Incorrectly or simply not annotated data is largely increasing in most public databases, undoubtedly caused by the rise in sequence data and the more recent boom of genomic projects. Molecular biologists and bioinformaticists should join efforts to tackle this issue. Practical challenges have been experienced when studying the alternative oxidase (AOX) gene family, and hence the motivation for the present work. Commonly used databases were screened for their capacity to distinguish AOX from the plastid terminal oxidase  and we put forward a simple approach, based on amino acids signatures, that unequivocally distinguishes these gene families. Further, available sequence data on the AOX family in plants was carefully revised to: (1) confirm the classification as AOX and (2) identify to which AOX family member they belong to. We bring forward the urgent need of misannotation awareness and re-annotation of public AOX sequences by highlighting different types of misclassifications and the large under-estimation of data availability. It has become clear that the coding portions of the genome are organized hierarchically in gene families and superfamilies. Groups of genes encoding proteins showing similarity with each other, traditionally defined by >50% pairwise amino acid similarity  is a terminal quinol oxidase found in the mitochondria of a wide variety of species from different kingdoms  are the membrane-bound members of this superfamily. Whereas AOX is present in almost all phyla, PTOX appears limited to organisms capable of oxygenic photosynthesis . This was spread to more recent data originated from Medicago genome sequencing (see accession AES64415) because both traditional sequencing, and certainly genome sequencing projects, use as reference previously annotated sequences.In a quick survey on sequences deposited at NCBI (using a known AOX sequence and the BLAST tool) at least seven protein sequences, from either gene-discovery sequencing projects or gene re-sequencing, and six sequences originating from whole genome analysis, are annotated as AOX but they are indeed PTOX , while more closely related species have large differences in AOX family composition . In the case of AOX, the most commonly used primers are the ones described by Saisho et al. . These sFocusing on the membrane-bound di-iron carboxylate proteins, we show that a re-annotation is needed. We have reviewed a large quantity of data present in different databases, identified an easy way (based on signatures) of distinguishing between the two mis-classified gene families—AOX vs. PTOX—and presented the largest phylogeny to date that comprises curated and annotated AOX amino acids sequences (based on the system developed by Costa et al., Misannotation in superfamilies containing multiple families that catalyze different reactions is an issue needing to be addressed as it can have serious repercussions on data interpretation and ultimately on our understanding of the systems. It is a shared responsibility, of researchers working with these superfamilies and bioinformaticists to tackle this challenge. The current identification and classification system is prone to error propagation and an increase in annotation errors over time is to be expected. Researcher's awareness can minimize error propagation and certainly errors in data interpretation. It is also a dynamic process that needs to be revised and updated in frame of the continuous new flow of data. The ones with deeper knowledge on the specific genes families can positively contribute to data revision and collaborate with bioinformaticists to the updating of the classification systems.TN and BA-S conceptualized the manuscript. TN, MDC, and EL-M collected and analyzed the data. TN designed and wrote the manuscript and all the authors revised and approved it.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


HTLV-1 and HTLV-2 are highly related retroviruses that transform T-lymphocytes in cell culture, but display distinct pathobiology in vivo. HTLV-1 is the causative infectious agent of adult T-cell leukemia/lymphoma (ATL) and a neurodegenerative disease (HAM/TSP), whereas HTLV-2 is nonpathogenic. HTLV-1 encodes a protein on the antisense strand of its proviral genome called HTLV-1 basic leucine zipper (bZIP) factor (HBZ), which inhibits Tax-mediated viral transcription and is required for high proviral load and efficient viral persistence. Studies have shown that HBZ also modulates several cellular pathways that include activating protein-1 (AP-1), NF-κB, and innate immune responses. HTLV-2 also encodes a protein on the antisense genome strand named antisense protein of HTLV-2 (APH-2). Like HBZ, APH-2 also inhibits Tax-mediated viral transcription. However, we show that unlike HBZ, loss of APH-2 results in enhanced viral replication and viral persistence in infected rabbits. This led us to hypothesize that HBZ and APH-2 modulate cellular pathways differently, which translates to the distinct HTLV-1 and HTLV-2 pathobiology. In this study we directly compared APH-2 and HBZ biologic properties and functions on known HBZ-modulated pathways. We provide evidence that APH-2 protein is significantly less stable than HBZ protein . Despite the difference in protein half-life, HTLV-2 does not compensate for this instability by increasing APH-2 mRNA copy number. Additionally, APH-2 and HBZ share similar mRNA stability measurements. We further show that APH-2 inhibits the transforming growth factor β (TGF-β) signaling pathway in contrast to HBZ's enhancement. Like HBZ, APH-2 is able to inhibit the cellular transcription factors p65 (NF-κB) and interferon response factor (IRF)-1. Taken together our results indicate that APH-2 is limited in some functions it shares with HBZ. Further studies should focus on distinct HBZ functions and interacting pathways to find new potential therapeutic targets for HTLV-1 disease.


Cucumis sativus L.) abiotic stresses resistance to salt, polyethylene glycol (PEG), cold and the potential mechanisms were investigated in this work. Previous reports have indicated that BRs can induce ethylene production and enhance alternative oxidase (AOX) pathway. The mechanisms whether ethylene is involved as a signal molecule which connected BR with AOX in regulating stress tolerance are still unknown. Here, we found that pretreatment with 1 μM brassinolide  relieved stress-caused oxidative damage in cucumber seedlings and clearly enhanced the capacity of AOX and the ethylene biosynthesis. Furthermore, transcription level of ethylene signaling biosynthesis genes including ripening-related ACC synthase1 (CSACS1), ripening-related ACC synthase2 (CSACS2), ripening-related ACC synthase3 (CSACS3), 1-aminocyclopropane-1-carboxylate oxidase1 (CSACO1), 1-aminocyclopropane-1-carboxylate oxidase2 (CSACO2), and CSAOX were increased after BL treatment. Importantly, the application of the salicylhydroxamic acid  and ethylene biosynthesis inhibitor aminooxyacetic acid (AOA) decreased plant resistance to environmental stress by blocking BRs-induced alternative respiration. Taken together, our results demonstrated that ethylene was involved in BRs-induced AOX activity which played important roles in abiotic stresses tolerance in cucumber seedlings.Effects of brassinosteroids (BRs) on cucumber ( In addition, it played an important role in a variety of plant physiological processes and adaptation to various abiotic and biotic stresses  were surface-sterilized for 15 min in 1% (w/v) NaClO, and then germinated on water-moistened filter paper. The plants were grown in a growth chamber at a 16-h photoperiod (100 μM m−2 s−1), temperature of 25°C/17°C (day/night).Plants growth as described  with 0.1, 0.5, 1, 5, or 10 μM solutions on leaves, while the control seedlings were sprayed with distilled water. Twelve hours after spraying, all the seedlings were exposed to salt (200 mM NaCl), PEG (16% PEG 6000), and cold stresses (at 4°C in a controlled growth chamber with a relative humidity of 70%) for 3 d. The third leaf of cucumber seedlings was used for the following experiments.2O2 scavenger), DPI (an NADPH oxidase inhibitor), aminooxyacetic acid , and ethylene were purchased from Sigma . 1 mM SHAM inhibits the AOX activity, as this concentration is sufficiently low to avoid the possible side effects , dimethylthiourea  was defined as O2 uptake in the presence of both 1 mM KCN and 0.5 mM nPG. The capacity of the cytochrome pathway (Vcyt) and the alternative pathway  were calculated by the formula: Vcyt = Vt–V0; Valt = V0–Vres. The Vres in our experiment was always low, and was negligible relative to other respirations. Therefore, the Vres was not shown.Respiratory oxygen consumption was measured using Clark-type electrodes  as previously described  was measured during the weak measuring pulses, and maximal fluorescence (Fm) was measured by a 0.8 s pulse of light at about 4000 μmol m−2 s−1. An actinic light source was then applied to obtain steady-state fluorescence yield (Fs), after which a second saturation pulse was applied for 0.7 s to obtain light adapted maximum fluorescence  were calculated as Fm– Fo/Fm and . For measurement of Total RNA was extracted from cucumber leaves according to a previously described method  and 3, 3-diaminobenzidine (DAB), respectively, as previously described  solution for 2 h, or DAB (2 mg mL−1) solution for 8 h. Leaves were then decolorized in boiling ethanol (95%) for 20 min.Superoxide and H2O2 content of leaves was measured as described  was tested as previously described  level as described previously  were heated in a boiling water bath for 2 min. The samples were then decolorized in boiling ethanol (95%) for 15 min.Cell death was showed by trypan blue staining. Leaves submerged in trypan blue  containing 0.2 mM EDTA, 2 mM AsA, and 2% PVP. The homogenates were centrifuged at 4°C for 20 min at 12,000 g, and the resulting supernatants were used for the determination of enzymatic activity  are indicators of PSII photochemistry. So, we investigated the function of photosystem II (PSII) under different abiotic stresses treatments. As shown in Figures Fv/Fm showed that compared with control, plants with BL treatment showed an increase in Fv/Fm in all three abiotic stresses treatments. In contrast, the non-photochemical quenching (NPQ) declined in the BL-pretreated seedlings in comparison with control plants under the same stress treatments for 3 d , which suggested the potential crosstalk between BRs and ABA or auxin in stress responses (Choudhary et al., Xia et al.  have pro Figures , 7. Furts Figure . Accordis Figure , we pres2 Figure . BRs may2O2 were involved in BRs-induced AOX activity. Thus, our results reveal a novel role of BRs in plant against environmental stresses and clarify the relationships between ethylene, AOX and ROS during environmental stress, although the detailed mechanism relations need further investigation. Future, investigations should focus on whether BRs could directly induced AOX activity by BR signaling.In brief, suitable concentrations of BL positively affected the tolerance of plant to stress conditions. AOX may play an important role in the plant resistance to environment stresses. Our study showed that both ethylene and HThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Initial success of inhibitors targeting oncogenes is often followed by tumor relapse due to acquired resistance. In addition to mutations in targeted oncogenes, signaling cross-talks among pathways play a vital role in such drug inefficacy. These include activation of compensatory pathways and altered activities of key effectors in other cell survival and growth-associated pathways.p1-model to infer posterior probabilities of gene-pairs in networks derived from the gene expression datasets of ErbB2-positive breast cancer cell-lines . Using this computational framework, we searched for cross-talks between EGFR/ErbB and other signaling pathways from Reactome, KEGG and WikiPathway databases that contribute to lapatinib resistance. We identified 104, 188 and 299 gene-pairs as putative drug-resistant cross-talks, respectively, each comprised of a gene in the EGFR/ErbB signaling pathway and a gene from another signaling pathway, that appear to be interacting in resistant cells but not in parental cells. In 168 of these (distinct) gene-pairs, both of the interacting partners are up-regulated in resistant conditions relative to parental conditions. These gene-pairs are prime candidates for novel cross-talks contributing to lapatinib resistance. They associate EGFR/ErbB signaling with six other signaling pathways: Notch, Wnt, GPCR, hedgehog, insulin receptor/IGF1R and TGF- β receptor signaling. We conducted a literature survey to validate these cross-talks, and found evidence supporting a role for many of them in contributing to drug resistance. We also analyzed an independent study of lapatinib resistance in the BT474 breast cancer cell-line and found the same signaling pathways making cross-talks with the EGFR/ErbB signaling pathway as in the primary dataset.We propose a computational framework using Bayesian modeling to systematically characterize potential cross-talks among breast cancer signaling pathways. We employed a fully Bayesian approach known as the Our results indicate that the activation of compensatory pathways can potentially cause up-regulation of EGFR/ErbB pathway genes (counteracting the inhibiting effect of lapatinib) via signaling cross-talk. Thus, the up-regulated members of these compensatory pathways along with the members of the EGFR/ErbB signaling pathway are interesting as potential targets for designing novel anti-cancer therapeutics.The online version of this article (doi:10.1186/s12918-014-0135-x) contains supplementary material, which is available to authorized users. Cancer development involves a series of events, ranging from tumorigenesis to metastasis, each of which may be caused by perturbations in crucial signal transduction pathways. Recently, drugs (inhibitors) specifically targeting critical components of signaling pathways known to be up-regulated in specific cancers have been used in the clinic. However, success of these inhibitors is limited by the intrinsic potential of cancer cells to acquire drug resistance. Recent advances in both clinical and laboratory research have reported that cancer cells may adopt several mechanisms against particular treatments including adjusting the signaling circuitry, activation of alternative pathways and cross-talks among various pathways to overcome the effects of inhibitors ,2. Resisde novo and acquired  possibilities, we found 10,811 gene-pairs  in our primary dataset, have some overlap with our secondary dataset.To further illustrate our method, we analysed a second dataset GSE16179) containing gene expression profiles of breast cancer cell-line BT474 under two conditions  [179 contae Census , there wThere were 78 genes involved in these sets of 83, 133 and 277 putative cross-talks. We performed a similar Netwalker analyses with these 78 genes as we did for the dataset GSE38376, and found 37 genes  resistance. We hypothesized that gene-pairs  that can potentially cause drug-resistance have a high probability of occurring in the resistant condition(s) but a low probability in parental conditions. We employed a fully Bayesian statistical model: a special class of Exponential Random Graph Model known as the n values  of resisling see .Our results primarily focus on compensatory signaling pathways i.e. Notch signaling, Wnt signaling, GPCR signaling, and IR/IGF1R signaling, which cross-talk with EGFR/ErbB signaling to reduce the inhibiting effect of lapatinib. We present additional literature evidence that the identified cross-talks of the above compensatory signaling pathways with EGFR/ErbB signaling may contribute to drug-resistance by maintaining key cell survival and/or proliferation signals in common down-stream pathways, including PI3K/Akt signaling [et al. [MDM2:STK11 cross-talk between EGFR/ErbB and IGF1R signaling, where STK11  phosphorylates and activates AMPK in absence of glucose [et al. by finding signaling cross-talks between EGFR/ErbB and IGF1R pathways.Komurov et al.  hypotheset al. . Here, b glucose . Again,  glucose . Thus ouμM and 1.0 μM) with those of all the samples in resistant conditions . However, we conducted another experiment on both of our primary  and secondary datasets  in which we first identified genes dysregulated in treatment vs basal conditions in parental samples and then checked if those genes were reversely changed in treatment conditions in resistant samples. To that end, for each sample, first we calculated the fold-change(s) of parental treatment condition(s) compared to parental basal condition, and then we calculated the fold-changes of resistant basal and resistant treatment conditions compared to parental basal condition , and for that particular sample, expressions were reversely changed  in all the resistant treatment conditions , we compared the expression changes of all the samples in parental conditions (basal, 0.1 et al. [Currently, our network modeling only considers undirected edges among genes. In future we would like to generalise the approach to identify directed and indirect interactions among genes. In network modeling, a combination of both direct and indirect relationships among gene-pairs was found to provide better insights into biological systems in our previous studies . The ratet al. . Other hOur proposed computational framework is able to predict putative cross-talks among signaling pathways that play a role in drug resistance in two breast cancer cell-lines, SKBR3 and BT474. Our framework could also be useful for other types of cancer to enhance understanding of the role of signaling cross-talks in drug resistance. Most importantly, we believe our method can be used to find a range of compensatory pathways that nullify/reduce the inhibiting effects of drugs via cross-talk with targeted pathways. These novel compensatory pathways can be further considered as novel targets for single or combination therapies.


This article provides the first detailed account of recent fertility trends in Iraq, with a particular focus on the changes resulting from the 2003–2011 war and the factors underlying them. The study is based on retrospective birth history data from the 2006 and 2011 Iraq Multiple Indicator Cluster Surveys (I-MICS). Estimates from the two surveys indicate that total fertility remained stable from 1997 to 2010, at about 4.5 children per woman. However, examination of the age patterns of fertility reveals an abrupt shift in the timing of births, with adolescent fertility rising by over 30 percent soon after the onset of the war. A decomposition analysis shows that the rise in early childbearing is due to an increased prevalence of early marriage among less-educated women. The prevalence of early marriage and childbearing among women with secondary or higher education is relatively low and has not increased after 2003.


Results: Among 2031 samples, 294 were reactive by screening. Of them, 48 (16.3%) were WB indeterminate and of those 15 (31.3%) were PCR+. Quantitative real-time PCR (qPCR) was performed to 52 HTLV-1+ samples, classified as Group 1 (G1): 25 WB+ samples from individuals with pathologies; Group 2 (G2): 18 WB+ samples from asymptomatic carriers (AC); and Group 3 (G3): 9 seroindeterminate samples from AC. Median PVL was 4.78, 2.38, and 0.15 HTLV-1 copies/100 PBMCs, respectively; a significant difference (p=0.003) was observed. Age and sex were associated with PVL in G1 and G2, respectively. Mutations in the distal and central regions of Tax Responsive Elements (TRE) 1 and 2 of G3 were observed, though not associated with PVL.The 8403A>G mutation of the distal region, previously related to high PVL, was absent in G3 but present in 50% of WB+ samples (p = 0.03). Conclusions: indeterminate WB results confirmed later as HTLV-1 positive may be associated with low PVL levels. Mutations in LTR and tax are described;  their functional relevance remains to be determined.Background: indeterminate Western blot (WB) patterns are a major concern for diagnosis of human T-cell lymphotropic virus type 1 (HTLV-1) infection, even in non-endemic areas. Objectives: (a) to define the prevalence of indeterminate WB among different populations from Argentina; (b) to evaluate if low proviral load (PVL) is associated with indeterminate WB profiles; and (c) to describe mutations in LTR and  Human T-cell lymphotropicvirus type 1 and 2 (HTLV-1/2) are distributed worldwide. HTLV-1 infects an estimated 20 million people in the world and is considered the etiologic agent of adult T-cell leukemia/lymphoma (ATLL), HTLV-associated myelopathy/tropical spastic paraparesis (HAM/TSP), and HTLV-1 uveitis ,4,5. In . According to the current algorithm, a serological confirmation, usually by Western blot (WB), should be performed after reactive screening results . As shown in p = 0.07) was observed when samples from acute leukemia  were compared with HAM/TSP ones (n = 21).Individual PVL values are shown in Regarding WB indeterminate samples, all successfully detected PVLs (8/9) were lower than 1 HTLV-1 copy/100 cells. Concerning G1 and G2 samples, an overlap in the range of PVL values was observed . In threp = 0.01) (n = 6/7), when compared with plasma samples from G1 and G2 selected randomly.A moderate correlation (S=0.56) between PVL values and age at the moment of the sample extraction was observed in G1, which included patients with pathology who were not in treatment. Meanwhile, there was a significant association between PVL and gender in G2 ( = 0.01) . No corrn=4; G2: n = 4; G3: n = 6); three G3 sequences could not be obtained. Cosmopolitan subtype HTLV-1a was clearly separated from HTLV-1 subtypes b, c, d, e, f, and g, with a bootstrap value of 69%. Within the cosmopolitan subtype, five subgroups were identified as previously described *100 and expressed as the number of HTLV-1 copies/100 cells.Absolute quantitation of PVL was performed by real-time SYBR Green PCR, using an ABI Prism 7500Prism System .The HTLV-1 MT2 cell . All staas >0.99 . The acctax genes. Amplification of the 3’ LTR region was performed using 8200LA (5’-CTCACACGGCCTCATACAGTACTC -3’) and R2 (5’-GTGCTATAGGATGGGCTGTCGC-3’) as outer primers and 3VINT (5’-GAACGCRACTCAACCGGCRYGGATGG-3’) and 3LTRf (5’-TCCCCATTTCTCTATTTTTAACG-3’) as inner primers . Amplification of the tax region was carried out with outer primers HFL75 (5’-GCTATAGTCTCCTCCCCCTGC-3’) and 3VINT (5’-GAACGCRACTCAACCGGCRYGGATGG-3’) and inner primers TaxF (5’-ATGGCCCACTTCCCAGGGTT-3’) and TaxR (5’-TCAGACTTCTGTTTCTCGGA-3’), specific for HTLV-1. A slight modification was made to the protocols described elsewhere, in order to amplify tax and LTR genes in seroindeterminate samples by enhancing the DNA amount ..i.e., thThere are few reports suggesting that low HTLV-1/2 PVL could cause indeterminate WB patterns in samples from infected individuals ,34. Regaet al. observed no significant association between PVL in HAM/TSP patients and healthy carriers with age and gender, it has been reported that for both HTLV-1 and 2 infections, women have lower PVL levels than men, consistent with our results [et al. showed no gender differences in PVL [et al. reported in 1999 that the anti-HTLV-1 antibody titer had a positive correlation with PVL levels, and years later, Akimoto et al. confirmed these results [et al., the optical density values obtained were lower for most of the seroindeterminate profile plasma samples when compared to HTLV-1 WB-positive ones [Regarding age and gender, it was determined that these variables were associated with PVL levels among G1 and G2, respectively. While Vakili  results ,39,40. Is in PVL . Thus, f results ,43. In tive ones .As for phylogeny, all 14 new strains described in this study belonged to the cosmopolitan subtype and most of them classified within the transcontinental subgroup A (one sample classified as divergent). Half of the G3 sequences did not group in any specific cluster, while two grouped in the small Latin American cluster  and one in the South African cluster (FSW7), together with other sequences previously reported by our group (FSW1 and BD1) . These dtax genes were linked to geographical subtypes, others were further analyzed in order to establish their relevance, as it is well known that during replication and transcription the LTR/Tax system is extremely important. The promoter region LTR responds to the transactivation mediated by the Tax protein, which directly interacts by binding the DNA or indirectly by binding cellular transcription factors, in the Tax regulation elements known as TRE-1 and TRE-2 [Even though some of the observed mutations in LTR and nd TRE-2 . Althougnd TRE-2 ,47, consnd TRE-2 . Interesnd TRE-2 . Regardind TRE-2 . Whetheret al. have recommended different strategies for reducing costs and improving the accuracy of the diagnosis [et al. reported the utility of digital droplet PCR (ddPCR) in the quantitation of HTLV-1 PVL [Considering diagnosis, indeterminate WB results cannot be avoided without an improvement of serological commercial kits aimed at enhancing sensibility and specificity. Thus, and taking into account the high prevalence of these seroindeterminate cases worldwide ,11,49, tiagnosis ,50. Whiliagnosis . Neverthiagnosis ,51. In tLV-1 PVL . They poLV-1 PVL . On the LV-1 PVL . Based oTherefore, we propose to re-evaluate the diagnostic algorithm, considering molecular confirmation by n-PCR for reactive samples, instead of qPCR, right after the combination of two screening tests. This alternative would avoid serological confirmation by WB, the most expensive stage of the diagnosis algorithm, until a better confirmation technique is available and standardized. Further studies with significant panels including HTLV-1, HTLV-2, and indeterminate samples should be carried out in order to establish whether it is time saving, effective, and less expensive.tax have been described among both indeterminate and positive HTLV-1 cases, highlighting 8403A>G in the distal region of TRE-1, already related to high PVL. Still, the functional relevance of these mutations remains to be determined.This study describes the prevalence of indeterminate WB patterns among different populations from Argentina and demonstrates that in some cases these profiles may be associated with low HTLV-1 PVL. Mutations in LTR and


Recently, a study by Morgan et al. identified that the white matter lesions are detectable with MRI study in HTLV-1 carriers at the same frequency as HAM/TSP patients. Little is known about the nature of the brain lesion and its relation to the spinal cord lesion. We assessed the inflammatory change including HTLV-1-specific cytotoxic T lymphocytes in the brain lesion of the patient with HAM/TSP, in the spinal cord of HTLV-1 carrier immunohistochemically. Fresh frozen samples including one brain from the patient with HAM/TSP and four spinal cords from HTLV-1 carriers were for use in this study. Inflammatory cells in the sections of the brain and spinal cords were stained with anti-CD4, anti-CD8 antibodies. HTLV-1-specific CTLs were detected with either phycoerythrin (PE)-conjugated HLA-A*0201/Tax11-19 tetramer or HLA-A*2402/Tax301-309 tetramer, and followed by rabbit anti-PE antibody as the secondary antibody. The signals from the third antibody with fluorescence were captured by confocal laser scanning microscopy (CLSM). HTLV-1-specific CTLs were frequently detected in the brain lesion of the patient with HAM/TSP. The CTLs were also detected in all the spinal cords of four HTLV-1 carriers. Interestingly, inflamed lesions of HTLV-1 carrier were distributed mainly in the lower thoracic level of the spinal cords like the spinal cord of HAM/TSP. These results suggest that inflammatory changes occur simultaneously in the spinal cord and the brain of the patients with HAM/TSP, and that latent inflammatory change may occur in asymptomatic HTLV-1 carriers.


In this paper, we aim to assess the extent to which individual-level completed fertility varies across contexts characterized by policies supporting different gender division of labor models. We examine key labor market and care policies that shape gender relations in households and in the public domain. We also consider the role of gender norms, which can act as both a moderator and a confounding factor for policy effects. We hypothesize that, by facilitating role compatibility and reducing the gendered costs of childrearing, policies that support gender equality lead to an increase in fertility levels and to a reduction in fertility differentials by the level of education. Using individual-level data from the European Union Survey on Income and Living Conditions for 16 countries, combined with country-level data, we analyze completed fertility through multilevel Poisson’s models. We find that the national level of childcare coverage is positively associated with fertility. Family allowances, prevalence of women’s part-time employment and length of paid leaves were also found to be positively associated with completed fertility, though the associations were not statistically significant. These variables show a significant positive pattern according to education. A high number of average working hours for men are negatively associated with completed fertility and show a strong negative pattern by educational level. The prevalence of gender-egalitarian norms is highly predictive of fertility levels, yet we found no consistent evidence of a weaker association of gender-equality policies in countries where egalitarian values are less prevalent. Gender systems are increasingly recognized as a critical component in explanations of fertility levels and differentials, in both advanced and less economically advanced societies  for women between ages 36 and 44 during the years 2004–2009. Our empirical analyses focus on 16 Western and Southern European countries showing a variety of fertility levels and policy patterns. Lack of comparable contextual data precluded us from including several Central and Eastern European countries in the analyses.We apply multilevel regression models to explain completed fertility. Our focus on completed fertility is due to its high political and scientific relevance (despite the fact that it is seldom studied), but timing and parity differentials could not be investigated in this paper due to space and data limitations. It should be taken into account that data limitations prevent us from making strong causal statements, and thus, we examine whether different policy and normative contexts are associated with fertility outcomes at the individual level. As we discuss later in the paper, our findings are highly consistent with theories relating gender relations and fertility levels. Furthermore, our results suggest that, in the context of low fertility societies, welfare state support to gender equality is a crucial factor for a recovery of fertility levels.Perhaps the most compelling and influential arguments linking gender relations to fertility are those made by McDonald , b, 2006laissez-faire state policy characterize liberal regimes. Conservative regimes boost the principles of status segmentation and familism .McDonald’s theory links fertility levels and patterns with the functioning of welfare regime institutions. The welfare regime approach provides a suitable framework for understanding relationships between institutions and is the central theoretical framework for international comparative social-policy research  parental leaves and the expansion of formal childcare provision. The goal has often been to accommodate or promote women’s employment, resulting in the creation of an adult worker model for both genders Orloff . As timeIn the third stage, institutional adaptation begins to focus more on men’s roles. Men take an increasing responsibility for care and domestic work, and their labor market involvement starts to change, for instance, by increasing their use of paternity leave. Policies supporting these changes include the promotion of fathers’ leaves and adapting the work environment to the growing caring role of men—such as shorter working hours or more flexible time schedules. Even if gender-equality values are highly prevalent, it is not yet clear what economic or social incentives men may have to adopt what have traditionally been women’s roles . No society has reached this stage yet. Increasing rates of female employment have narrowed the gender gap in labor force participation, but gender differences persist in such areas as career breaks, occupational segregation, working hours or pay. And although men’s engagement in domestic work and caregiving has increased in many countries, nowhere does it match women’s influx into paid employment. In reality, the stages overlap, and the timing of changes is likely to be particularly important for fertility outcomes. If change comes late but very rapidly, as in Southern Europe, this might provoke very low fertility. If institutions begin to adapt early, as in Scandinavia, then a higher fertility level is easier to sustain.public policies that support egalitarian gender relations have an overall positive effect on fertility (Hypothesis 1). Conversely, policies centered on the male breadwinner model are likely to be counter productive for rising fertility levels. In order to empirically examine this hypothesis, we use several indicators of policies that are expected to reflect key dimensions of the effect of the gender system on fertility . In particular, we focus on “work-family reconciliation” polices, for both men and women, in the areas of working-time regulations and childcare (including policies that provide time or money to care). Of course, we acknowledge that many other polices have a potential impact on fertility or on gender relations. For instance, health and education subsidies decrease the cost of children for parents, and elderly care policies may influence women’s involvement in paid work, etc. Nevertheless, here we focus on polices that directly influence the gender division of labor for parents, which is considered a critical element by the theoretical arguments cited above.In the theoretical framework sketched above, gender-equality polices have the role of providing concrete resources and opportunities to women and families as well as the role of influencing norms in a society. These policies mitigate the gendered costs of childbearing by supporting particular models of families and gender relations. Thus, the institutional context, including policies, helps to determine the structure of costs and rewards of fertility. To the extent that particular policies reduce the direct and especially the indirect economic costs of childbearing, they can have a positive effect on fertility  more than others. In particular, the decline in women’s status that is often observed upon the birth of a child is closely related to her employment situation and educational level. The economic and psychic costs of having children tend to be particularly high for highly educated women if the combination of parenthood and paid work is difficult. As a consequence, fertility drops are generally severe for them in the early stages of the gender transition, while educational differentials tend to disappear, or even reverse, in more advanced stages. Opportunity costs, i.e., the loss of income related to time out of the labor force to care for children, have been rightly highlighted in the literature, while the direct economic costs seem to be relatively less important for women with high market productivity . This logic is applied to specific contextual factors or policies, to the extent that they give support to a particular gender arrangement. For example, childcare services favor the combination of work and parenthood and therefore should have a generally positive effect on fertility, following our first hypothesis. Additionally, we hypothesize that the availability of formal childcare should have a stronger positive effect on the fertility of highly educated women relative to the low-educated women. Improved access to childcare means that wage-dependant opportunity costs are substituted by childcare costs, which are generally less dependent on income  tend to negatively affect labor force attachment. Additionally, short leaves have been associated with poorer health and developmental outcomes for children , i.e., in statistical terms, we expect a positive interaction between policies and macro-level gender equality. By the same token, policy measures that conflict with a gender-equality model or favor gender role specialization, such as short-paid parental leaves, certain types of family benefits, or even part-time work, could be negatively associated with fertility levels when combined with a high proportion of adherence to gender-egalitarian norms in the population.If the effects of policies can vary according to the prevailing We use the EU-SILC longitudinal data for the years 2004–2009 for 16 Western and Southern European countries for which we also have contextual information. We restrict our analyses to the most recent observation of women aged 36–44 years, yielding a working sample of 69,213 women . The dependent variable used in all the analyses is the total number of own children living in the same household as the mother at the time of interview. This variable approximates the completed fertility of women. In Table The explanatory variables include, firstly, the respondent’s age at the time of the survey and the highest educational level attained. We have coded education into three categories: some secondary education and less, completed secondary and/or post-secondary non-tertiary education (the reference category that includes the range from upper secondary studies to post-secondary education that cannot be regarded as university-level studies) and university studies or higher.We complement the micro-level data with country-level data on policies, labor market conditions and norms. In particular, we consider data on family benefits, leaves, availability of formal childcare, labor market conditions and the prevalence of gender-equitable attitudes (as an indicator of norms). We use contextual information reflecting the situation in the mid-1990s 1992–1998), when most of the women in our sample had their children. When data are not available for this period, we consider the oldest data available. In any case, variations in country-specific indicators over time are very limited. The values of our macro-level indicators are reported in Table –1998, whfamily allowances), years 1992–1998. Values are divided by 1000 when using the variable in the regression models.Monthly family allowances in US dollars (PPP adjusted) for the second child , years 1992–1998.Sum of weeks of maternity, paternity and childcare leave weighted by the level of cash benefits paid during each type of leave , year 2004.Number of places in public (or publicly subsidized) childcare facilities as a share of the number of children aged 0–2 years , year 2004.Children aged 0–2 years cared for in formal childcare arrangements as a percentage of all children in the same age group , years 1992–1998.Standard number of working hours per week for men (women on part-time), years 1992–1998. Part-time is defined as less than 30-week-usual hours worked in the main job.Share of women working part-time out of the total number of employed women  and the European Values Study (EVS). We include data from the oldest wave for which our indicator is available (1999). Our measure of gender-egalitarian norms is based on a widely used item in gender studies , we employ Poisson’s regression models to estimate the association between individual and contextual independent variables and fertility.ij indicates a woman belonging to country j, yij denotes the responses , xij represents the independent variables with (fixed) coefficients β and uj represents country-level random effects (“random intercepts”). For each country, uj is a realization from a normal variable with mean 0 and standard deviation to be estimated. Independent variables can be included at the both individual and country level. Multilevel models can handle unbalanced data structures characterized by different cluster sizes  as a function of a set of covariates and random effects:One limitation for the application of a multilevel model in our case is the limited number of countries available in our dataset (16). In fact, multilevel modeling requires a sufficient sample size at each level of the hierarchy. Simulation studies, as summarized in the literature review by McNeish and Stapleton , have shmeqrpoisson in Stata13. In all models considered, we controlled for age and education. Education was entered as an individual-level covariate. To take into account that the amount of exposure time over which the dependent variable is observed for each women in the sample varies (our sample include women aged 36–44 years), we included the variable age in the exposure option. In this way, the estimates are adjusted for the length of time a woman was at risk of having children even though the coefficient of the variable age is not estimated and therefore will not be reported in the tables [log(age) is included in the fixed-effects portion of the model with the coefficient constrained to be 1]. Using age as a covariate yielded results similar to those presented here.All models have been estimated using the command To test our hypotheses, the country-level indicators described in the previous section are also considered as independent variables together with individual controls. A key advantage of using multilevel models in our analysis is that they recognize that the relevant sample size at the country level for estimating coefficients of the country-level variables is the number of countries in the dataset and not the overall sample size. However, having a low number of countries implies that we have limited degrees of freedom at the country level, limiting the number of country-level variables we can simultaneously enter into the model. Moreover, our country-level variables show a quite high degree of correlation . In Table p < 0.01), which is consistent with Hypothesis 1 for this variable. Moreover, we found a significant interaction between childcare coverage and education levels in the direction predicted by Hypothesis 2. In fact, Model 3 in Table p < 0.001) and weaker for the lower-educated , as compared with women with a medium level of education.Starting with formal childcare coverage for children under 3, an overall significant positive association with fertility is found . Overall, the results provide no evidence that gender policies are more effective if egalitarian norms are highly prevalent in the population Table . This maUsing data from the EU-SILC database for sixteen European countries, we have analyzed the relationship between gender policies and individual-level completed fertility. Our results provide evidence in two areas: First, policies that support particular gender relations models are associated with fertility and, second, these associations are strongly heterogeneous in the population by the level of education.Framing the analyses in the welfare regime perspective has been useful in identifying key policies that shape gender relations and understand their relative role in the institutional context. This perspective focuses on how regulations and welfare provision affect the functioning of each institution, thus avoiding partial views restricted to welfare provision only (services or cash). Our results suggest that policies that promote de-familialization of care, via direct provision or by creating the conditions for market provision, are powerfully associated with higher fertility. A universal day care policy is likely to be a pre-condition for gender equality by reducing the total amount of care done by families and favoring a more gender-equal sharing of care and wage work.Labor market policies that support a familialization of care offer less clear-cut results. Policy strategies centered on women’s primary care responsibility, such as part-time work, maternity leave or monetary support, show a weaker relationship with fertility. Welfare state support to family care, even if they are temporary in women’s life courses, still implies gendered costs of fertility. On the other hand, a reduction in men’s standard working hours is associated with a sharp increase in completed fertility, suggesting that working-time regulations have an important role in men’s involvement in care. Institutional contexts and policies that provide support to a dual-earner/dual-carer model are positively related to a higher fertility level, with respect to policies that maintain a preferential care role for women. These results also highlight that existing policies support a variety of gender relations, which cannot be reduced to a continuum between a male breadwinner–housewife model and a dual-earner/dual-carer model.In trying to discern the relationship between public policies and fertility, we should pay attention to the existence of differentials in these associations by population subgroup and the mechanisms involved in explaining such differentials. Here we have hypothesized that the higher the level of gender-equality prevalent in the welfare regime institutions, the less a woman’s educational level will be negatively associated with fertility. The results of the analyses of the differential association between policies and fertility according to the level of education show theoretically consistent and statistically significant patterns. A positive association between education and fertility was found for all of the indicators analyzed, which were particularly strong for formal childcare, men’s standard working hours and gender norms. However, contrary to our expectation, we find no evidence that a high prevalence of gender-egalitarian norms in the population enhances the fertility impact of policies. This result suggests that policies and norms are closely interrelated and that no strong inconsistencies exist in the countries and cohorts studied . Most countries seem to be in a stage of the gender transition in which the breadwinner model has been modified to accommodate women’s labor market participation, but have not yet accommodated men’s caring role.Overall, our findings are quite consistent with our theoretical expectations about the impact of gender policies on fertility. Of course, our data had several drawbacks, concerning in particular the suitability of the indicators available and the time lag between the measurement of the context indicators and completed fertility. The cross-sectional nature of the data used implies that no close relationship could be made between changes in polices and changes in fertility behavior. This approach involves the assumptions that our contextual variables do not change and that they apply to the moment when women (or couples) decide whether to have a child. As we note in the section on data, the policies included in the study are highly stable for the period considered. Furthermore, our choice of analytical approach precluded us from undertaking an analysis of the timing of births and involved that no distinctions between different parities could be made. Some studies have suggested that first births are influenced by policies to a lesser extent than subsequent births (Harknett et al. Analyzing policy indicators one by one has provided clear-cut results, in particular concerning their interactions with education. However, the high correlation between policy indicators suggests caution in interpreting each of their individual effects in causal terms, since they are likely to be overestimated. An alternative empirical strategy focusing on the effects of specific constellations of variables would necessitate analyses of the situations of particular countries, which was beyond the scope of our study.In spite of these limitations, our analyses reveal that, depending on the policy environment, fertility differentials reached about half a child, most notably when educational groups were considered. This is the order of magnitude of the existing gap between countries with very low levels and those with sustainable fertility levels. Although all educational groups seem to benefit from policies enhancing gender equality, the highly educated benefits more. The rapid increase in women’s educational levels observed in many European countries suggests a parallel increase in the demand for gender-egalitarian polices, which is consistent with economic considerations and with the higher prevalence of gender-egalitarian values among the highly educated. The diffusion of egalitarian behavior from the highly educated to the less educated could reinforce this trend. Furthermore, gender-egalitarian policies provide an explanation for the convergence in fertility levels that has been observed across educational levels in several countries. Following our arguments, this convergence can be expected to continue. As we have shown, it is a process that is fueled by policies that support the gender transition.


Many extracellular and intracellular signals promote the c-Abl tyrosine kinase activity. c-Abl in turn triggers a multitude of changes either in protein phosphorylation or in gene expression in the cell. Yet, c-Abl takes part in diverse signaling routes because of several domains linked to its catalytic core. Complex conformational changes turn on and off its kinase activity. These changes affect surface features of the c-Abl kinase and likely its capability to bind actin and/or DNA. Two specific inhibitors  regulate the c-Abl kinase through different mechanisms. NMR studies show that a c-Abl fragment (SH3–SH2-linker–SH1) adopts different conformational states upon binding to each inhibitor. This supports an unconventional use for allosteric compounds to unraveling physiological c-Abl signaling circuits. Caenorhabditis elegans; Proteomics has revealed a rather complex picture underlying cellular signaling circuits. Recent studies have indicated an extensive overlap between diverse cellular responses. DNA damage-induced stress response overlaps with pathogen infection response and with heat stress in intact animals  are crucial for the network. Colocalization of the binding partners converts protein interactions into functional consequences . CrosstaPosttranslational modifications are dynamic and necessary for assembling a temporary platform of local signaling circuits. PTMs work as an allosteric device both for the modified protein and for the assembly/(or reshaping?) of multiprotein complexes. Enzymes are often “switchable,” with their activities controlled by many targets/effectors. The tyrosine kinases are themselves regulated by phosphorylation through various allosteric mechanisms. Non-receptor tyrosine kinases (RTKs) have a conserved catalytic domain (kinase domain = SH1), auto-inhibited by the binding of regulatory domains . Such an allosteric regulation links the enzymatic activity of the kinase with the colocalization of its substrate. Allosteric auto-regulation seems to be a recurring feature in cell signaling . ProteinNon-receptor Abl tyrosine kinases regulate a diverse range of cellular signaling paths. Recent reviews have discussed both the biological functions of the mammalian c-Abl tyrosine kinase  and the Here, I will recall the mechanisms of c-Abl auto-inhibition. The purpose is to highlight the effects of small molecule inhibitors on the conformation of c-Abl.Drosophila, D-abl signaling is linked to actin dynamics and cell adhesion  and Abl2 (Arg). Both kinases have redundant and unique roles due to their conserved sequence/domain structures. c-Abl and Arg have two different variants (1a and 1b). Both variants are ubiquitously expressed. Abl kinases share a conserved assembly of amino-terminal regulatory and catalytic domain (SH3–SH2-linker–SH1 domain). At the carboxyl terminus region, Abl kinases contain a filamentous (F)-actin-binding domain ABD; . c-Abl aoutcomes . These routcomes . Likely The auto-inhibited conformation of c-Abl is controlled through SH3–SH2-linker unit as in the Src family tyrosine kinases. In c-Src, the SH2 domain interacts with the C-terminal tail phosphotyrosine residue (Y527). By contrast, in c-Abl, the SH2 domain interacts more intimately with the large C-terminal lobe of the kinase domain SH1). Interestingly, the tight interactions of the SH2–SH1 domain are induced by the binding of the myristoylated residues of the N-terminal region into a hydrophobic pocket of the kinase  of a c-Abl fragment (SH3–SH2-linker–SH1 domains) provide the first structural information of apo form of c-Abl in the absence of small molecule inhibitors . The apoper se a signaling circuitry.It has been proposed that the ABD may stabilize the auto-inhibited conformation of the kinase by binding to F-actin . InteresAn unproductive conformation (“open” inactive state) of the catalytic site induced by Imatinib may promote the availability of the regulatory domains (SH3–SH2-linker–ABD) with profound effects on c-Abl-interactome.Molecular switches like c-Abl have modular domains required for their assembly into multiprotein complexes. Yet, c-Abl has also a modifier/kinase domain to regulate scaffold/signaling dynamics. The challenge now is to understand how such a complex signaling assembly is regulated in time and space . Of noteThe c-Abl kinase was early discovered as the oncogene in the Abelson murine leukemia virus  and thenCell signaling relies on PTMs for its regulation. The interplay and the crosstalk between phosphorylation and ubiquitination represent a recurrent theme in cell signaling . We haveCompelling evidence indicates that c-Abl modulates the degradation of two proteins implicated in the pathogenesis of Parkinson’s disease . A speciin vivo supports such a model (Compelling evidence indicates the physiological relevance of the interface between c-Abl signaling and stress response, metabolic regulation mediated by transcription factors . A small a model . c-Abl i a model , in line a model . Surely,The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Work completed during the strength training session was completed with equipment that had an integrated linear force transducer . During the placebo trials concentric mean power significantly (p < 0.05) decreased from sets 1–6. Accumulated power output during the AOX HTS was 6746 ± 5.9 W which was significantly greater compared to the placebo HTS of 6493 ± 17.1 W . Plasma growth hormone (GH) concentration was significantly less immediately following AOX supplementation . This study demonstrates ingestion of an AOX cocktail prior to a single bout of resistance training improved muscle contractile performance and modulated the GH response following completion of the resistance exercise. Future studies should explore the mechanisms associated with the performance modification and specific muscle adaptations to AOX supplementation in conjunction with heavy RT.Antioxidant supplementation is known to increase human endogenous antioxidant (AOX) capacity providing a means of blunting exercise induced reactive oxygen species (ROS). The purpose of this study was to compare the effects of a single acute dose of an AOX (vs blinded placebo) on muscle contractile performance and hormonal responses to a single bout of lower limb ‘hypertrophic’ resistance training (RT). Fifteen resistance trained subjects (age 23 ± 4 years: body mass 86 ± 6 kg) volunteered to participate in the study. Each subject attended the laboratory on three occasions, firstly to determine three repetition maximum (3-RM) isotonic strength in the back squat and perform a familiarisation of the experimental task. On the second/third visits subjects completed the hypertrophic training session (HTS) which consisted of six sets of 10 repetitions of 70% of a predicted 1 RM load (kg). Four hours prior to the HTS the subjects consumed 2 ml#x2219;kg Reactive oxygen species (ROS) have been implicated as one of the causes of skeletal muscle fatigue during both aerobic and anaerobic exercise . AlthougTo maximise gains in muscular hypertrophy an RT session would typically involve exercising at a moderate intensity, defined as lifting a load between 65-85% of an individual’s one repetition maximum (RM), and using a high volume, typically 3–6 sets of 6–15 repetitions of the exercise . GoldfarIt has been suggested that a supplementation regime of antioxidants could reinforce the body’s endogenous antioxidant system providing a means of blunting exercise induced ROS molecules ,16. SeveMost AOX studies have focused on the effects of vitamin C and/or E supplementation to attenuate the oxidative stress caused by RT ,18-20. TFifteen healthy subjects volunteered to participate in the study (age 23 ± 4 yr: body mass 86 ± 6 kg: height 179.4 ± 6.1 cm). Each subject had been resistance training for a minimum of 2 yr prior to recruitment for the study. All the subjects were familiar with the back squat exercise (BS) and could perform the activity satisfactorily from a technique perspective. Assessment was carried out by the primary researcher who was a certified strength and conditioning coach. Each subject completed a consent form and pre activity screening questionnaire to identify any musculoskeletal and orthopaedic problems that could affect performance of the exercise. The study gained approval from the University of New South Wales, Human Research Ethics Committee.A double blind repeated measures design was employed where the subjects ingested either the AOX treatment or a placebo version prior to completing the training session. In the 48 h leading up to these sessions the subjects were instructed to refrain from intense physical exercise in order to eliminate residual fatigue. The supplements were provided using a randomized and counterbalanced design. The subjects visited the laboratory on three occasions, firstly to record their physical characteristics and determine their 3 RM BS which was used to predict 1RM strength [1.06 × 3RM (kg) ]. On the1 for five min. The determination of the 3RM was followed according to methods previously described  containing 2.4 g#x2219;L of PYC in a randomised order. The placebo and AOX mixtures tasted and appeared the same. The participants and researchers were not aware of which substance was supplement or placebo until after the completion of the study when details were released by an independent person. The placebo mixture contained the exact energy content (315 KJ) and constituents  as the Lactaway© mixture (315 KJ) but it did not include any of the active AOX ingredient PYC.Four hours prior to the HTS the subjects consumed 2 ml#x2219;kgAfter a brief cycling warm up, the subjects completed a warm up set consisting of 10 repetitions at 50% of the actual load to be used during the work sets. After a two min rest period the subjects performed the second warm up set at 80% of the load to be used during the work sets. After a three min rest period, subjects completed six sets, separated by 2 min rest periods. The subjects were instructed to lower the barbell under control (eccentric) and then verbally encouraged to “drive” the barbell upwards in as short as time possible (concentric). The squat training session lasted ~18 min. After the completion of each set the subjects were also asked their rate of perceived exertion (RPE) using the Borg scale . Five miAn integrated linear force transducer  was used to determine barbell displacement for each repetition and set completed. This system allows for the determination of concentric mean power (W), and concentric velocity (m·s) to be determined. The system was set up according to the manufacturer’s guidelines and has been shown to provide a reliable (Coefficients of variation (CV) = 3.3%) and valid estimate of power during resistance training .Venous blood was withdrawn via venepuncture before, immediately after and twenty min after the HTS. Blood was collected from a vein in the cubital fossa in ethylenediaminetetraacetic acid (EDTA) (10 ml tube) vacutainers . The samples were then centrifuged at 3000 rpm for 10 min, at 4°C. The plasma top layer was placed into Eppendorf tubes  and snapped frozen and stored at −80°C until analysis.Plasma GH, an indicator of the anabolic hormonal milieu during RT  was detePlasma cortisol (CORT) was measured as an indicator of the catabolic hormonal environment during RT , and wasXanthine oxidase (XO) was measured because it is involved in free radical production and its elevation contributes to oxidative stress ,13. The d[A two way repeated measures analysis of variance (ANOVA) was used to evaluate changes over time and condition for power and velocity along with lactate, RPE, GH, CORT and XO. If a significant F value was achieved the Bonferroni post hoc test was performed. The level of significance was set at p ≤ 0.05. All data was analysed using SPSS for Windows version 16. Data are presented as mean ± standard error of the mean (SEM). Where relevant effect size ratios (ES’r) were calculated using Cohens d. An ES’rlac) increased significantly after both AOX supplementation; 1.23 ± 0.08 to 7.68 ± 3.01 mmol.l−1 (p < 0.05) and placebo supplementation; 1.79 ± 0.30 mmol.l−1 to 8.11 ± 2.98 mmol.l−1 (p < 0.05). Blood lactate continued to be significantly elevated twenty min post-exercise for both groups, but there was no significant difference in Blac levels between the two conditions at any time point (p > 0.05). The RPE was significantly increased in both groups for sets three to six compared to set one. There were however no significant differences in RPE between the AOX and placebo conditions at any point during the HTS (p < 0.05).The pre to post HTS, blood lactate concentrations . Similarly average velocity during the AOX was higher compared to placebo. Accumulated power output during the AOX HTS was 6746 ± 5.9 W which was significantly greater compared to the placebo HTS of 6493 ± 17.1 W .The concentric mean power and velocity are presented in Figures .ml−1) and AOX condition’s . The difference between the two conditions was not statistically significant (p > 0.05). Circulating GH levels increased significantly after both trials, however the increase was significantly less immediately following AOX supplementation; 6.65 ± 1.84 ng#x2219;ml−1 compared to the placebo trials;16.08 ± 2.78 ng#x2219;ml−1 . GH continued to be significantly elevated 20 min after the HTS for both treatments, and was still significantly greater following the placebo trial in comparison to the AOX trial (p < 0.05) . Cortisol was still significantly elevated 20 min post exercise for both treatments (p < 0.05) however there was no significant difference between the AOX and placebo HTS at any time point (p < 0.05).The HTS resulted in a significantly elevated XO in both the placebo , and generate greater mean concentric power and velocity throughout the HTS after consuming the AOX mixture. An additional aim was to establish the physiological, endocrine and oxidative stress response to a HTS. There were no significant differences between RPE, Bntensity ,37.The attenuation of power decrement during the AOX trial agrees with previous studies that have shown that PYC AOX supplementation improves high intensity cycling and running performance ,29. ThisThe present study also found the oxidative stress response as measured by the marker XO was significantly increased after the HTS following both the placebo and AOX trials. This is similar to other studies which also observed an elevated XO response following strenuous exercise ,39. The p = 0.069). There was also no difference in blood lactate concentration between the two conditions suggesting that differences in anaerobic fatigue were not the cause for the disparity in performance. This data suggests other mechanisms of muscular fatigue may have been involved in the performance changes observed. One possible mechanism is a decrease in Na+/K + ATPase pump activity [Yet, there was no significant difference in XO levels between the placebo and AOX trials, although a slight trend towards a reduction in XO following the AOX trials was observed  was not significantly reduced in the AOX treatment, as hypothesised. Future studies should incorporate further measures of oxidative stress, particularly GSH, and muscle blood flow which may help determine the biochemical and physiological mechanisms that led to the results in this study. Furthermore, GH secretion was significantly attenuated in the AOX trial compared to the placebo. The mechanisms that led to these results are not fully understood, but further research is required as GH secretion is involved in MH and strength development and its attenuation may negatively impact training adaptations.The authors declare they have no competing interests.DB and LRM conceived the concept for the investigation and contributed significantly to the drafting of the manuscript. JA was primary investigator in this study conducted the majority of testing and biochemical analysis. TC and DB assisted in data collection and provided a significant contribution to composition and review of the manuscript. All authors read and approved the final manuscript.


Within a few years of the discovery of Toll-like Receptors (TLRs) and their role in innate immunity, viral and bacterial proteins were recognized to antagonize TLR signal transduction. Since then, as TLR signaling networks were unraveled, microbial systems have been discovered that target nearly every component within these pathways. However, recent findings as well as some notable exceptions promote the idea that more of these systems have yet to be discovered. For example, we know very little about microbial systems for directly targeting non-cytoplasmic portions of TLR signaling pathways, i.e. the ligand interacting portions of the receptor itself. In this review, we compare and contrast strategies by which bacteria and viruses antagonize TLR signaling networks to identify potential areas for future research. Innate immunity is mediated by specialized proteins called pattern recognition receptors that sense microbial invaders and guide our immune systems to eradicate infections. These receptors detect pathogen associated molecular patterns (PAMPs), which are structures common to many microbial species, such as viral nucleic acids or bacterial lipopolysaccharides (LPS). The first identified and most studied group of these receptors, Toll-like-Receptors (TLRs), are displayed at the cell surface and within endosomal compartments where they act as molecular sentinels to detect invading microbes. Upon recognition of a pathogen, in either infected or non-infected cells, TLRs initiate a signaling cascade that leads to expression and release of pro-inflammatory cytokines, chemokines, and Type-I interferons. Cytokine and interferon expression leads to recruitment and activation of immune cells to promote clearance of the infectious agent, but also stimulates expression of genes to block entry and/or replication of microbes. This elegant system is adapted to detect and eliminate almost any threat. However, pathogenic microbes are masters of evading host innate immunity and have evolved a multitude of mechanisms for preventing the antimicrobial activities of TLR signaling pathways. Microbial manipulation of host TLR signaling comes in the form of three main strategies: 1) directly antagonize signaling components 2) avoiding detection by altering their PAMPs to be less immunogenic 3) disrupting cellular organelles/induce cell death. Here we will focus our discussion on direct antagonism approaches.module, of TLR receptor pathways that they antagonize (receptor module (Supramolecular Organizing Centers (SMOC) . SMOCs are multiprotein complexes that have been proposed to serve as organizing centers that coordinate the multitude of cellular responses to microbial infections and cytokines . The best-characterized SMOC is that formed between the aforementioned TIRAP and MyD88 adaptors and IRAK kinases. This SMOC has been dubbed the myddosome [TRAF6 and the TAK1 complex (Mitogen Activated Protein Kinases (MAPKs) and the NEMO complex , from btagonize . The recr module  consistss (SMOC)   domains present on both TLRs and adaptor proteins . Sortingl emerge , 12. Intl emerge , 14. Thiradation . This meradation . Interesradation , 18. Theradation . Future S3 of HSV-1 was shown to reduce levels of polyubiquitination on TRAF6, resulting in inhibition of TLR2 signaling during viral infection [S3 is dependent on its kinase activity and found to be downstream of MyD88. However, how this protein affects TRAF6 ubiquitination remains an open question [Shigella flexneri is an example of a bacterial protein that inhibits TRAF6 polyubiquitination [Escherichia coli (EPEC) and YopJ from Yersinia pestis [Myddosome formation involves the recruitment of IRAK family kinases, which in turn activate a module consisting of the E3 ubiquitin ligase TRAF6 and the MAP3K, TAK1. TRAF6 polyubiquitinates itself and recruits TAK1 regulatory components called TAK1-binding protein (TAB) 1–3. Binding of TAB2 and TAB3 to ubiquitin chains made by TRAF6 is required to activate TAK1 . Active nfection . Inhibitquestion . Next, rquestion , 23. Furtination . Howevertination . Enzymesa pestis , 26. Nlea pestis . In contThe MAPK cascade is a phospho-relay system that signals through ERK, JNK and p38 to activate the transcription factor AP-1. Previously, only bacterial systems were known to block MAPK cascades (reviewed ). HoweveDownstream of MyD88 and TAK1 is a signaling module governed by the protein NF-κB essential modulator (NEMO). NEMO controls the activation of IKKs, such as IKKα and IKKβ. NEMO binds non-degradative polyubiquitin chains that act as a scaffold for TAK1 phosphorylation of IKKα/IKKβ . IKKα/IKS. flexneri utilizes a strategy similar to viruses by promoting the degradation of NEMO. This example of bacteria using a strategy similar to that of viruses promotes the idea that knowledge of viral strategies can be used as a framework for future investigation of novel bacteria proteins that antagonize this signaling module.Diverse microbial strategies are used to target the NEMO complex including degrading, binding, and even deubiquitinating host enzymes and accessory proteins. Recent discoveries revealed that the 3C proteases from hepatitis A virus and foot-and-mouth disease virus can inhibit TLR signaling by cleaving NEMO , 32. AddS3 of HSV-1, which phosphorylate IRF3 to block its proper activation by host kinases [S3 was also shown to hyperphosphorylate NF-κB, blocking its activation [Transcription factor activation involves nuclear translocation of AP-1, NF-κB and the IRFs, which lead to transcriptional responses and the production of pro-inflammatory cytokines, chemokines, and interferons. Targeting the transcription factor module is a widely used strategy for viruses. Viral mechanisms for targeting this module include degrading and mimicking transcription factors and have been extensively reviewed elsewhere , 35; the kinases , 40. Rectivation . Thirdlytivation , 43. Rectivation .Escherichia coli (EHEC), EPEC, and S. flexneri. EHEC and EPEC encode effectors NleH1 and NleH2 that block NF-κB by binding to NF-κB subunit, RPS3 [S. flexneri encodes OspZ, which is interchangeable with the C-terminus of NleE, and thus likely acts via a similar mechanism [S. flexneri encoded effector, OspG, acts similar to VACV A49 discussed above. OspG prevents degradation of IκB by binding and interfering with the E2 ubiquitin-conjugating enzyme, UbcH5b [Despite the abundance of viral species that block this module, only a few bacterial species are known to possess this ability. These include enterohemorrhagic it, RPS3 , 46. Nleit, RPS3 –50. Two it, RPS3 . The tarechanism . A secon, UbcH5b , 53. ThiS. flexneri expresses OspF which blocks MAPKs, OspI which blocks TRAF6, and IpaH9.8 which promotes degradation of NEMO. Other examples include Salmonella spp. which express TlpA, a TIR domain containing protein that interferes with adaptor proteins [Yersinia spp., which acetylates several MAPKs and IKKβ [Y. pestis which only secretes 6 known effectors [A common tactic utilized by viruses and bacteria is to attack several modules of TLR signaling simultaneously. For example, VACV expresses N1L to block IKKs but also expresses A46R and A52R to target adaptor proteins and IRAK kinases , 19, 54,proteins , and Spvproteins , 57. It proteins , 23, ICPproteins , 42, andand IKKβ , 58, effand IKKβ . Howeverffectors , this stS3) can promote down-regulation of TLR4 and TLR3, respectively [In our search for microbial systems that antagonize specific components of TLR signaling networks, we find that viruses are not known to antagonize TLR signaling at the module of the receptor. Perhaps this is due to a lack of effective tools, such as antibodies, for detecting changes in TLRs on the surface of cells during infections. We also note that this module of TLR signaling is the least understood. Assays to monitor inducible (microbe-triggered) interactions between TLRs and their ligands or co-receptors are very much lacking. Despite these technical challenges, it is possible that viruses may not interfere with TLR detection directly. One reason to consider this possibility is that it may be more efficient for them to target downstream signaling components that are used by multiple innate immune pathways than to antagonize individual receptors. Nevertheless, there are some viral strategies that could antagonize TLRs. For example, viral proteins in both human T-cell leukemia virus type 1 (p30) and HSV-1  SSL3 and SSL4 from  of TLR2 , 64. Inceceptors . Thus, SStreptococcus pneumoniae, Haemophilus influenzae, and Neisseria spp., [Porphyromonas gingivalis have been shown to preferentially cleave CD14, an extracellular co-receptor vital to TLR4 signaling, from phagocytes [Porphyromonas spp. remains unclear. This example suggests that bacterial proteases have the potential to counteract TLR function and inflammatory responses through direct cleavage of receptor complexes on the surface of host cells. Perhaps future investigation of viral or bacterial proteins and their interaction with TLRs will reveal novel systems for targeting the receptor module.Other bacterial strategies also suggest possible mechanisms for direct TLR antagonism, although these strategies have never been directly implicated in antagonizing these receptors. For example, bacteria can secrete proteins to degrade host immune components such as the IgA proteases of ia spp., , 67 and agocytes , though Recent years have seen an explosion in findings of bacterial and viral systems to antagonize TLR signaling. Here we have compiled a wide-ranging list of these known strategies with the intent of providing insight into where future research will be best applied. Notably, we have identified gaps in our knowledge, such as the lack of systems for targeting the receptor module, which present some interesting challenges for the field. Overall, our findings indicate that viruses and bacteria have evolved similar mechanisms for antagonizing TLR signaling. This evolutionary convergence of strategies suggests that the information presented here can be used to guide future discovery of novel microbial factors that antagonize TLR signaling, both viral and bacterial.


Appropriately regulated signaling through each pathway is necessary for normal development and health, while dysregulation can lead to developmental defects and disease. Though both pathways have been vigorously studied, there is relatively little research exploring the possibility of crosstalk between these pathways. In this review, we provide a brief background on (1) the roles of both AHR and Wnt signaling in development and disease, and (2) the molecular mechanisms that characterize activation of each pathway. We also discuss the need for careful and complete experimental evaluation of each pathway and describe existing research that explores the intersection of AHR and Wnt signaling. Lastly, to illustrate in detail the intersection of AHR and Wnt signaling, we summarize our recent findings which show that 2,3,7,8-tetrachlorodibenzo- Ahr) and wingless-related MMTV integration site (Wnt) genes are found in animals as diverse as worms, flies, fish, and humans, which not only hints at their importance throughout evolution, but also provides multiple model organisms in which they can be studied . Th. Th44].  process ,113,114 neration . Thus, iCtnnb1 mRNA levels increase after 10 days of differentiation, but in cells with AHR knocked down, Ctnnb1 mRNA levels decrease after 10 days of differentiation  rapidly induced WNT5A expression and increased mRNA levels of FZD1, FZD3, and LEF1  affect mRNA levels of the same two target genes in the liver . C5. C528]. testines ,125, whil tumors ,127,128.in vivo in the rat cortex, in primary cortical neurons isolated from rat, and in the rat PC12 cell line . T. T131]. Wnt5a. Both WNT5A and LEF1 expression were reported to be downregulated in developing mouse palate after exposure to TCDD  . Nu. NuWnt5aThe adult mouse prostate consists of four pairs of bilateral lobes—the anterior, dorsal, lateral, and ventral lobes. Each develops via a highly orchestrated budding process from the embryonic urogenital sinus (UGS), which is situated between the bladder and pelvic urethra A. The UGin vitro with 5α-dihydrotestosterone  plus XAV-939, a chemical inhibitor of canonical Wnt signaling [Lef1 and Lgr5, were decreased in the XAV-939-exposed UGSs, confirming that XAV-939 downregulated canonical Wnt signaling [Canonical Wnt signaling is also essential for prostatic bud formation. UGSs cultured ignaling , had greignaling . Transcrignaling . UGSs cuignaling , also deignaling . Furtherignaling ,66,144. in vitro in prostate cancer cell lines. The extent to which these pathways crosstalk in vivo in the UGM and/or UGE is not well defined, and, therefore, it is not known if these pathways function independently or in tandem to orchestrate prostatic budding initiation and elongation.Interestingly, there are abundant data demonstrating crosstalk between androgen signaling and canonical Wnt signaling, including a physical interaction between androgen receptor and CTNNB1, and transcriptional regulation of one pathway by the other ,147,148.Ahr gene retain normal bud formation and develop functional prostates [in utero development can inhibit prostatic budding. A single dose of TCDD given to pregnant C57BL/6J mice  within the E13.5 to E15.5 developmental window, which is prior to the initiation of prostatic budding, reduces the number of dorsal and lateral buds and completely prevents ventral bud formation , male UGSs were placed in culture containing DHT and either vehicle or TCDD. After four days, prostatic budding was visible in all UGSs, but those exposed to TCDD developed about 50% fewer buds than controls , and Wnt inhibitory factor 1 (Wif1) [in vitro.controls  [143,1531 (Wif1) ,158,159.in vivo, transcription of AHR target genes was assessed in male UGSs exposed to TCDD in utero. TCDD increased transcription of Cyp1a1, Cyp1b1, and an AHR reporter gene in the UGE and/or UGM [To demonstrate activation of AHR signaling d/or UGM . Becaused/or UGM .in utero. As discussed above, activation of canonical Wnt signaling—which is characterized by stabilization, cytoplasmic accumulation, and nuclear localization of CTNNB1—is a process that must occur in the UGS to initiate prostatic budding [in utero exposure to TCDD blocks this activation  in the UGE of E16.5 control UGSs, but was greatly reduced in the ventral epithelium of E16.5 TCDD-exposed UGSs  an as yet unidentified activating signal triggers the stabilization and nuclear localization of CTNNB1, (2) this nuclear CTNNB1 is functionally relevant since it upregulates expression of downstream target genes Lef1 and Wif1, and (3) activation of AHR signaling in the UGS by TCDD can block the CTNNB1-stabilizing paracrine signal.Altogether, the in utero TCDD exposure inhibits canonical Wnt signaling in basal epithelial cells of the ventral UGS by preventing accumulation and nuclear localization of CTNNB1, the question remains, how? We performed a thorough examination of the Wnt signaling pathway by assessing the RNA abundance of over 40 genes that act upstream of CTNNB1. Pregnant C57BL/6J dams were treated with either vehicle or TCDD on E15.5, and UGSs were analyzed by ISH on E16.5 for differences in transcript abundance. The study revealed that RNA abundance of Rspo2, Wnt10a, Wnt16, Ror2, and as already discussed, Wif1 was altered in TCDD-exposed UGSs  at all times tested [Rspo2 transcript abundance was reduced in the VMP at E16.5, but not E17.5 or E18.5, while Rspo3 transcript abundance was unchanged in the VMP at E16.5, but decreased at E17.5 and E18.5 (R-spondins (RSPOs) have been shown to activate the Wnt signaling pathway by multiple mechanisms, including synergism with Wnt ligands, activation of noncanonical Wnt signaling through heparin sulfate proteoglycan binding, competition with DKKs  for receptor binding, and activation of LGR receptors to enhance canonical Wnt signaling . Rspo2 as tested ,152. Thes tested . In TCDDnd E18.5 A–H [143,in vitro. Male UGSs were harvested at E14.5 and cultured with or without TCDD for 2, 3, or 4 days. Rspo2 transcript abundance was reduced in TCDD-exposed UGSs after two days in culture (similar to E16.5), but was not different from control UGSs after three and four days in culture [Rspo3 transcript abundance in TCDD-exposed UGSs was not different from control UGSs after two days in culture, but was reduced compared to control UGSs after three and four days in culture  [These findings were confirmed  culture . Conversctively) . Exogenoctively) . Furtherctively) .These findings are significant for several reasons: (1) TCDD-induced activation of AHR downregulated expression of RSPO2 and RSPO3, two potentially key paracrine regulators of ventral prostate bud development; (2) this downregulation occurred in the UGM which is known to be the site of action for TCDD-induced, AHR-dependent inhibition of prostatic budding; and (3) addition of exogenous RSPO2 and RSPO3 to UGS organ cultures countered the effects of TCDD-induced activation of AHR signaling by upregulating canonical Wnt signaling and increasing prostatic budding. We have already established that TCDD-induced activation of AHR signaling can inhibit canonical Wnt signaling; these findings elucidating RSPO regulation and function upstream of CTNNB1 provide one possible mechanism by which this inhibition occurs.Wnt10a in the UGE, and Wnt16 in the UGM [Wnt10a transcript was observed in basal epithelial cells of the urethra, with little to no transcript present in basal epithelial cells of the ventral UGS  [Multiple studies have shown that CTNNB1 signaling is required in the UGS for prostatic budding ,121,144.1, Wif1) . The ups1, Wif1) . WNT5A i1, Wif1) . We hypo1, Wif1) .Interestingly, during normal development, both WNT5A and WNT16 are expressed in the mesenchyme around the urethra where no buds develop, while only WNT5A is expressed in the ventral UGM. After exposure to TCDD both WNT5A and WNT16 are expressed in the ventral UGM and no prostatic buds develop .Both AHR and Wnt signaling have been rigorously studied for many years, but not until relatively recently was there evidence of crosstalk between these two pathways. Our review of literature exploring the intersection of AHR and Wnt signaling in vertebrates found a majority of these studies reported that activated AHR downregulated canonical Wnt signaling . Careful


Insight into the exact regulation of hypertrophy by the signaling network is necessary for the efficient application of MSCs for articular cartilage repair and for developing novel strategies for curing OA. We focus on articles describing the role of the main signaling pathways in regulating chondrocyte hypertrophy-like changes. Most studies report hypertrophic differentiation in chondrogenesis of MSCs, in both human OA and experimental OA. Chondrocyte hypertrophy is not under the strict control of a single pathway but appears to be regulated by an intricately regulated network of multiple signaling pathways, such as WNT, Bone morphogenetic protein (BMP)/Transforming growth factor-β (TGFβ), Parathyroid hormone-related peptide (PTHrP), Indian hedgehog (IHH), Fibroblast growth factor (FGF), Insulin like growth factor (IGF) and Hypoxia-inducible factor (HIF). This comprehensive review describes how this intricate signaling network influences tissue-engineering applications of MSCs in articular cartilage (AC) repair, and improves understanding of the disease stages and cellular responses within an OA articular joint.Hypertrophic differentiation of chondrocytes is a main barrier in application of mesenchymal stem cells (MSCs) for cartilage repair. In addition, hypertrophy occurs occasionally in osteoarthritis (OA). Here we provide a comprehensive review on recent literature describing signal pathways in the hypertrophy of MSCs-derived  Osteoarthritis (OA) is a multifactorial complex and chronic disease characterized by progressive degradation of joint cartilage. The underlying molecular mechanisms involved in the pathogenesis and progression of OA are still largely unknown, and currently no disease-modifying therapy is available for OA.in vivo and in vitro [in vivo, hypertrophic differentiation towards the osteogenic lineage is observed. Prevention of hypertrophy is becoming increasingly important for clinical application of MSCs in cartilage tissue engineering [In cell-based cartilage regeneration therapies, the use of mesenchymal stem cells (MSCs) has shown promising results. Evidence showed that MSCs can be differentiated into chondrocytes (marked by Sex determining region Y box 9 (SOX9); Aggrecan (ACAN); Collagen type II Col2A1)) after a condensation state (marked by Cyclic adenosine monophosphate (cAMP), Transforming growth factor-β (TGFβ), Fibronectin, Neural cell adhesion molecule (N-CAM) and N-cadherin) ) after ain vitro  /TGFβ, Parathyroid hormone-related peptide (PTHrP), IHH, Fibroblast growth factor (FGF), Insulin like growth factor (IGF) and Hypoxia-inducible factor (HIF) signaling pathways , Figure + pathway [WNT signaling pathways are highly evolutionarily conserved pathways with crucial roles in embryonic development, patterning, tissue homeostasis, growth, as well as in the onset and progression of a variety of diseases . There a pathway . The can pathway . HoweverNumerous studies have revealed a central role of WNT signaling in cartilage homeostasis. In cartilage, moderate activity of WNT is essential for chondrocyte proliferation and maintenance of their typical characteristics , but excin vivo and overexpression methods in vitro suggest distinct roles of different WNTs in the control of chondrogenic differentiation and hypertrophy. WNT4 and WNT8 block chondrogenic differentiation but promote hypertrophy [There are several types of WNT ligands, which play different roles in the chondrogenic differentiation and cartilage development. Experiments using retroviral misexpression ertrophy ,26. WNT9ertrophy . WNT3a aertrophy . WNT16 tertrophy . WNT5a eertrophy .Interestingly, the expression of hypertrophy-related markers in chondrogenesis of MSCs is decreased in the presence of Dickkopf (DKK1), which acts as WNT signaling inhibitor (antagonist) by binding to low density lipoprotein receptor related protein (LRP5/6) through cartilage protective mechanisms . ActuallBMPs are multi-functional cytokines that belong to the transforming growth factor-β (TGF-β) superfamily. BMP signaling is mediated primarily through the canonical BMP-Smad pathway in chondrocytes . The patet al. showed that BMP2 and BMP4 induce hypertrophy during the chondrogenic differentiation of human MSC in vitro [In vivo studies showed that overexpression of BMP4 in cartilage of transgenic mice resulted in an increased hypertrophic zone, indicating increased differentiation of hypertrophic chondrocytes [Although BMPs have a protective effect in articular cartilage, they are also involved in chondrocyte hypertrophy and matrix degradation ,40,41,42in vitro . In anotin vitro . In vivodrocytes . As the drocytes . Therefodrocytes . Our predrocytes .in vitro [TGFβ is a potent inducer of chondrogenesis in vitro ,49. Duriin vitro ,53,54,55in vitro . Althougin vitro ,11.Most recently, several lines of evidence have suggested that the TGFβ/Smad pathway played a critical role in the regulation of articular chondrocytes hypertrophy and maturation during OA development ,58,59. Zin vivo. However, the continuous co-activation of two signaling pathways resulted in hypertrophy inhibition, characterized by the suppressed expression of collagen type X, RUNX2, and ALPL, and did not lead to ossified tissue in vivo [β-catenin crosstalk with TGFβ was reported in hypertrophy regulation in MSCs . In the  in vivo .It was demonstrated that the crosstalk between WNT and BMP plays key roles in regulating chondrocyte activity in pathogenesis of osteoarthritis, which may be cell type-specific . PapathaThis report adds to the accumulating evidence that increased or excessive activation of canonical WNT signaling in chondrocytes is detrimental and contributes to OA cartilage degradation. Recently, studies from our group also indicated that the natural WNT and BMP antagonists DKK1, FRZB and GREM1 inhibit hypertrophic differentiation of hMSCs during chondrogenesis by blocking WNT and BMP pathways . Therefoin vivo. In another cell study, it was demonstrated that PTHrP from human articular chondrocytes inhibits hypertrophy of MSCs during chondrogenesis in co-culture, and intermittent supplementation of PTHrP also improves chondrogenesis of MSCs and reduces the hypertrophy [PTHrP is a member of the parathyroid hormone (PTH) family that blocks hypertrophy by stimulating NK3 homeobox 2 (Nkx3.2)  and prevertrophy ,84. A siertrophy . Howeverertrophy . Howeverertrophy ,87. Thisertrophy . PTH1R kertrophy . The choertrophy .in vivo [Cell studies have shown that FGF2 combined with PTHrP inhibited the TGFβ responsive COL2A1 and COL10A1 expression and ALPL induction. However, calcification of implanted pellets was not prevented by PTHrP in vivo . In anotin vivo . In addiin vivo . Therefo−/− mice have normal limb bud development and mesenchymal condensations, but show decreased chondrocyte proliferation in stylopod elements, delayed initiation of chondrocyte hypertrophy and abnormal osteogenesis in skeletal vascularization [−/− mice, the phenomenon of delayed mineralization was observed, which was found to be closely associated with delayed initiation of chondrocyte hypertrophy, decreased chondrogenesis proliferation of early stages, delayed skeletal vascularization and delayed osteoclast and osteoblast recruitment to the growth plate [FGF signaling plays a critical role in controlling chondrocyte differentiation . Specifirization . In the th plate . Furtherth plate . The FGFth plate . Recentlth plate .The interplay of WNT and FGF signaling is important to determine the fate of MSCs and their subsequent differentiation. FGFR1 appears to act downstream of the β-catenin pathway and serves as a key determinant in the lineage decision of skeletal precursors . HypertrIGF-1 has been identified as an important growth factor for skeletal development by promoting chondrocyte proliferation and maturation, while inhibiting apoptosis to form bones with appropriate size and strength. IGF-1 transmits signals via the type 1 IGF-1 receptor (IGF1R), which is expressed in the proliferating and prehypertrophic zone chondrocytes of growth plates . Evidenc3) in the growth plate, and the chondrocytes undergo proliferation and differentiate to prehypertrophy. It seems that chondrocyte proliferation may be triggered by the IGF-1/IGF1R-mediated PI3K/Akt/GSK3β pathway, while cell hypertrophy is likely due to activation of Wnt/β-catenin signaling, which is at least in part initiated by IGF-1 signaling or the IGF-1-activated PI3K/Akt signaling pathway [−/− mice long bones, whereas expression of PTHrP was increased, suggested that IGF-1 signaling is also required to maintain the IHH-PTHrP loop during skeletogenesis [IGF-1 signaling also is involved in the interaction between the thyroid hormone and the WNT/β-catenin signaling pathways in regulating growth plate chondrocyte proliferation and differentiation. Evidence showed that IGF-1 and the IGF-1 receptor (IGF1R) stimulate Wnt-4 expression and β-catenin activation in growth plate chondrocytes. Chondrocyte proliferation and terminal differentiation induced by IGF-1/IGF1R can be partially inhibited by the Wnt antagonists FRZB and DKK1 . The IGF pathway . The facogenesis .Healthy articular cartilage is a typical avascular tissue, and chondrocytes are able to survive in low oxygen environments . HypoxiaEpas1, was identified as a regulator of endochondral bone formation, and appears to be a central positive regulator of collagen X, MMP13 and VEGF expression by enhancing promoter activities through specific binding to the hypoxia-responsive elements [in vivo and in vitro, which need to be clarified. The balance between HIF-1α/HIF-2α activities clearly contributes to the control of cartilage homeostasis.Under hypoxic conditions, the transcription factor HIF-1α accumulates and activates the transcription of genes, which are involved in energy metabolism, angiogenesis, vasomotor control, apoptosis, proliferation, and matrix production. In subcutaneous stem cell implantation studies, HIF-1α was shown to potentiate BMP2-induced SOX9 and cartilage formation, while inhibiting RUNX2 and endochondral ossification during ectopic bone/cartilage formation. In the fetal limb culture, HIF-1α and BMP2 synergistically promoted the expansion of the proliferating chondrocyte zone and inhibited chondrocyte hypertrophy and endochondral ossification . Howeverelements . Inflammelements ,125. Furelements . RUNX2 aelements ,127. Theelements . There ielements . Howeverelements . The seeChondrocyte differentiation is regulated by multiple signal transduction pathways. Maintaining a normal chondrocyte phenotype and avoiding hypertrophy is important for cartilage repair. SOX9 and RUNX2 are two typical markers in chondrocyte development. SOX9 is expressed in chondrocytes, while RUNX2 is highly expressed in hypertrophic chondrocytes. In most cases, a hypertrophic phenotype was accompanied by high expression of RUNX2 through activation of either of the WNT, BMP, IHH, FGF and HIF signaling pathways. However, TGFβ, IGF-I and PTHrP promote the proliferation of chondrocytes. Here we propose a model in which the balance of these signal pathways adjusts the state of chondrocyte proliferation or hypertrophy through the shifting between SOX9 and RUNX2 transcriptional activities. In the WNT pathway, the LEF/TCF/β-catenin complex can promote RUNX2 expression. BMP/TGF-β signaling has a dual role in the chondrocyte development. TGF-β induces collagen II and SOX9 deposition through Smad2/3 phosphorylation pathway, while BMP2/4 promotes chondrocyte hypertrophy and cartilage mineralization via Smad1/5/8 phosphorylation. PTHrP represses hypertrophic cartilage differentiation whereas IHH signaling positively regulates the hypertrophic phenotype by high transcription and expression of collagen type X and RUNX2. IGF-1 signaling stimulates chondrocyte proliferation by the IGF-1/IGF1R-mediated PI3K/Akt/GSK3β pathway, while cell hypertrophy is likely due to activation of Wnt/β-catenin and IHH signaling by IGF-1. HIF-1α and HIF-2α have a distinct role in the chondrocyte development. The former inhibits the RUNX2 expression, while the latter enhances the expression of collagen X, MMP13 and RUNX2 and promotes the hypertrophic differentiation of chondrocytes. The fine balance of the crosstalk between these signaling pathways is a requirement for normal chondrocyte differentiation and cartilage development.