites such as Ser32 of Prdx1 on redox relays is also unknown but may perhaps oppose peroxidase inactivating phosphorylation sites.The highly sensitive peroxidatic cysteine thiol of Prdx proteins makes them well suited to sense and control cell signaling due to minor changes in HPost-translational modifications of Prdx family members have been identified to play a significant role in several signaling cascades, but there remains to be a complete understanding of the function of several putative sites in many family members. The introduction of phosphomimetic and non-phosphorylatable mutants via genomic editing through systems such as CRISPR/Cas9 would be useful to determine the functional consequences of Prdx phosphorylation in normal non-transformed cells as well as cancer cells. Candidate phosphorylation sites to be studied further could be identified from proteomic screens in cancer cells such as those indicated in bold in


RNA binding proteins (RBPs) are strongly linked to the pathophysiology of motor neuron diseases. Recent studies show that RBPs, such as TIA1, also contribute to the pathophysiology of tauopathy. RBPs co-localize with tau pathology, and reduction of TIA1 protects against tau-mediated neurodegeneration. The mechanism through which TIA1 reduction protects against tauopathy, and whether TIA1 modulates the propagation of tau, are unknown. Previous studies indicate that the protective effect of TIA1 depletion correlates with both the reduction of oligomeric tau and the reduction of pathological TIA1 positive tau inclusions. In the current report, we used a novel tau propagation approach to test whether TIA1 is required for producing toxic tau oligomers and whether TIA1 reduction would provide protection against the spread of these oligomers. The approach used young PS19 P301S tau mice at an age at which neurodegeneration would normally not yet occur and seeding oligomeric or fibrillar tau by injection into hippocampal CA1 region. We find that propagation of exogenous tau oligomers (but not fibrils) across the brain drives neurodegeneration in this model. We demonstrate that TIA1 reduction essentially brackets the pathophysiology of tau, being required for the production of tau oligomers, as well as regulating the response of neurons to propagated toxic tau oligomers. These results indicate that RNA binding proteins modulate the pathophysiology of tau at multiple levels and provide insights into possible therapeutic approaches to reduce the spread of neurodegeneration in tauopathy.The online version of this article (10.1007/s00401-018-1937-5) contains supplementary material, which is available to authorized users. RNA binding proteins (RBPs) are strongly implicated in neurodegeneration. Mutations in RBPs are associated with motor neuron diseases and form a strong component of the pathology of these diseases. Recent studies demonstrate that RBPs, stress granules (SGs) and the translational stress response are also key pathways mediating the pathophysiology of tauopathy , 9, 43. The current study uses tau seeding and propagation models to investigate the mechanisms through which TIA1 impacts on tauopathy. In tauopathies, misfolding or aggregated tau forms oligomers and fibrils . EmerginThe current study investigates the activities of tau species in propagating tau pathology and neurodegeneration and demonstrates a key role for TIA1 and SGs in neurodegeneration mediated by extracellular, propagated tau oligomers. The critical role of TIA1 enabled further investigation into the pathophysiological mechanisms through which TIA1 contributes to tauopathy. This study begins by comparing the effects in vivo and in vitro of different tau species extracted from P301S tau mouse brains and demonstrates that tau oligomer and fibrillar fractions both propagate tau pathology in vitro and in vivo, but exhibit striking differences in the neurodegenerative outcomes. The oligomeric tau fraction induced tau pathology in neuronal soma that co-localized with SG markers and elicited profound neurodegeneration. In contrast, over the 3-month period of this study, the fibrillar tau fractions induced tau inclusions predominantly in synapses and dendrites, but showed no association with SG markers, nor did they elicit neurodegeneration. Next, we further use this model for mechanistic studies. We show that TIA1 reduction elicited neuroprotection through both cell autonomous and cell non-autonomous mechanisms. Mice with reduced TIA1 produce less toxic tau oligomers and also exhibit reduced vulnerability to toxic tau oligomers generated from elderly P301S MAPT mice. These results demonstrate that oligomeric tau propagates toxic tau pathology through a mechanism mediated by TIA1 and pathological SGs, suggesting a broad role for SGs in the mechanisms of tau-mediated neurodegeneration.Tia1−/− mice (B6.129S2(C)-Tia1tm1Andp/J) were generated in and obtained from Anderson lab in Harvard University, Dana Farber Cancer Institute . . 7]. OurTo further confirm the existence of oligomeric and fibrillar forms of tau in S1p or P3 injected mice brain, tau oligomer marker TOMA2 and fibrillar tau marker thioflavin S were used to detect tau inclusions in CA3 and LEnt, respectively. The data indicate that the S1p fraction primarily induced oligomeric tau aggregation, while the P3 fraction primarily induced fibrillar tau aggregation . We also examined a cohort of injected PS19 mice that were killed at 15 days after injection. In both cohorts, the fractions containing tau aggregates exhibited only modest diffusion into CA3 and no diffusion into distant LEnt region . The limited diffusion of the tagged tau suggests that the tau aggregation present in CA3 and LEnt over both short- and long-term models resulted predominantly from the templating of human tau synthesized in the recipient neurons.The mechanism of propagation and toxicity in neurons is largely unknown. The localization of propagated tau oligomers to neuronal soma raised the possibility that they might interact with the TIA1/SG pathway, which also localizes to the neuronal soma. To test for co-localization of tau oligomers with TIA1 positive SGs, brain tissue sections from mice treated with the S1p or P3 fractions were probed with anti-TIA1 antibody and the oligomeric tau-specific antibody TOMA2  tau than fibrillar tau (P3 fraction). Neurodegeneration associated with propagation of the oligomeric or fibrillar tau was assessed by immunohistochemical labeling with neuronal marker NeuN in CA3 and LEnt regions Fig. a. The reWe hypothesized that the neurodegeneration induced by the propagated oligomeric tau might elicit a stress reaction in the affected neurons and that this stress response might be associated with cytoplasmic translocation of TIA1 and SG formation. This is important because recent studies indicate that tau pathology associates with TIA1 positive SG responses in neurons , 43, 44.Tia1/++ and PS19::Tia1/−+ mice and harvested at 6 months. The TOMA2 staining data showed that TIA1 reduction greatly reduced the basal level of TOMA2 oligomeric tau in PS19::TIA+/− mice, as well as blocking the propagation of oligomeric tau in S1p-injected mice  via AAV9-mediated transduction in hippocampal neurons over-expressing human WT or P301L tau. The efficiency of TIA1 knock down was greater than 50% of TIA1 as detected in cell lysate by immunoblot . We documented propagation of S1p-induced tauopathy (CP13 staining) in these cultures . We proceeded to test whether the S1p fraction from PS19::Tia1/−+ mice was less toxic than the equivalent fraction from PS19::Tia1/++ mice. We exposed DIV14 cultures of hippocampal neurons to samples of S1p fraction isolated from PS19::Tia1/++, PS19::Tia1/−+ and WT C57BL/6 mice. Medium was collected after 24 h. Analysis of LDH levels from the medium of the neurons demonstrated a 30% decrease in LDH from cultures exposed to PS19::Tia1/−+ S1p compared to PS19::Tia1/++ , TIA1 and SGs. Indeed, binding of TIA1 to tau oligomers generated by templating of extracellularly propagated tau could promote their stabilization and accumulation . This suTia1/−+ mice were highly resistant to neurodegeneration mediated by the S1p oligomeric fractions from PS19 mice, and S1p oligomeric fractions from PS19::Tia1/−+ mice exhibited reduced toxicity in cultures of hippocampal neuron over-expressing tau. Although the relative contributions to neurodegeneration from cell synthesized tau and intercellular propagated tau are not well understood for PS19 P301S tau mice aging normally, the current study used templated tau fractions to greatly accelerate production of tau pathology, which means that most of the tau pathology studied resulted from tau propagation. The neuroprotection provided by TIA1 reduction demonstrates its requirement in neurodegeneration mediated by propagated, templated tau oligomers. Thus, the current study demonstrates for the first time that TIA1 regulates neurodegeneration mediated by both intrinsic and extrinsic tau, with TIA1 regulating toxicity by determining the amount of toxic tau oligomers and the response of neurons to the toxic tau oligomers. The discovery that these neurodegenerative pathways share a unified mechanism mediated by tau oligomers, TIA1 and SG biology, suggests that therapeutic approaches targeting this pathological mechanism will be able to inhibit both the cell synthesized (intrinsic) and intercellular propagated tau pathways in tauopathy, leading to increased therapeutic efficacy. We suggest that inhibiting the interaction of TIA1 with tau might be one such approach.The direct role of TIA1 in the mechanism of toxicity mediated by propagated tau was proven by showing that TIA1 reduction decreased degeneration associated with propagated tau. PS19::Supplementary material 1 (DOCX 11731 kb)Below is the link to the electronic supplementary material.


Concerns have been raised about several results reported in this article :The second and last FACS plots in Fig 2C (U251/Empty Vector and A172/Notch2) appear similar, although the listed percentages are different.In Fig 3, U251 NF-κB panel lanes 1, 2 appear similar to A172 PCNA panel lanes 2, 1, respectively, when flipped vertically and horizontally. Additionally, the U251 MMP2 panel appears similar to the A172 PI3K panel, the U251 Cyclin D1 panel appears similar to the A172 Bcl-2 panel, and there are similarities between the U251 Caspase 3 panel and the A172 Notch2 panel.In Fig 4, several mice appear to be included in multiple panels as representing different experimental results. The authors reported that errors were made in generating Fig 4 such that images from control experiments were used as representing Scramble Notch1 siRNA (Scr siRNA) and Empty Vector results, and images from the Scramble Notch1 siRNA (Scr siRNA) group were used as representing Notch2 results.The tumor sizes reported in Fig 4 exceed sizes commonly accepted for mouse tumor studies. In response to journal queries, the authors did not provide scientific or ethical justification for the tumor sizes, and they clarified that no specific criteria  were used to determine when animals were euthanized. They explained that they typically terminated the experiment after 3–4 weeks, when quality of life was “seriously affected” in the group bearing the largest tumors.In Fig 6, there is overlap between the lower half of the Notch1 siRNA and the upper half of the Notch2 plasmid images. The authors commented that this resulted from a figure preparation error and that the images are both from the Notch2 siRNA experiment.In the published Fig 6, there are areas where the TUNEL staining pattern (green) appears similar although the DAPI staining (blue) differs, when comparing the Control, Scramble Notch1 siRNA (Scr siRNA), and Empty Vector panels; and when comparing the Notch1 siRNA and Notch2 plasmid panels. There are also areas in the Control and Scr siRNA panels where DAPI staining patterns appear similar, although regions adjacent to these areas differ.The authors offered updated versions of Figs 4 and 6 in which the panels in question were replaced to address image duplications, but they noted that the original data supporting results reported in this article are no longer available.PLOS ONE Editors retract this article in light of the above issues which call into question the validity and reliability of the reported results and raise concerns about animal welfare considerations in the study design.The AZ agreed with the retraction. The other authors did not respond or could not be reached.


They arrived to the laboratory 10 hrs and 34 hrs after the final football session and blood samples were collected at fasted (0 min) and 45, 90, 240 and 360 min post a high fat load meal. There were non significant increase for postprandial TG AUC  and iAUC  between 10 and 34 hrs after the 1FOOT. For the 3FOOT, there was a non significant decrease in postprandial TG AUC  and iAUC  from 10 to 34 hrs, respectively. Performing three consecutive days of football exercise may offer no greater protective effect for postprandial TG before a period of reduced activity, compared to a single session.Elevated postprandial triglyceride (TG) is associated with increased risk of cardiovascular disease. The time window for the last bout beneficial effect on postprandial lipaemia after football play is unknown. The aim of the present study was to examine whether playing affects postprandial TG during 1.5 day of reduced activity. Eighteen males were randomly allocated to perform either 1 (1FOOT; n = 9; age = 33.0 ± 5.0 yrs; body mass index = 24.2 ± 3.6 kg/m Sedentary behavior is an important issue in public health . ProlongSeveral different models of sedentary behaviour exist, including training cessation for athletes, increasing sitting time, reducing daily ambulatory activity and bed rest. All of these likely induce negative physiological effects that may provide information relating to metabolic dysfunction. The negative effect of low activity levels on postprandial TG have been reported in some studies ,8,9,10. Several studies have shown endurance, resistance and high intensity exercise to independently lower postprandial TG –16. Howe2) or 3 football  sessions across a 5-day study period. Participants arrived to the laboratory 10 hrs (day 4) and 34 hrs (day 5) after the final football session and blood samples were collected at fasted (0 min) and 45, 120, 240 and 360 min post a high fat load meal. The study was approved by the Qatar Anti Doping Laboratory ethics committee (number F2015000112), and was in accordance with the Declaration of Helsinki. Written informed consent was obtained from all study participants prior to commencing.Eighteen males were randomly allocated to perform either 1  cardiovascular, pulmonary or metabolic disease, (2) absolute contraindications to exercise testing as established by the American College of Sports Medicine, (3) dietary restrictions regarding the meals provided. In order to control for confounding variables, participants were instructed to (1) fasting was standardized for 10 and 34 hrs before the fat meal, (2) abstain from caffeine, tobacco, and vitamin supplements for 24 hrs before blood sampling, and (3) awake between 06:00 and 07:00 hrs prior to laboratory testing. Participants were asked to refrain from any other physical activity, besides that of the exercise intervention, for the 36 hrs before the start of pre intervention measures.th rib landmarks (waist) and the girth of the buttocks at the level of the posterior protuberance. Body fat was estimated using the InBody Bioelectrical Impedance Analysis Systems .Body mass and stature were recorded using a combined scale and stadiometer  with body mass index calculated. Waist and hip circumferences were measured at visual narrowing of the waist between the iliocristale and 10All participants performed the Yo Yo intermittent endurance test level 1 (YYIETL1), in an air-conditioned sports hall on artificial turf to establish maximal heart rate (HR) and cardiorespiratory fitness. The YYIETL1 test consists of 2 x 20 m shuttle runs performed at increasing running speeds, interspersed with 5 s of active recovery, during which the participants jogged around a cone placed 2.5 m behind the start/finishing line. The speeds were controlled by audio and the test was terminated when the participant was no longer able to maintain the required speed for 2 consecutive runs. The total distance (metres) covered represented the test result.On day 1 participants arrived to the laboratory at approximately 0800 hrs after an overnight fast (10 hrs) . FollowiParticipants were instructed to wear an Actigraph activity monitor  across the 5-day experimental period to monitor step counts, this included the three days of the intervention  as well as the two days of trials (10 and 34 hrs). The activity monitor was placed on the hip as this position has shown to be the most reliable for estimating energy expenditure . ParticiAll sessions for both groups consisted of 60 min (2 x 30 min) small sided game (5 vs 5) performed on an artificial outdoor court (36.5 x 27.5 m). All matches were played at the same time of day (20:00 hrs) and each player in turn played as goalkeeper for the matches. The activity profiles during the football match were recorded using the same Actigraph activity monitor and session rating of perceived exertion (sRPE) was recorded within 5 min of exercise completion using the CR-10 Borg scale and player load calculated (sRPE x minutes) . PerceivFollowing the last football session and the evening prior to the first visit to the laboratory, all participants consumed a standardised meal  providing 960 kcal; 36 g fat, 120 g carbohydrates and 42 g protein. Participants were instructed to consume the sandwich, and nothing else, after the last football session. Participants consumed the same meal (Subway sandwich) the evening of trial one (day 1) to ensure the dietary intake was replicated prior to the second trial (day 2) experiment.g and at room temperature) and was collected and stored at -80 oC for further analysis. Analyses of all samples from the same participant were done in a single batch. The spectrophotometric analyses were performed on an automated clinical chemistry system  to determine the level of TG, insulin, glucose and non-esterified fatty acids (Abbott-Cell Dyn 3700).Blood samples were collected into 9-ml potassium-EDTA Monovettes  or serum separate tubes (SST)-gel/clot activator. Serum was obtained by centrifugation . Incremental area under the curve was calculated using the trapezoid rule and calculated by subtracting, from the postprandial area, the mean baseline value extrapolated over 6 hrs; thus they reflect changes occurring after the meal. Whole-body insulin sensitivity was assessed with the homeostasis model assessment (HOMA) of insulin resistance index from serum fasting data as: insulin (μU/ml) × glucose (mmol/l)/22.5)  and withData were tested for normality with the Kolmogorov-Smirnov test, with all data shown to be normally distributed. A 2x2x5 (group x hours after the match x blood sampling time points) analysis of variance (ANOVA) was used for all comparisons between the two trials. When significant overall differences between the trials was found, a Bonferonni post hoc test was applied. Students’ unpaired t-test was used to compare baseline characteristics between 1FOOT and 3FOOT groups. Statistical significance was set at p< 0.05 and 95% confidence intervals (CI) and Cohen’s d effect sizes (ES) were calculated. The criteria to interpret the magnitude of the ES were: > 0.2: small, > 0.6: moderate, > 1.2: large and >2.0: very large) . All staEstimated energy expenditure was greater for day two football session  than day one  and day three  in the 3FOOT group. No other difference was found between the 3FOOT consecutive days of football session for any of the metrics relating to time in different zones for energy expenditure. In the 3FOOT condition we found no difference for sRPE , although a large effect size was calculated for day one vs day three; ES = 1.17. There were no differences for PRS  between the 3 days for the 3FOOT group. We found no differences for sRPE between 1FOOT (282 ± 56) and 3FOOT (313 ± 16) (ES = 0.75) for the only (1FOOT) and day 3 of football matches (3FOOT). There were no differences between groups for the number of steps for the three days prior to the first laboratory visit and there was no difference for the number of steps between 1FOOT (4897 ± 1504 steps/day) and 3FOOT (4753 ± 2071 steps/day) during day 4 of the experiment (10 hrs), inferring that participants performed a similar activity level prior to day 5 of the experiment (34 hrs).There was a non-significant increase in postprandial TG AUC  and iAUC   between Insulin iAUC increased from 34 to 10 hrs  in the 3The aim of the present study was to examine whether different frequency of playing football (1 session vs 3 sessions) affects postprandial TG during 1.5 day of reduced activity. We found no difference for postprandial TG incremental area under the curve from 10 to 34 hrs following either one single or three consecutive 60 min sessions of playing football. Our findings have important clinical implications, notably that there does not appear to be an additive effect from three consecutive days of playing football in the evening 10 and 34 hrs post the last bout of exercise, compared to solely one football session.In comparison to our study, Zhang et al.,  reportedThe fact we showed no difference between the 10 and 34 hrs may be explained by several reasons. While we did not perform pre intervention measure of postprandial TG, several studies, including previous work from ourselves, have shown the lowering effects of exercise from control to post training intervention. Energy expenditure and lipoprotein lipase (LpL) are suggested to be important factors for postprandial TG reduction, however, evidence has shown a dissociation of energy expenditure from the magnitude of postprandial TG attenuation . While t2max every 30 min, improved postprandial TG area under the curve [The light ambulatory contractions of habitual daily activity performed during the 10 and 34 hrs time period may have been enough to maintain lower levels of postprandial TG, since LpL has shown to be sensitive to small changes in ambulatory daily activity, albeit this was reported in rats . While the curve . Thus, sIt is important to consider the participant characteristics, as well as the synergistic interaction between previous training experience and acute exercise, may be a factor for the results in our study. Though our participants were not highly trained individuals, they had previous experience playing football. Research has shown that trained individuals may have a low chylomicron-TG half-life which may partly be due to a reduction in the fasting TG pool size and partly to a direct effect of chronic exercise on the TG removal system . It is aThe 3FOOT group showed a substantial increase ~108%) in insulin incremental area under curve from 10 to 34 hrs, compared to the 1FOOT group (~ 16%). This change in the 3FOOT is probably a manifestation of the 40% lower concentrations at 45 min on day 4 (10 hrs). The negative effect of low levels of activity on insulin response have been shown after only a few days 3. Pafili et al 8% in ins. The altThe different changes in insulin from day 4 to 5 seen between 1FOOT and 3FOOT may be a combination of a decreased concentration on day 4 , but also a large increase on day 5 (after the period of inactivity). We speculate a greater muscle damage in 3FOOT which may have been detected 34-hour post. Paschalis et al.,  demonstrOur results may also be explained by the composition of the high fat meal consumed. Chong et al.,  demonstrIn conclusion there was no difference for postprandial TG incremental area under the curve from 10 to 34 hrs following either one single, or three consecutive days of playing 60 minutes of football. Performing three consecutive days of football exercise may offer no greater protective effect for postprandial TG before a period of reduced activity, compared to a single session. Two-hour insulin response increased from 10 to 34 hrs, suggesting impaired sensitivity.


TNFAIP3 gene encodes A20, an inhibitor of nuclear factor-κB pathway, and is a susceptibility gene for autoimmune diseases. We investigated deleterious variants in the coding regions of TNFAIP3 gene of Japanese AIH patients or those with cirrhosis. The deleterious variants in the coding regions of TNFAIP3 gene were analyzed by the cycle sequencing method and the frequencies of deleterious TNFAIP3 alleles of AIH or AIH with cirrhosis were compared with those of Japanese controls. The deleterious alleles in TNFAIP3 were not associated with AIH. A significant association was shown for the deleterious alleles in TNFAIP3  4.28, 95% confidence interval (CI) 1.53–11.95) with AIH with cirrhosis at presentation. The serum IgM levels in AIH patients with deleterious alleles in TNFAIP3 were tended to be lower than those without . The frequency of deleterious alleles in TNFAIP3 was higher in the AIH subset without the DRB1 risk alleles than that with . The deleterious alleles in TNFAIP3were associated with AIH with cirrhosis.Autoimmune hepatitis (AIH) is an autoimmune liver disease and cirrhosis is sometimes complicated with AIH at diagnosis, influencing its prognosis.  Liver cirrhosis is sometimes complicated with AIH at diagnosis and influences its prognosis. AIH patients with cirrhosis had a worse survival2. The genetic and environmental factors are involved in the pathogenesis of AIH. A genome-wide association study (GWAS) on European population revealed that human leukocyte antigen (HLA) is the strongest genetic risk factor for AIH3. HLA-DRB1*03:01 and DRB1*04:01 were associated with AIH in European populations4. DRB1*04:01 and DRB1*04:05 were associated with AIH in Japanese populations8. DRB1*08:02 and DRB1*08:03 were also predisposing for the disease, when these alleles were possessed by individuals with DRB1*04:058. The previous GWAS also suggested associations of single nucleotide variants in other genes outside of HLA3. Several candidate-gene approach studies reported weak genetic associations of single nucleotide variants with Japanese AIH in other genes than HLA; STAT49, PTPN2210, ICOS11, TNIP112, and SH2B313.Autoimmune hepatitis (AIH) is an autoimmune liver disease with chronic progressionTNFAIP3 (tumor necrosis factor-α induced protein 3) gene encodes A20, an inhibitor of nuclear factor-κB (NF-κB) activation, and is a susceptibility gene for autoimmune diseases including systemic lupus erythematosus15 or rheumatoid arthritis17. A20 is a negative regulator of the NLRP3 inflammasome and myeloid cell specific deletion of A20 caused spontaneous arthritis in mice18. Analogically, inflammasome was activated in monocytes of non-transplanted AIH children and liver-transplanted children with de novo autoimmune hepatitis, but increased expression of A20 was observed in monocytes of liver-transplanted children without de novo autoimmune hepatitis19. Thus, A20 plays some important roles against autoimmune diseases. Recent studies reported that loss of function (nonsense or frameshift variants) or deleterious missense variants (variants that changed amino acid residues on positions highly conserved across orthologs) in TNFAIP3 dominantly caused an autoinflammatory disease with Behçet’s disease-like symptoms, the haploinsufficiency of A20 syndrome (HA20)22. However, the symptoms of HA20 also included those of autoimmune diseases, arthritis, nephritis, vasculitis, or hepatitis23. Thus, we investigated the variants in the coding regions of TNFAIP3 gene of Japanese AIH patients by the cycle sequencing method and tried to compare the frequencies of deleterious TNFAIP3 alleles of AIH or AIH with cirrhosis with those of Japanese controls.TNFAIP3 gene were amplified by 9 primer sets to detect variants by the direct sequencing method. Seven single nucleotide variants were found in the coding regions of TNFAIP in 360 AIH patients. No variant was detected in the splice sites of TNFAIP3 gene and no insertion or deletion was found. Two (rs200595071 and rs769014911) of these variants were synonymous and the other five were non-synonymous. Of these five non-synonymous variants, three variants  were predicted to be deleterious  by all five protein prediction algorithms, and two [c.380T > G  and c.2140C > T ] were predicted to be neutral  by all. Thirteen alleles of these three deleterious variants  4.28, 95% confidence interval (CI) 1.53–11.95, Table Deleterious allele frequencies in the AIH patients and the Japanese controls are shown in Table TNFAIP3 were analyzed .The demographic features of AIH patients with or without deleterious alleles in ed Table . The serTNFAIP3 and DRB1 was also investigated .The gene-gene interaction between TNFAIP3 were predisposing for AIH with cirrhosis in a Japanese population. TNFAIP3 encodes A20, an inhibitor of the NF-κB signaling pathway, and is a susceptibility gene for autoimmune diseases and HA2023. A20 is a negative regulator of the NLRP3 inflammasome and plays some important roles against autoimmune diseases19. TNIP1 is a predisposing gene in AIH12, and encodes an adaptor protein binding to A20. These data suggested the common signaling pathways in the pathogenesis of AIH, other autoimmune diseases, and HA20.The present study revealed that deleterious variants in 2. Cirrhosis at presentation was also a risk factor of development of hepatocellular carcinoma24. However, an age at onset was older in AIH patients with cirrhosis at presentation2, but that of AIH patients with deleterious variants in TNFAIP3 was not older  were detected in Japanese AIH patients, but none of them were reported to cause HA20. One of the deleterious variants in TNFAIP3 (c.305A > G) was appeared to be related to the poor clinical outcome of rheumatoid arthritis16, suggesting that deleterious variants in TNFAIP3 are modulating symptoms of autoimmune diseases.The progression pattern of AIH patients with cirrhosis at presentation would be smoldering and latent and the prognosis of AIH with cirrhosis was worseis Table . Thus, dTNFAIP3 was also analyzed between patients with or without the DRB1 risk alleles. The frequency of deleterious variants in TNFAIP3 was higher in the AIH patients without the DRB1 risk alleles than those with. These data suggested that deleterious variants might not predispose AIH in individuals with the strongest genetic risk factor, the DRB1 risk alleles. It was also possible that deleterious variants would be readily found in the individuals without the DRB1 risk alleles. Serum IgM levels in AIH with deleterious variants in TNFAIP3 were tended to be lower than those without , linkage disequilibrium was not strong between these two loci , suggesting that deleterious variants in TNFAIP3 and the DRB1 risk alleles would be independently associated with AIH. However, the independence could not be confirmed by logistic regression analysis, because the genotypes of the Japanese controls were not available.The association of deleterious variants in ut Table . Since ATNFAIP3 with Japanese AIH with cirrhosis. Since the sample size of this study was limited and the frequencies of deleterious variants were low, the association was modest. The associations should be confirmed in future large scale studies.To the best of our knowledge, this is the first report of the association of deleterious variants in 24. All the AIH patients were native Japanese living in Japan and satisfied the criteria of International Autoimmune Hepatitis Group for diagnosis of AIH25. The allele frequencies of single nucleotide variants in TNFAIP3 gene in Japanese population were referred to 3.5KJPN panel from the genome cohort study of Tohoku Medical Megabank Organization (https://ijgvd.megabank.tohoku.ac.jp/)28. The distribution pattern of the age and gender of the controls was described elsewhere . This study was reviewed and approved by NHO Central IRB and University of Tsukuba Research Ethics Committee. Written informed consent was obtained from each individual. This study was conducted in accordance with the principles expressed in the Declaration of Helsinki.A total of 360 AIH patients were recruited from National Hospital Organization (NHO) hospitals. The clinical information of the enrolled patients was obtained and 38 had cirrhosis at presentationTNFAIP3 gene was performed by the direct sequencing method of PCR products amplified with previously reported primers16 for exon 2 to exon 9 and BigDye Terminator v3.1 Cycle Sequencing Kit  using Applied Biosystems 3130xl Genetic Analyzer (Thermo Fisher Scientific Inc.). Deleterious alleles were defined by missense variants annotated as deleterious by all five protein prediction algorithms of PolyPhen-2 HumDiv , PolyPhen-2 HumVar , SIFT , Mutation Taster , and Likelihood Ratio Test Predictions 34. DRB1 genotyping results of the AIH patients were reported previously8. DRB1*04:01, *04:05, *08:02, or *08:03 were designated as the DRB1 risk alleles for AIH8.Genotyping of 35. The clinical phenotypes of AIH patients with deleterious alleles were compared with those without by Fisher’s exact test using 2 × 2 contingency tables or Mann-Whitney’s U Test. The distribution of deleterious allele frequencies was compared between AIH patients with or without the DRB1 risk alleles by Fisher’s exact test using 2 × 2 contingency tables under the allele model. Correction for multiple testing was performed by calculating false discovery rate Q-value36.The distribution of deleterious allele frequencies in AIH patients or AIH patients with cirrhosis was compared with those in Japanese controls by Fisher’s exact test using 2 × 2 contingency tables under the allele modelSupplementary Figure S1


The opposite syndrome (microduplication) has not yet been characterized. Our main objective is the recognition of a new clinical entity - 12q14 microduplication syndrome. - as well as confirming the role of HMGA2 gene in growth regulation.Array Comparative Genomic Hybridization (CGH), Karyotype, Fluorescence in situ Hybridization, Quantitative-PCR analysis and Whole exome sequencing (WES) were performed in a girl presenting overgrowth and obesity. Array CGH identified a 1.5 Mb 12q14.3 microduplication involving HMGA2, GRIP1, IRAK3, MSRB3 and TMBIM4 genes. Karyotype and FISH showed that duplication was a de novo insertion of 12q14.3 region on chromosome 9p resulting in an interstitial microduplication. Q-PCR confirmed the duplication only in the proband. WES revealed no pathogenic variants.Phenotypic comparison with patients with 12q14 microdeletion syndrome showed a reciprocal presentation, suggesting a phenotypically recognizable 12q14 microduplication syndrome as well as confirming the role of HMGA2 gene in growth regulation. It is also indicative that other genes, such as IRAK3 and MSRB3 might have of role in weight gain and obesity. HMGA2, GRIP1 and in some cases LEMD3, IRAK3 and TMBIM4 genes [HMGA2 gene gains of function were acquired rearrangements related with potentially malignant conditions. The majority were HMGA2 fusions with partner genes and subsequently formation of chimeric transcripts with gain of HMGA2 gene function and tumor formation [Copy number variations on chromosome 12q14 have been mainly reported as a microdeletion syndrome OMIM #166700) first described by Menten et al. in 2007 and classified as an autosomal dominant bone dysplasia. The patients showed failure to thrive in infancy evolving to proportionate short stature, mild intellectual disability and osteopoikilosis [6700 firsormation .HMGA2 and IRAK3 genes [HMGA2 gene as a common feature in the 12q14 microdeletion syndrome [HMGA2, GRIP1, IRAK3, MSRB3 and TMBIM4 genes and the phenotype associated with the duplication apparently mirrors the microdeletion syndrome phenotype.Previous reports on microdeletions within 12q14 region ranged from 1.83 Mb to 10.12 Mb with a 378Kb smallest region of overlap containing the syndrome . There aOur patient was born to non-consanguineous 37 year-old Human Immunodeficiency Virus (HIV) positive mother and 22 year-old healthy father. She has an 18 year-old healthy sister on her mother side. Anti-retroviral medication was carried out throughout both pregnancy and delivery according to national guidelines, and this treatment was not complicated by any adverse effects. The girl was born by caesarean section at 38 and 2/7 weeks, and Apgar scores were 9 and 10 at 1st and 5th minutes respectively. Birth weight was 4015 g (P95), length 50.5 cm (P75–90) and OFC 36 cm (P95). She was admitted to the neonatal intensive care unit soon after birth for moderate respiratory distress. She had mild hypotonia and feeding difficulties. Head ultrasound was normal. Supraventricular extrasystoles conditioning tachyarrhythmia were noted on electrocardiogram, and she received propranolol treatment transiently. Echocardiogram was normal. No other anomalies were noted. Upon follow up after discharge she was noted to have increased growth parameters with marked obesity, weighing 12.7Kg at six months (+ 6.7SD). At 8 months she was referred for genetic evaluation. She had developed mild dysmorphic features, with slight bilateral ptosis, scarce eyelashes, anteverted nostrils and macroglossia. She had inverted nipples and increased inter nipple distance. Both hands and feet seemed edematous and she had tapering fingers. She had normal skin. Follow up studies at 12 and 18 months showed cholesterol, apolipoprotein A1 and B, and endocrine studies  were normal. Serological markers for HIV were negative. She had no signs of urogenital abnormalities, visceromegaly or lipomatosis on abdominal ultrasounds. Radiologic studies did not show any signs of skeletal dysplasia. At 18 months her weight was 20.2Kg (+ 5.1SD), height was 91 cm (+ 3.6SD), occipitofrontal circumference (OFC) 50 cm (+ 2.8SD) and body mass index (BMI) 24.4 (+ 6.7SD)  according to manufacturer recommendations Results were analyzed using Cytogenomic software 2.9.2.4 and Human Genome 19 (GRCh37) assembly.HMGA2 gene. Applied Biosystems CopyCaller v2.0 software was used to calculate the copy number probe and the confidence of the statistical method.Copy number quantification was carried out by quantitative real time PCR (qPCR) on a StepOnePlus™ Real-Time PCR System  using the taqman Hs01969532_cn probe (Applied Biosystems) locate within exon 6 of Conventional G Banding with Trypsin and Leishman (GTL banding) and Fluorescence in situ hybridization (FISH) studies were performed on the proband and progenitors peripheral blood lymphocytes using standard protocols and according to the International System for Human Cytogenetic Nomenclature (ISCN) 2016 .green color) and 12 (aqua color) (Abbot®) and also a SureFISH probe specific for 12q14.3 HMGA2 gene region (red color)  were used.Centromeric FISH probes for chromosomes 9 (Whole exome sequencing (WES) was performed as a trio on proband and progenitors using Agilent SureSelect Human All Exon V6 and Illumina Hiseq2000 plataform. GRCh37 was used as Human Genome Reference.Standard deviation scores were calculated according to the World Health Organization (WHO) Growth Reference Data Z-scores .HMGA2, IRAK3, GRIP1, MSRB3, TMBIM4, HELB, RPSAP52 and LLPH . The duplication region contains 8 genes, including HMGA2 gene revealed one signal in each q14.3 arm of both chromosomes 12 and a third signal on chromosome 9p arm  encodes a protein that belongs to the non-histone chromosomal high mobility group (HMG) protein family. These proteins function as architectural factors and contain structural DNA-binding domains that may act as transcriptional regulating factors. The HMGA2 gene consists of five exons spanning a genomic region of 160 kb. Exons 1–3 encode A-T hook domains that bind to the minor grove of AT- rich DNA. In humans, haploinsufficiency of theHMGA2 gene is capable of causing a poor growth phenotype with failure to thrive and short stature, as seen in the 12q14 microdeletion. The association between growth retardation and short stature and the loss of the HMGA2 gene has been established [HMGA2 presenting exclusively short stature and none of the other 12q14 microdeletion syndrome features. HMGA2 deletion was also present in this patient’s mother and maternal grandmother, and also in a maternal aunt, all presenting short stature as well. The patient and his relatives therefore did not fulfill the criteria described for a 12q14 microdeletion syndrome, suggesting instead a strong association between the HMGA2 gene deletion and short stature [The ablished , 12. BuyHMGA2 [Ligon et al. by other side described a patient with overgrowth and advanced bone age who had a de novo pericentric inversion of chromosome 12, with breakpoints at p11.22 and q14.3, apparently truncating HMGA2 . The pathmga2 inactivation are born with reduced body size and extremely decreased fat levels [HMGA2 is an early event in the pathway to tumor formation. Normally, the expression of HMGA2 is restricted to undifferentiated mesenchymal cells. Chromosomal rearrangements frequently result in the activation of the normally silent HMGA2 allele in terminally differentiated cells. It has been suggested that such activation can lead to mesenchymal tumor formation [HMGA2 3′-UTR contains target sites for the let-7 miRNA and some rearrangements may lead to increased levels of HMGA2 protein due to the loss of mi-RNA-mediated repression [In vivo data shows that mice with homozygous t levels  whereas t levels . It is gormation , 16. It ormation . Additiopression .HMGA2 is expressed either solely from the translocated allele associated with a gain of function or from the nondisrupted allele when the disrupted allele does not acquire a regulatory domain [HMGA2 has been identified as the target gene of in mesenchymal tumors, namely in lipomas, the exact mechanism by which HMGA2 overexpresssion drives benign tumorigenesis in a target cell is still widely unknown [Ashar et al. suggest that in lipomas y domain , 16. Alt unknown .HMGA2 seems to act in a narrow window early in the adipogenesis where it is involved in expanding the population of preadipocytes and keepping the cells in an undifferentiated state. The authors concluded that in an in vitro model both wild type and truncated HMGA2 resulting in an overexpression of the gene abrogated growth inhibition and adipogenesis [HMGA2 gene develop undifferentiated lipomas and abundant adipose tissue [HMGA2 gene rather a rearrangement (truncated HMGA2 gene) could also involve different regulatory mechanisms. Publications of new constitutional cases with similar phenotypes will be necessary to confirm this possible new entity.According to Heriksen et al. ogenesis , which se tissue . This coHGMA2 with a dosing effect could explain the overgrowth of our patient.Taking into account the clinical data, all previous reports and the phenotype associated with the microdeletion, the duplication of The absence of lipomas or other tumors thus far in our patient remains to be elucidated. Nonetheless, given the patient’s young age these features can still arise later in life, and follow up medical screening is mandatory.HMGA2 gene and FISH demonstrated that the third copy was inserted on chromosome 9p. The presence of an insertion on p arm of this chromosome could also interfere with gene overexpression or subexpression. Literature revision on genes located chromosome 9p and association for overgrowth or obesity showed no results except a paper suggesting a susceptibility loci on 9p22 for adiposity phenotypes [In our patient array CGH revealed the presence of three copies of the enotypes . AlthougGRIP1 (OMIM#604597)gene has been implicated in the regulation of glutaminergic and perhaps also GABAergic signaling, both of which are involved in many neuronal mechanisms, and it is known that bialelic mutations of this gene cause Fraser syndrome [GRIP1 is also necessary for dendritic development [GRIP1 variants have been described in patients with neuronal and behavioral autistic phenotypes [GRIP1 gene only one did not present developmental delay [GRIP1 is involved in our patient duplication and apart from neonatal hypotonia, development and language have occurred within the normal timing frame.syndrome . Additioelopment . Furtherenotypes . Of the IP1 OMIM#04597geneIRAK3 and increased obesity [IRAK3 is downregulated in obesity, but also that its expression is increased following weight loss [IRAK3 duplication are not known. Dosing of blood adiponectin was not performed in our patient. As our patient presents severe obesity, implication of IRAK3 gene cannot be excluded.IRAK3 (OMIM#604459) encodes a member of the interleukin-1 receptor-associated kinase protein family. Members of this family are essential components of the Toll/IL-R immune signal transduction pathways and mutations in this gene have been associated with a susceptibility to asthma . It also obesity . Hulsmanght loss . ClinicaMSRB3 (OMIM#613719) is predominantly located to the endoplastic-reticulum and is thought to play a role in cell growth. Bialelic mutations of this gene cause autosomal recessive deafness 74 [MSRB3 protects mammalian and Drosophila cells against endoplastic-reticulum stress and has been shown to increase the longevity of Drosophila [MSRB3 deficiency induces cell growth inhibition in mouse embryonic fibroblasts. Haploinsufficiency of MSRB3 could therefore contribute to the growth restriction seen in all patients with 12q14 microdeletion. Correspondingly, duplication of MSRB3 with a simultaneous gain of function could cause overgrowth, either by inducing growth itself or by not regulating it.fness 74 . Overexposophila . These aTMBIM4 is a candidate gene for susceptibility to type 2 diabetes and obesity [TMBIM4 duplication can put our patient at a higher risk of type 2 diabetes. obesity . As metaLLPH is an intrinsically disordered protein predicted to function in nucleolus, possibly as a molecular hub for protein-protein interaction and regulating neuronal morphogenesis and synaptic transmission [HELB is a DNA Helicase  and it has been hypothesized that HELB is recruited to sites of DNA damage and has a role in recovery from replication stress [LLPH and HELB genes have not been published.smission . HELB isn stress . NeverthHMGA2 gene in growth regulation and suggest that other genes, such as IRAK3 and MSRB3 might have a role in weight gain and obesity.Patients with 12q14 microdeletion syndrome present low birth weight and failure to thrive, proportionate short stature and developmental delay, and in some cases osteopoikilosis. Our patient, presenting overgrowth, obesity and tall stature with advanced bone age, is the first described case with 12q14 duplication. Phenotypical comparison of both entities shows that they mirror each other. We propose that this phenotype could represent a new microduplication 12q14 syndrome. WES studies showing no other known pathogenic variants support the causative effect of the duplication. In conclusion, our results strongly support the role of


Following the publication of this article , the folFigure 3A panel IV and Figure 3B panel IV appear to contain areas of similarity;Figure 3A panels I, V, and VI appear to contain areas of similarity;Figure 3B, panels I and II contain areas of similarity;Figure 3B, parts of panels V and VI contain areas of similarity;Journal of Food and Chemical Toxicology [Figure 6 of a previously published article in the xicology  by four xicology  appear txicology  appears xicology ; Figure xicology  appears xicology ;Repeated regions of similarity in flow cytometry plots within Fig 6 IV, VI, and II;Figure 2A ERK1/2  bands in lanes 5 and 6 appear similar when adjusted for brightness/contrast;Figure 2A p38  panel lanes 2 and 6 appear similar when adjusted for brightness/contrast;Figure 2A ERK1/(P) panel contains vertical discontinuities across all lanes when adjusted for brightness/contrast;Figure 5 p53 contains background irregularities when adjusted for brightness/contrast such that lane 2 appears to have a background different to other lanes.The authors have been unable to provide any primary data underlying the figures. The authors commented that the regions of similarity in histopathological images arose due to similarity in lesions studied. For the similarities noted in flow cytometry plots, the authors commented that this could have arisen due to similarity in equipment and protocols.PLOS ONE Editors retract the article.In the absence of the data underlying the figures and in light of the above concerns, the The authors did not comment on the retraction decision.


The role of many lncRNAs in cancer remains elusive including that for a Prostate Cancer Associated Transcript 92 (PCAT92). PCAT92 shares the locus on chromosome 13 with ABCC4 gene, known to be implicated in prostate cancer. It has been shown that PCAT92 and ABCC4 are up-regulated in prostate cancer samples from multiple transcriptome datasets. Among the prostate cancer cell-lines LNCaP showed maximum overexpression of PCAT92 compared to control cell-line RWPE-1. We have shown that knockdown of PCAT92 in LNCaP cells reduces cell viability and proliferation and down-regulates ABCC4 transcript/protein expression. The shared region between PCAT92 and ABCC4 has a binding site for an oncogenic transcription factor (ZIC2) which is also upregulated in the majority of datasets studied here. ZIC2 binding to the predicted ABCC4 promoter has been confirmed using pull-down assay. Interestingly, under PCAT92 knockdown condition, there is a reduction in the ZIC2 binding to ABCC4 promoter indicating the potential involvement of PCAT92 in the recruitment of ZIC2. We have identified distinct regions on PCAT92 with potential to bind to ZIC2 non-DNA binding Zinc-finger domain and potential for triplex formation near ABCC4 promoter region, which have been experimentally validated. Together, these observations and localization in the nucleus suggests that PCAT92 may play a role in prostate cancer by increasing the local concentration of ZIC2 by forming RNA-DNA triplex near ABCC4 promoter thus helping in recruitment of ZIC2 for ABCC4 regulation. Long non coding RNAs (lncRNAs) are bringing a new level of understanding of cancer biology. Using high-throughput sequencing, a large number of cancer-associated lncRNAs are being discovered and involvement of many of them in cancer is slowly emerging. The mechanism of MALAT1, one of the earliest lncRNAs associated with cancer, is still emerging. Using mouse knockout models, it was shown that a set of metastasis associated genes were deregulated, strongly indicating its basis as a regulator of gene expression –4. OvereMore recently, using high-throughput transcriptome sequencing, it has been shown that almost a quarter of abundant transcripts in prostate cancer are lncRNAs with many that are still unannotated . This isOne of more established mechanism of action of lncRNAs is by regulation of genes within the same locus. For example, PCA3, located in intron 6 of the PRUNE2 gene, is co-expressed along with PRUNE2 in the nucleus to form a double-stranded RNA . The lncWe have previously shown that PCAT92 is located closely to ABCC4 gene on chromosome 13 . ExpressZinc-finger transcriptional regulators ZIC1 through ZIC5 have been associated with increase in transcriptional activity of many genes . ZIC2 isHere our work demonstrates how PCAT92 transcript is regulating the expression of ABCC4 by recruiting ZIC2 at the ABCC4 promoter region and forming triple helix near the promoter site to stabilize the binding of ZIC2 at ABCC4 promoter region.PCAT92 and ABCC4 are only 4305 bases apart on chromosome 13 in a divergent configuration sharing the 5’ regulatory region Figure . PromoteIt was reported that PCAT92, ABCC4 and ZIC2 are overexpressed in multiple prostate cancer datasets in the public repository (SRP002628 and ERP000550) . The oveFigure Binding affinity of ZIC2 at the ABCC4 promoter region in cell line is studied using ChIP-RTPCR with anti-ZIC2 antibody. Primers flanking the predicted ZIC2 binding site on ABCC4 promoter region was made. As shown in Figure in vitro. The transcribed RNA was then titrated onto the purified zinc finger domains of ZIC2 using Nano Isothermal Titration Calorimetry (NanoITC). This validated the interaction between the predicted PCAT92 RNA strand and the zinc finger domains of ZIC2. Antisense strand to the predicted PCAT92 RNA region was also cloned and transcribed to be used as negative control. As shown in Figure in-vivo validation of interaction between PCAT92 and ZIC2, we performed RNA Immunoprecipitation (RIP). The cells were fixed and RNA and protein were crosslinked. Using anti-ZIC2 antibody RNA-protein complexes were pulled down. RNA-Protein complex were broken by reverse crosslinking and the resulting RNA was subjected to cDNA synthesis. HEIH, one of the well-established non-coding RNA  ATP using T4 Polynucleotide Kinase . Zinc finger domains of ZIC2 protein were PCR amplified and cloned in pET28a expression vector. The clone was sequenced verified and the induced using 0.5mM IPTG, at 16°C overnight. Induced protein was purified using His Tag affinity chromatography and eluted with the buffer containing 200 mM imidazole. 2μg of ZIC2-HIS protein and 100pmol of radiolabeled target dsDNA, was mixed in the binding buffer. The reaction solutions were incubated for 30 minutes at 37°C and subject to electrophoresis on 6% polyacrylamide gels prepared in 1×TBE (Tris-borate-EDTA) buffer for 16 to 18 hours at 100V at 4°C. Imaging was done using phospho imager .To find all putative regions within PCAT92 transcript with triplex forming potential against the 4kb shared 5’UTR region on the chromosome, Triplexator computational pipeline was used  with all*DD) triple helical structure comprising U*AT and C*GC (Sequence 1) base triplets is generated conforming to a 12-fold helix [A parallel RNA.DNA.DNA  experiments were performed at 25°C using Nano ITC . The protein sample was prepared as explained in Electrophoretic Mobility Shift Assay study. The predicted region of PCAT92 was cloned in pBS-SK plasmid followed by synthesis of sense and antisense RNA from the clone using T3 RNA polymerase  and T7 RNA polymerase . 50μl of each sense RNA (60μM) and antisense RNA (60μM) was titrated into 300μl protein solution (5μM) with a fixed stirring speed of 300 rpm at 25 °C. Both RNA and Protein were dialysed in the same buffer . Similarly to under the interaction between the PCAT92 RNA and ABCC4 DNA we performed ITC for the predicted PCAT92 and ABCC4 region. The oligos corresponding to both were obtained. 50μl of each PCAT92 RNA (10μM) and random RNA (10μM) was titrated into 300μl ABCC4 DNA solution (5μM) with a fixed stirring speed of 300 rpm at 25 °C. Both RNA and DNA were dialysed in the same buffer . For both the titration experiment, 5μL of RNA was dropped for 10 injections. To achieve complete equilibration, the spacing time between each injection was set to 300 seconds. All measurements were repeated at least twice. Using Nano ITC NanoAnalyze software, the raw data was integrated, corrected for nonspecific heats and analysed according to independent model.


AZFc microdeletion in their blood leukocytes. However, if AZF genes were involved in impaired spermatogenesis, a higher frequency of chromosomal microdeletions was expected. In this study the frequency of AZFc microdeletion was compared with TTY2 gene family, i.e., TTY2A2A and TTY2A12A in blood leukocytes of NOA patients and normal fertile control. In the present study 30 normal fertile individuals with mean age of 35.0 ± 6.0 and 30 NOA patients with mean age of 34.0 ± 7.0 were screened for microdeletion of TTY2L2A and TTY2L12A at Yq11 and Yp11 respectively and sequence-tagged site (STS) markers for AZFc gene using multiplex PCR technique. At the first step karyotyping was done for all subjects using standard G-banding technique to identify patients with normal karyotype as well as non-affected normal controls for molecular analysis.About 10–15% of non-obstructive azoospermia (NOA) patients show AZFc microdeletion in normal and NAO patients whereas one TTY2L2A microdeletion in normal control (3.3%) and 4 in NOA (13.3%) was observed (p < 0.05). However our data indicated that 6 of 30 NOA patients (20%) showed TTY2L12A microdeletion whereas there was no observed microdeletion in normal control (p < 0.01).Results showed no AZF genes in infertile men. Therefore, screening these genes along with AZF genes might be valuable for infertile patients. The reason why these genes are deleted from Y chromosome is not known but might be associated with genomic instability induced by environmental physico-chemical genotoxic agents.Results indicate that the studied genes might be involved in impaired spermatogenesis more effective than the routinely screened  Therefore the aim of this study was to evaluate the frequency of microdeletion in TTY2 genes versus AZFc in peripheral blood leukocytes of NOA patients.As known, sperm DNA damage is clearly associated with male infertility and abnormal spermatogenesis 2.2.1.This experimental study was performed on whole blood samples obtained from 30 non-obstructive azoospermia patients and 30 normal men candidate for Assisted Reproductive Technology (ART) referred to Fertility and Infertility Center of Shariati Hospital . Normal and patients' demographic data is presented in 2.2.Karyotype analysis was performed to exclude those individuals with chromosomal abnormalities especially those involved in male infertility such as Klinefelter's syndrome from molecular analysis. Whole blood obtained from normal individuals and non-obstructive azoospermia patients in heparinized tubes was cultured in RPMI-1640 supplemented with 10% fetal bovine serum (FBS), antibiotics (penicillin (100 U/mL)/streptomycin (100 µg/mL), and 0.1 mL phytoheamaglutinin . Cells were exposed to thymidine 17 hours prior to colcemid treatment at final concentration of 0.4 µg/mL one hour before harvesting. Cells were harvested at 72 h after culture initiation. Harvesting, slide preparation and G-banding were performed according to routine standard procedure. At least 20 metaphases were analyzed for each subject. The resulting karyotypes were described according to the norms established by the International system for Human Cytogenetic Nomenclature 2.3.AZFc and TTTY2 microdeletion analysis. Peripheral blood samples were collected in tubes containing EDTA from cytogenetically normal infertile patients. All tests were performed on genomic DNA that was extracted from peripheral blood leukocytes using a commercially available DNA-isolation kit for mammalian blood . For each participant, AZFc-specific STSs that spanned the sub regions (sY254 and sY255) as well as STS for SRY  as internal controls were used to amplify specific regions of the Y chromosome using multiplex PCR TTY2L12A and TTY2L2A regions with SRY  as internal control. The primers used in this study have been previously used by Yapijakis et al. AZFc and TTY2L2A and TTY2L12A gel electrophoresis images in normal and azoospermia patients respectively.The patients and normal individuals with 46,XY, normal karyotypes were selected for 2.4.U-test as well as one way analysis of variance (ANOVA) was used to compare differences between the study groups. Sigma Plot (2004) for Windows was used to draw figures, p < 0.05 was considered as statistically significant.Statistical analysis was done using SPSS software . The Mann-Whitney 3.p < 0.001) clearly indicates the status of fertility in both groups. Both normal individuals and non-obstructive azoospermia patients did not show any microdeletions in their AZFc reigon . In non-obstructive azoospermia group six patients showed deletion in TTY2L12A (20%), whereas four patients had deletions in TTY2L2A (13.3%). In addition, none of the patients showed deletions in both studied TTY2 genes simultaneously. Results obtained for TTYA2L2A and TTY2L12A microdeletion were significantly different compared to control fertile group (p < 0.05 and p < 0.01 respectively). There was no correlation between microdeletions and age of the patients.Results are summarized in c reigon  and 4. N4.AZF region is mainly done with the use of PCR on blood leukocytes In the diagnostic work-up of infertile men, screening of Y chromosome microdeletion usually in AZF genes, AZFc is the most commonly deleted interval in men with azoospermia or severe oligozoospermia AZFc deletion frequency in azoospermia patients is reported to be 10–15%. Although, recent report indicates that a high frequency of microdeletions in AZFc region especially DAZ gene might occur de novo during spermatogenesis when sperm nuclei are studied AZF) region of Y chromosome especially in the DAZ genes was shown previously to occur in lymphocytes of men exposed to low dose and background level of ionizing radiation in vitroAmong TTY2L2A and TTY2L12A genes, expect one individual who exhibited microdeletion for TTY2L2A. The reason for this observation is not understood, but might be a de novo chromosomal alteration due to environmental exposure. Similar observation was previously reported for DAZ deletion in normal population exposed to high natural background radiation TTY2-associated deletions observed in leukocytes of infertile azoospermia patients might suggests that TTY2-like transcripts may play a significant role in the complex process of spermatogenesis. As shown in TTY2L2A and TTY2L12A respectively which is very high frequency compared to the overall frequency reported for AZFc microdeletion and more importantly in those patients with no deleted AZFc. These results might clearly indicate the involvement of the studied genes in spermatogenesis failure and male infertility. Perhaps their implication in male infertility might be more important than AZF genes which are routinely screened. A similar frequency of microdeletions of TTY2L2A and TTY2L12A genes is reported in oligoazoospermia and azoospermia patients form Greek patients TTY2L2A and TTY2L12A genes in azoospermia patients with no AZFc microdeletion and higher frequency of microdeletions in azoospermia patients with unkown AZF situation TTY2L2A and TTY2L12A genes are related to AZF deletion, then only in a lower percentage of patients' deletion in TTY2 related genes could be detected. Moreover the deletion in TTY2L12A gene has nothing to do with AZF genes because it is located on the short arm of Y chromosome. Another possibility of lower frequency reported by Shaveisi-Zadeh et al. TTY2 gene family is in its early stage. There are only limited reports associating these genes with male infertility. Although the possible causes of different frequency of microdeletions reported for these genes has already been discussed but for implementing testing these genes in paraclinic along with other Y microdeletions, there is a need for more researches and more data from different populations with different ethnicity and geographic distributions. So that we could be able to make a meta-analysis from different reports to provide a frequency range of microdeletions for TTY2 gene family for clinical use. As known for routinely screened Y microdeletions among infertile or subfertile men, there is a very big variation in the frequency of occurrence from 1–55% TTY2 gene family leading us to observe these microdeletions with different frequencies.As shown in TTY2 genes is in line with our previous observation for DAZ microdeletion and indicate that there are hot spots in these genes that make them vulnerable to damage and eventually deletion from entire DNA in Y chromosome Our observations regarding to AZFc microdeletion might clearly indicate involvement of TTY2 genes in spermatogenesis failure and male infertility.In conclusion a high frequency of microdeletions observed in our study on non-obstructive azoospermia patients without


Uncovering how new members of multigene families acquire new functions is an important topic in evolutionary and developmental genetics. CORL proteins  are a family of CNS specific proteins related to mammalian Sno/Ski oncogenes. Drosophila CORL (dCORL) participates in TGF-β and insulin signaling during development and in adult homeostasis but roles for the two mouse CORL proteins (mCORL) are essentially unknown. A series of studies were conducted to test the hypothesis based on previous results that mCORL1 is more similar to dCORL than mCORL2. Neither an updated alignment nor ectopic expression in adult wings were able to distinguish mCORL1 or mCORL2 from dCORL. Transgene experiments employing a dCORL endogenous function in mushroom body neurons showed that mCORL1 is distinct from mCORL2 and dCORL. mCORL1 and mCORL2 are also distinct in biochemical assays of Smad-binding and BMP signaling. Taken together, the data suggests testable new hypotheses for mCORL2 function in mammalian TGF-β and insulin signaling based on known roles for dCORL. Overall, the study reiterates the value of transgenic methods in Drosophila to provide new information on multigene family evolution and the function of family members in other species. The dominant model for the growth of multigene families and the acquisition of novel functions by new genes is based on gene duplication with one copy of the pair maintaining the original function while the other copy accumulates advantageous mutations leading to a novel function. We recently explored the consequences of this ‘duplication then neofunctionalization’ model for new genes in mammals and flies. For each, we employed the conserved Dpp/BMP signaling pathway (subfamily of the TGF-β family) that regulates embryonic dorsal-ventral axis formation in flies and vertebrates as our assay. Neofunctionalization for two new genes contrasted with the conservation of a third new gene.lolal is not present in mammals. It encodes a BTB domain protein that functions in a chromatin-remodeling complex maintaining Dpp transcription during dorsal-ventral axis formation  in the larval brain. In Drosophila mushroom body neurons dSmad2 signaling upstream of EcR-B1 transcriptional activation is facilitated by the Smad binding dCORL protein (mCORL1 (mSKOR1) was identified as a Sno/Ski family member that functions as a transcriptional co-repressor in cell culture. In embryos mCORL1 is expressed only in dorsal interneurons of the cerebellum  and both rescue Dpp mutants during dorsal-ventral patterning. Underlying the rescue, BMP2 is 82% and BMP4 is 81% similar to Dpp in the ligand region , mCORL1 (AK049035) and mCORL2 (NM_001109743) generated with Clustal Omega at escribed .Wing experiment stocks were: MS1096.Gal4 (Df(4)dCORL / ciD (Df(4)dCORL/Dci, 2) UASt.mCORL1 on III; Df(4)dCORL/Dci or 3) UASt.mCORL2 on III; Df(4)dCORL/Dci for rescue and overexpression experiments. Fly crosses for rescue and overexpression experiments were maintained at 25° prior to heat shock. Larvae were heat shocked 40-64 hr after egg lay for 1 hr @ 37°. After one hour of recuperation at 18° they were returned to 25°. Wandering 3rd instar larvae were picked 6 days after egg lay and checked for GFP before dissection (Df(4)dCORL status . This allowed the interpretation of the lobe with a clone in the MB as either mutant rescue or wild type repression of EcR-B1. At least seven brains were examined per genotype . The number of brains with clones in the MB for each genotype is reported in the figure legend for each experiment.Flip out clone stocks were: yw hs.FLP; AY.Gal4 UASt.GFP; yw) were picked, sorted and their brains dissected as in Post-heat shock larvae that have stopped wandering but not yet begun pupariation . A schematic of the structure of CORL proteins and an assessment of the similarity of dCORL, mCORL1 and mCORL2 in the Smad-binding Sno homology domain based on a new alignment is in The premise that similar sequences lead to similar functions is the reason investigators use BLAST to search for proteins similar to their favorite protein with the hope that functional data exists for a match, pointing to a specific hypothesis they can test in their system. A tree of the CORL/Sno/Dac family that share the Smad-binding Sno homology domain was valuable in suggesting we look in mushroom body neurons for dCORL function as this was the only established location for Activin subfamily signaling at the time , between the two mCORL proteins and dCORL in the Sno homology domain is greater than initially reported due to updated methods underlying the new alignment. In this region mCORL1 and mCORL2 (94% similar with 14 differences over 195 residues) are almost as similar as human BMP2 and BMP4 (96%) in the ligand region. The conservation of these two with dCORL, 89% for mCORL1 (27 differences) and 90% for mCORL2 (24 differences) is very high. This is greater than the conservation of Dpp with BMP2 (82%) or BMP4 (81%), proteins that can reciprocally substitute for each other across species dCORL mutant brains was due to the loss of dCORL, we rescued EcR-B1 expression with heat shock driven flip-out clones of UASt.dCORL (Df(4)dCORL mutants expressing UASt.GFP or UASt.GFP with either UASt.dCORL or UASt.mCORL1 or UASt.mCORL2 .Previously, to show that the loss of EcR-B1 mushroom body (MB) expression in Df(4)dCORL brains are easily visible in the presumptive EcR-B1 domain of the MB body. This location is characterized by proximity to Tailless expressing MB neuroblasts that are unaffected in Df(4)dCORL brains (Df(4)dCORL brains fully rescues EcR-B1 dCORL; Pursuing another line of experimentation employed in our prior analysis of dCORL, we examined flip-out clones expressing dCORL, mCORL1 and mCORL2 in the MB of wild type siblings of The data are shown at high magnification in dCORL mutants. Genetic evidence from both assays of an endogenous function of dCORL (regulation of EcR-B1 expression) support the alternative hypothesis that mCORL1 has a divergent function with mCORL2 and dCORL more similar.In this assay, MB clones of mCORL1 did not repress EcR-B1 expression ; Table 1dCORL mutants  consistent with loss of EcR-B1 expression in The binding data shows that mCORL2 does not bind Smad3  even in Consistent with the binding assay, parallel studies of TGF-β and BMP signaling showed that mCORL1, mCORL2 and c-Ski can repress TGF-β signaling  but onlyTaken together the sequence, genetic and biochemical data support the alternative hypothesis that dCORL and mCORL2 are the closest functionally with mCORL1 divergent. The data does not address which mCORL came first, as either of the two duplicates could diverge while the other maintained the ancestral function.dCORL mutants, 2) repression of EcR-B1 when overexpressed in wild type, 3) mCORL2 failure to bind the mCORL1 partner mSmad3 and 4) mCORL1 inability to antagonize BMP signaling in luciferase assays. We recognize that the data are not black and white with similarities between mCORL1 and mCORL2 in the wing assays and modest rescue of EcR-B1 by mCORL1 in dCORL mutants. However, the preponderance of evidence indicates that mCORL2 and dCORL share the ancestral function and that mCORL1 has a distinct function.None of the data supported the initial hypothesis that mCORL1 is more similar to dCORL than mCORL2. Instead most experimental data were consistent with the alternative hypothesis that mCORL1 has a distinct function from mCORL2 and dCORL: 1) rescue of EcR-B1 in From a larger perspective the data informs our understanding of multigene family evolution, specifically the accumulation of advantageous mutations. The six differences between the mCORL genes in the Sno homology domain are quantitatively similar to the eight differences between human Smad2 and Smad3 in the DNA-binding domain (Regarding functions for mCORL2 based on its biochemical properties and the reported roles of dCORL (In summary, our data that mCORL2 and dCORL share functions while mCORL1 has diverged reinforces the current working hypothesis that neofunctionalization of a new gene can be achieved with a handful of advantageous amino acid changes in a highly conserved functional domain. We also encourage our colleagues to experimentally test roles for mCORL2 in development and adult homeostasis that reflect functions for dCORL. Overall, the study reiterates the value of transgenic methods in Drosophila to provide new information on multigene family evolution and the function of family members in other species.