TNFRSF10D DNA-methylation status (TNFRSF10D methylated vs. unmethylated) as a prognostic marker for overall (p = 0.001) and for relapse-free survival (p = 0.008) in the training set. This finding was confirmed in the independent test set . In a multivariate Cox-regression analysis including all patients, the TNFRSF10D DNA-methylation status remained as the most significant prognostic parameter for overall and relapse-free survival (relative-risk (RR) of death, 4.6 , RR of relapse, 7.2 ). In this study, we demonstrate that TNFRSF10D DNA-methylation analysis of a small tissue-punch from archival FFPE melanoma tissue is a promising approach to provide prognostic information in patients with melanoma.In this retrospective pilot study, the DNA-methylation status of genes that have been demonstrated to be involved in melanoma carcinogenesis was analyzed in order to identify novel biomarkers for the risk assessment of melanoma patients. We analyzed DNA extracted from punch-biopsies from 68 formalin-fixed paraffin-embedded (FFPE) melanoma specimens. Using MethyLight PCR, we examined 20 genes in specimens from a training set comprising 36 melanoma patients. Selected candidate genes were validated in a test set using FFPE tissue samples from 32 melanoma patients. First, we identified the  The incidence of melanoma is increasing in white populations worldwide . In advaIn the present pilot study, we aimed to explore whether differences in the DNA-methylation pattern from formalin-fixed paraffin-embedded (FFPE) tissues may be useful as a prognostic marker for the further outcome of non-metastatic melanoma patients.APC, CDH13, CDKN2A, CYP1B1, ENC1, ESR1, LOX, MAGEA1, MIR34A, PPP1R3C, PYCARD, RARB, RARRES1, RASSF1, SFN, SOCS1, TIMP3, TNFRSF10C, TNFRSF10D and TP73) [MAGE1A showed higher DNA-methylation values  in women (p = 0.001). In this set, we identified age, the Clark level, ulceration, tumor thickness, mitotic rate and TNFRSF10D DNA-methylation status (TNFRSF10D methylated vs. unmethylated) as univariate prognostic markers for overall survival . The Clark level, ulceration, tumor thickness and TNFRSF10D methylation status were found as univariate prognostic markers for relapse-free survival  ,16,17,18TNFRSF10D DNA-methylation status in an independent test set consisting of FFPE tissues of 32 melanoma patients. Furthermore, in this analysis, TNFRSF10D DNA-methylation was confirmatively shown to be highly significantly associated with a poor overall and relapse-free survival , the Clark level, mitotic rate and TNFRSF10D DNA-methylation status as prognostic parameters for poor overall survival  and a 7.2-fold higher risk of relapse  than patients with unmethylated TNFRSF10D  and relapse-free survival (p = 0.001).Finally, we analyzed all patients, including the variables, sex, age, Clark level, presence of ulceration, tumor thickness, interferon alpha therapy and NFRSF10D . After eThe prognostic biomarkers currently used in melanoma do not adequately predict disease recurrence and overall survival in a significant subset of patients. Therefore, novel biomarkers are highly required to overcome these problems.TNFRSF10D DNA methylation status in paraffin-embedded melanoma tissues to be an independent prognostic biomarker for relapse-free survival and overall mortality in non-metastatic melanoma patients. Surprisingly, ulceration and tumor thickness were significantly associated with survival only in the training set, whereas the invasion level (Clark’s level) was a significant, prognostic feature consistently in all analyses performed in this work. Due to the small sample size of this pilot study, a subsequent validation study in a larger, independent patient cohort must be performed. In our study, the prognostic value of TNFRSF10D concerns mainly the group of patients with methylated TNFRSF10D in the tumors. Recently, TNFRSF10D DNA-methylation has also been shown to be an independent prognostic and predictive marker in the serum of patients with MYCN nonamplified neuroblastoma [TNFRSF10D is epigenetically silenced in human melanoma [et al. found that 72% of the analyzed melanoma cell lines had no TNFRSF10D mRNA expression and that the transcript level of TNFRSF10D was correlated inversely with promoter methylation [TNFRSF10D DNA-methylation in only 16% of all analyzed specimens. This discrepancy probably reflects the different levels of TNFRSF10D DNA-methylation in melanoma cell lines, metastatic tumor tissues and primary tissues. Bonazzi et al. observed a five-fold average increase in TNFRSF10D mRNA expression in five melanoma cell lines after treatment with the DNA-demethylating agent, 5-aza-2'-deoxycytidine [TNFRSF10D belongs to the tumor necrosis factor (TNF) receptor superfamily. This receptor contains an extracellular tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) binding domain, a transmembrane domain and a truncated cytoplasmic death domain. The second known TRAIL decoy receptor, TNFRSF10C, lacks this intracellular death domain completely. Therefore, both receptors appear unable to induce apoptosis. Considering that TNFRSF10D, as well as TNFRSF10C have been presumed to function as oncogenes, because of their postulated anti-apoptotic effect [et al. have shown that an ectopic overexpression of TNFRSF10C and/or TNFRSF10D in melanoma cell lines led to a significant reduced growth rate and a clear increased apoptotic response [TNFRSF10D may represent a special feature of more aggressive cancer cells.In this pilot study, we identified blastoma . Interesmelanoma ,16, as wmelanoma . Bonazzic effect , our datresponse . In the a priori randomly split into a training and a test set, consisting of 36 and 32 patients, respectively. The study was approved by the local medical research ethics committee  and conducted in accordance with the Declaration of Helsinki. Reporting Recommendations for Tumor Marker Prognostic Studies (REMARK) were adhered to where applicable [We retrospectively analyzed prospectively collected melanoma specimens (FFPE tissues) from melanoma patients treated at the Department of Dermatology and Venereology, Innsbruck Medical University, Innsbruck, Austria. Samples have been collected during primary surgery. Sixty-eight patients (32 women and 36 men) diagnosed between 1983 and 2004 with primary, invasive, non-metastasized melanoma were included in this study  classification American Joint Committee of Cancer 2001). Patients had no metastases at the time of diagnosis or surgery, respectively. Thirty-four melanomas were located on the trunk, 34 on the limbs. The tumor thickness was 0.5–2 mm (T1/T2), 2.01–4 mm (T3) and >4 mm (T4) in 25, 23 and 18 patients, respectively; tumor thickness was unknown in two patients. Twenty-three patients showed ulcerated melanomas. All patients underwent surgery with 1–2 cm excision margins, according to standard guidelines, and 16 patients received adjuvant interferon alpha. The median age was 53.4 years (21.9–90.7 years). Thirty and 32 patients relapsed/died, respectively, due to the consequences of the melanoma after a median follow up period of 2.0 (interquartile (IQ)-range 6.2) and 3.4 (IQ-range 5.5) years, respectively. The patient study cohort was plicable ,23.DNA was isolated from punches gained from FFPE melanoma specimens using the DNeasy Tissue Kit  in order to assure that mainly melanoma tissue was collected.Sodium bisulfite-modification of genomic DNA (700 ng) was performed using the EZ DNA Methylation-Gold Kit , according to the manufacturer’s instructions.COL2A1), to normalize for input DNA. Primers and probes for APC, COL2A1, CDH13, CDKN2A, CYP1B1, ENC1, ESR1, LOX, MAGEA1, MIR34A, PYCARD, PPP1R3C, RARB, RARRES1, RASSF1, SFN, SOCS1, TIMP3, TNFRSF10C, TNFRSF10D and TP73 are listed in Tables S1 [Thirty nanograms of bisulfite-modified DNA were analyzed by means of MethyLight analysis, as described previously . Brieflyables S1 ,26.COL2A1 DNA sequence lacking CpG dinucleotides. The specificity of the reactions for methylated DNA was confirmed separately using SssI -treated human white blood cell DNA (heavily methylated). The SssI treated DNA was additionally used for the standard curve preparation, which is required for the quantification. The percentage of fully methylated molecules at a specific locus was calculated by dividing the GENE:COL2A1 ratio of a sample by the GENE:COL2A1 ratio of SssI-treated white blood cell DNA and multiplying by 100 . PMR values have been calculated for all analyzed genes. If less than 50% of the samples were methylated for a specific gene , we categorized the samples in “unmethylated” and “methylated” and performed the analyses with these dichotomized variables. Only in 3 genes were more than 50% of the samples methylated . For these genes we used the PMR values for the following statistical analysis.To control for the amount of input bisulfite-modified DNA, this value was normalized to the extent of amplification of a The mitotic rate per square millimeter of tumor tissue was evaluated by counting mitotic figures on hematoxylin and eosin (H&E)-stained tissue sections.etc.) or DNA-methylation status was performed using a two-sided log rank test. A multivariate Cox proportional hazards model was applied to estimate the prognostic effect of TNFRSF10D DNA-methylation. A p-value <0.05 was considered statistically significant. SPSS 19.0 was used for all statistical analyses .Disease-free and overall survival were calculated from the date of diagnosis to the date of relapse, death or last follow-up. Disease-free and overall survival curves were calculated with the Kaplan–Meier method. Univariate analysis of overall survival according to clinicopathological factors (age, Clark-level, tumor thickness, presence of ulcerations, TNFRSF10D promoter from a small tissue punch from archival paraffin-embedded melanoma tissue is able to provide independent prognostic information in order to identify patients with a higher risk for aggressive progress.Our data demonstrate that DNA-methylation analysis of the TNFRSF10D promoter hypermethylation is associated with poor prognosis and aggressive proliferation in melanoma. Additionally, further research needs to be conducted to assess if TNFRSF10D hypermethylation in serum samples from melanoma patients could be an indicator of poor prognosis in melanoma, as has been shown in neuroblastoma patients [Further studies are needed to elucidate how patients .


Moreover, immunohistochemical investigations have helped defining the lesional and cellular distribution of iron in MS brain tissue. Imaging studies in MS patients and of brain tissue combined with histological studies have provided important insights into the role of iron in inflammation and neurodegeneration in MS.Iron is considered to play a key role in the development and progression of Multiple Sclerosis (MS). In particular, iron that accumulates in myeloid cells after the blood-brain barrier (BBB) seals may contribute to chronic inflammation, oxidative stress and eventually neurodegeneration. Magnetic resonance imaging (MRI) is a well-established tool for the non-invasive study of MS. In recent years, an advanced MRI method, quantitative susceptibility mapping (QSM), has made it possible to study brain iron through  Iron as an electron facilitator is involved in healthy brain functions including myelin production, oxygen transport and neurotransmitter synthesis ,2,3. Excin vivo study of brain iron, which are important for translating tissue studies into clinical practice and for in vivo biological and pathophysiological investigations.We briefly review the tools that can be used to detect iron in tissue, including histological techniques and elemental analysis tools to quantitatively measure iron in tissue samples. We also review non-invasive MRI techniques for ex vivo methods for studying tissue iron, which can be considered as the gold standard for studying iron chemistry and quantity.We first discuss qualitative and quantitative 3+ staining (Perls’ Prussian blue) and Fe2+ staining (Turnbull’s blue) have been used for qualitatively exploring the distribution of iron in brain tissue. Because most iron in the brain is present as ferric (Fe3+) iron, Perls’ staining is the most commonly used technique to visualize iron histologically. In addition, immunohistochemical labeling is used for iron-related proteins, including ferritin [Feferritin ,47,48, tferritin ,50 and cferritin . Howeverferritin . i.e., Fe2+ or Fe3+, thus quantifying the total iron content. Here is a brief outline of commonly used iron quantification methods:Colorimetry: This iron-detecting approach requires a standard of the measured absorbance for known concentrations, thus making it problematic for comparing tissue samples of different measurements [urements ,54,55.Atomic Absorption SpectrometryorSpectrophotometry (AAS): The technique equally requires standards with known analyte content to establish a relationship between measured absorbance and analyte concentration [ntration ,57,58.Instrumental Neutron Activation Analysis (INAA): The intensity and wavelength of the signal is element-specific and can be used to characterize the examined sample [d sample ,60,61.Inductively Coupled Plasma Mass Spectrometry (ICP-MS) [(ICP-MS) ,62,63: I(ICP-MS) ,65,66,67(ICP-MS) ,69. LA-I(ICP-MS) ,70.X-ray Fluorescence Spectrometry (XRF) or Rapid Scan X-ray fluorescence spectrometry (RS-XRF) [(RS-XRF) ,72: This(RS-XRF)  or more (RS-XRF) .Proton-Induced X-ray Emission (PIXE) [n (PIXE) ,76,77: An (PIXE) ,79,80.A few spectrometric techniques are available to quantify iron in biological tissue, which are either based on the characteristic atomic mass  or elemeRS-XRF, PIXE and LA-ICP-MS are the only techniques that can been applied to correlate MR signal and local iron concentration voxel by voxel ,71,81. TNoninvasive MRIs are highly sensitive to tissue iron content and has become the method of choice for investigating brain iron, including iron in MS brains. We review first the qualitative MRI methods for detecting iron, semi quantitative methods for measuring iron, and then describe quantitative susceptibility mapping (QSM) for biophysics model based iron quantification.T2-weighted imaging (T2w). Iron appears hypointense on T2-weighted images because paramagnetic iron produces a field in its surroundings, contributing to the incoherent thermal dephasing, i.e., enhancing the transverse relaxation rate 1/T2 = R2. Iron R2 enhancement increases with magnetic field strength [strength . Iron costrength ,86,87,88T2* weighted imaging (T2*w). This magnitude gradient echo imaging (GRE) based T2* contrast is sensitive to the intravoxel variation of the iron induced magnetic field [in vivo iron detection [ic field , in addietection ,91,92. Aetection ,93. SWI MRI offers rich tissue contrasts to sensitize tissue compositions and biophysical processes. Traditional T2 and T2* relaxation contrasts have been used to detect brain iron; T1 relaxation has been shown to be slightly affected by iron, but not as strongly as T2 or T2* ,83,84,85i.e., depends on both local iron in a voxel and iron outside the voxel of interest.Magnetic Field Correlation Imaging (MFC) is directly related to iron-induced field variations and has been used to estimate iron concentration [ntration . MFC beantration ,95.Phase Imaging. The gradient echo MRI data contains both magnitude and phase images. Traditionally, the phase images have been discarded and only the magnitude images are saved on the MRI scanner. The phase shift divided by the gyromagnetic ratio is the echo time times the iron induced magnetic field, which is proportional to the iron concentration convolved with the dipole kernel [e kernel ,97.R2* mapping. A number of correlation studies show that iron content is linearly related to the 1/T2* = R2* in DGM nuclei where iron distribution is approximately constant [constant ,98. Howeconstant ,99, and constant ,103,104.Quantifying iron from MRI data has long been a desired goal. A number of MRI metrics have been investigated, which are proportionate to iron content in limited situations. However, these methods are essentially based on the magnetic field, which contains blooming artifacts, i.e., their values at a given voxel depend not only on the iron content in that voxel but also on iron concentration in the nearby voxels with a distance and direction dependent weighting [In summary, current traditional MRI quantification methods do not provide absolute quantification , and suffer from being non-local, eighting .The GRE phase images or, more precisely, the magnetic field can be deconvolved to accurately determine the distribution of magnetic source—tissue susceptibility, which is quantitative susceptibility mapping (QSM). QSM eliminates blooming artifacts and quantifies the local tissue magnetic properties ,106,107.Iron is the most abundant trace metal in the healthy human brain, at levels 20–30 times higher than all other trace metals combined ,129. Iro3+) stored in the spherical shell of protein ferritin [2+). Iron transporter proteins including transferrin, DMT1  and ferroportin can transport Fe2+ ions. When ferrous iron is not needed, the H-subunit of ferritin oxidizes ferrous iron into ferric iron and stores it in ferritin [The majority of brain iron is present in the inactive form of ferric iron , mature brain), red nucleus (19 ± 7 mg iron/100 g fw), substantia nigra (18 ± 7 mg iron/100 g fw) and putamen (13 ± 3 mg iron/100 g fw) . These mWM iron is more homogeneously distributed compared to the variations found in GM. The WM iron concentration is higher in the frontal lobe and lower in the occipital lobe compared to adjacent cortical GM indicating a gradient in iron concentration ,81,133. The pathological hallmark of MS is inflammatory demyelination in both white and gray matter. Demyelination is mediated by myelin protein-specific T cells and myelin-phagocytosing macrophages. It is well documented that iron is retained by macrophages during inflammation ; howeverM1 macrophages secrete high levels of proinflammatory cytokines and reactive oxygen species and have increased microbiocidal capability while M2 macrophages dampen proinflammatory cytokine levels, have a high scavenging capacity and are essential for late phase tissue repair ,137,138.WM lesions are characterized by an initial infiltration of activated T cells and macrophages, demyelination and blood-brain barrier (BBB) breakdown. Iron is absent from early active, gadolinium-enhancing lesions as suggested by imaging studies of MS patients and by MRI and histological analysis of MS brain tissue ,42,144. A retrospective QSM imaging study of MS patients showed that lesion susceptibility values increase sharply as lesions become non-enhancing and remain high for several years . Histoloi.e., longstanding lesions that no longer contain inflammatory cells have low susceptibility values, thus indicating low iron content [Finally, chronic silent lesions,  content .i.e., lesions that did not contain inflammatory cells, found that iron density was reduced compared to normal appearing GM [Iron content has also been examined in cortical lesions. We found that activated microglia at the border of chronic active cortical lesions contained high amounts of iron , consistaring GM . This ir2’ signal in NAWM of MS patients as compared to controls [A recent histopathological report suggests that iron is lost from Normal Appearing White Matter (NAWM) and that this loss becomes more pronounced with increasing disease duration . This obcontrols . Iron decontrols ,37.Increased iron deposition has been observed in the deep nuclei of MS patients, predominantly in the putamen ,34 Figu. MoreoveIn summary, brain iron homeostasis is profoundly perturbed in MS, involving different compartments and resulting in various pathologies. First, iron accumulates after initial acute demyelination in activated microglia at the rim of white matter lesions, where it might contribute to long-lasting  neurotoxic inflammation. Moreover, with increasing disease duration, iron is slowly depleted from normal appearing white matter, which potentially compromises oligodendrocyte functions including myelin maintenance and contributes to axonal damage outside of white matter lesions . Iron isEx-vivo and in vivo qualitative and quantitative detection and measurement of brain iron levels is very useful for understanding the role of iron in MS pathogenesis, and for translating in vivo MRI patient studies.3+ stain [2+ stain [A number of studies have used histology to characterize iron content in post-mortem brain tissue of MS patients, either employing Fe3+ stain ,41,147, 3+ stain ,144, or 2+ stain ,33,51 an2+ stain ,28. OnlyWe explored the role of lesional iron in multiple sclerosis using multiple approaches: immunohistochemical examination of autoptic MS tissue , iron-upIron deposits were more prevalent in WM lesions of patients with active relapsing-remitting MS than in patients with stable, longstanding disease. Together, these data suggest that iron accumulation in WM lesions are indicative of chronic, non-demyelinating inflammatory activity that occurs behind a closed blood-brain barrier and can be detected by QSM.Noninvasive MRI has been the main window for studying MS. In particular, non-conventional MRI techniques have been increasingly used to better probe the MS pathological substrate, including Gradient echo (GRE) imaging, which is highly sensitive to iron and myelin distribution.T2-weighted imaging (T2w). T2 relaxometry has been applied to detect iron accumulation in DGM of MS patients showing a shortening of the T2 relaxation time for MS patients [patients . Howeverpatients . Furtherpatients . WM lesiT2* weighted imaging (T2*w). Lesion magnetic susceptibility increases, as microglia degrade and remove diamagnetic myelin fragments [ragments ,162,163,ragments ,13,20,31ragments ,165,166.ragments , but botragments ,167 (shoSusceptibility Weighted Imaging (SWI) SWI has been utilized to see differences of iron content in healthy subjects vs. MS patients [patients  and to dpatients ,168,169.The magnetic susceptibility weighting in T2* contrasts becomes more prominent in Ultra-High Field MRI, allowing the study of DGM iron deposits with increased sensitivity for basal ganglia regions ,156,157 Phase Imaging. MS lesions may appear isointense or hypointense on phase images [e images ,166. It e images   MFC has been utilized to measure increased iron content in MS patients [patients ,34. HoweIron quantification is highly desirable for monitoring disease progression and therapeutic outcome as well as for enabling longitudinal or cross-center studies. Here, we summarize three semi-quantitative methods that have been used for measuring iron in MS:Important for MS applications, QSM is more sensitive than R2* in detecting pathological changes in the basal ganglia , allowinDifferentiating solid from shell lesions is important for understanding MS lesion pathology ,10, but p < 0.01) and 1.3 ± 2.3 ppb/m  for nonenhancing lesions at age = 0–4 y, and 4.67 ± 3.18 ppb and −0.11 ± 0.55 ppb/m for old nonenhancing lesions (age > 7 y). Examples are presented in Thirty-two clinically confirmed MS patients underwent two MR exams (denoted by MRI1 and MRI2) with 0.43 ± 0.16 year (y) interval on a 3T scanner. MRI included T2w, T1w, T1w+c and QSM . Ages of 162 lesions were measured by examining their first appearance on prior MRIs (over past 0.3–10.6 y) in PACS. The susceptibilities relative to NAWM and temporal rates of change in lesion susceptibility relative to CSF were 0.53 ± 3.34 ppb (parts per billion) and 9.29 ± 1.84 ppb/month(m) for new enhancing lesions (age = 0 y), 38.0 ± 13.6 ppb  , 42 relapsing-remitting MS) and 23 healthy control (HC) subjects underwent 3T MRI. QSM and R2* maps were reconstructed from the same 3D multiecho spoiled GRE sequence. QSM successfully detected the difference in caudate nucleus and putamen between HC and CIS subjects, and difference in globus pallidus between HC and MS subjects while R2* failed to do so. Susceptibilities were higher with increasing neurologic deficits ( < 0.01) .3 [Both iron deposition and demyelination in MS lesions can cause a susceptibility increase as measured by QSM , but theperature  and a tiature [3 . The myeIron in white matter lesions can be identified at the hyperintense rim on QSM, in the QSM lesion volume lying outside the lesion volume on T2-weighted imaging, or with the positive QSM value relative to the CSF ,181,182 In summary, iron-sensitive imaging of multiple sclerosis offers the possibility to monitor several aspects of MS pathology, in particular chronic microglial activation and shifts in iron concentrations in NAWM that cannot be measured by conventional MRI methods.Noninvasive MRI is critical for the diagnosis and surveillance of MS patients and is nowadays the main window for studying this neurodegenerative disease. Current inflammation assessment in clinical practice is based on gadolinium (Gd) enhancement on contrast enhanced T1-weighted (T1w+Gd) MRI . HoweverQSM can be used to alleviate this situation. Myeloid cells during the Gd-enhancing period phagocytose myelin fragments, a process that is reflected in the initial lack of change in the susceptibility of active lesions on QSM. After the BBB is reconstituted, myeloid cells metabolize diamagnetic myelin fragments, and at the same time or afterwards, microglial cells at the lesion periphery accumulate paramagnetic iron . Both myp < 0.005). Receiver operating characteristic (ROC) analysis for discriminating enhancing and non-enhancing lesions using susceptibility values showed an area-under-the-curve of 0.956. A cutoff-value of 11.2 ppb for QSM-measured susceptibility provided a sensitivity of 89.2% and specificity of 90.7%. Therefore, QSM can be used in routine MRI monitoring of new MS lesion activity to accurately identify the BBB leakage status of T2w lesions without gadolinium injection, which would be useful particularly for MS patients with contraindications for Gd injection.Using QSM and T2w together can allow for accurate identification of lesions in MS patients without Gd injection , utiliziIron homeostasis in the MS brain is profoundly perturbed, with iron-overload in lesional myeloid cells and in deep nuclei on one hand, and diminished iron concentrations in NAWM and chronic cortical lesions on the other. These shifts are likely to contribute to low-grade chronic inflammation, oxidative stress and neurodegeneration. QSM can be used to detect iron changes and may thus improve our ability to monitor therapeutic efficacy and progression in MS.In clinical practice, iron-sensitive imaging could therefore be used to aid treatment decisions in patients that appear clinically and radiographically stable but may have iron-positive, activated microglia within lesions. Moreover, accelerated resolution of iron accumulation could become an additional measure for treatment efficacy to reduce low-grade inflammation in established lesions.


Oral mucosa is a useful material for regeneration therapy with the advantages of its accessibility and versatility regardless of age and gender. However, little is known about the molecular characteristics of oral mucosa. Here we report the first comparative profiles of the gene signatures of human oral mucosa fibroblasts (hOFs), human dermal fibroblasts (hDFs), and hOF-derived induced pluripotent stem cells (hOF-iPSCs), linking these with biological roles by functional annotation and pathway analyses. As a common feature of fibroblasts, both hOFs and hDFs expressed glycolipid metabolism-related genes at higher levels compared with hOF-iPSCs. Distinct characteristics of hOFs compared with hDFs included a high expression of glycoprotein genes, involved in signaling, extracellular matrix, membrane, and receptor proteins, besides a low expression of HOX genes, the hDFs-markers. The results of the pathway analyses indicated that tissue-reconstructive, proliferative, and signaling pathways are active, whereas senescence-related genes in p53 pathway are inactive in hOFs. Furthermore, more than half of hOF-specific genes were similarly expressed to those of hOF-iPSC genes and might be controlled by WNT signaling. Our findings demonstrated that hOFs have unique cellular characteristics in specificity and plasticity. These data may provide useful insight into application of oral fibroblasts for direct reprograming. Oral mucosa is a convenient cell source for regenerative medicine, having the following advantages: 1) simple operation, 2) no cosmetic and functional problems after operation, 3) fast wound healing without scar formation [ simple o no cosme fast wouRecently, induced pluripotent stem cell (iPSC) technology has shown remarkable progress and has been applied to personalized medicine for diagnostics, drug screening, and regenerative therapy . We alsoSo far, two comprehensive transcriptome studies have been reported using oral mucosa. One included the comparison of the expression profiles between skin and oral mucosal tissue derived from wound healing mouse models . In this Krüppel-like factor 4 (KLF4) and v-myc avian myelocytomatosis viral oncogene homolog (c-MYC), which are the reprogramming factors for generating iPSCs, and maternally expressed gene 3 (MEG3), which is an imprinted gene and long noncoding RNA, were highly expressed in hOFs [ Meg3/Gtl2 is located within the delta-like 1 homolog 1 (Dlk1)-deiodinase, iodothyronine type III (Dio3) region and the activation of this region is associated with the level of pluripotency in iPSCs or ESCs [Additionally, we previously found that endogenous in hOFs . Meg3/Gt or ESCs . These fIn this study, we performed comparative analyses of gene profiles of hOFs, hDFs, and hOF-iPSCs to understand the molecular characteristics of hOFs. We chose hOFs derived from the buccal region, not other regions of oral mucosa  because of its superior accessibility as a cell source appropriate for future regenerative medicine. hOF-iPSCs were used as not only the alternative for fetal skin fibroblasts, but also pluripotent stem cells to find out the specificity in the gene signature of hOFs.hOFs were isolated from individually collected buccal mucosal tissues obtained from four healthy volunteers (26–35 years old) after receiving written agreement including an informed consent at the Tokushima University Medical and Dental Hospital. Approval from the Institutional Research Ethics Committee of the University of Tokushima was obtained (Project number 708). Details on hOFs isolation have been described previously [hOFs  were cultured in Dulbecco's Modified Eagle Medium  supplemented with 10% FBS . Three types of hDFs derived from individuals aged 33–36 years old were purchased from the Health Science Research Resources Bank . hDFs were cultured in Eagle's MEM  supplemented with 10% FBS (Nichirei Biosciences). solute carrier family 7, member 1 (mslc7a1), was introduced into hOFs using lentiviral infection. Then, four reprogramming factors, POU class 5 homeobox 1/octamer-binding transcription factor 4 (POU5F1/OCT4), KLF4, SRY (sex determining region Y)-box 2 (SOX2), and v-myc avian myelocytomatosis viral oncogene homolog c-MYC, were transduced by retroviral infection. Generated hOF-iPSCs were maintained in human ES medium  supplemented with 5 ng/mL of basic fibroblast growth factor (bFGF) on SNL feeder cells. The pluripotency of hOF-iPSCs was confirmed by the expression of the pluripotent cell markers and by in vitro differentiation through embryoid body formation.hOF-iPSCs were generated as shown previously . BrieflyRNA samples were prepared from three individual samples in each group . Total RNA was isolated using TRI Reagent , according to the manufacturer's protocol.μg of labeled DNA target was hybridized to the array for 17 h at 45°C on the GeneChip Hybridization Oven 640 (Affymetrix). After washing, arrays were stained on a GeneChip Fluidics Station 450 and scanned with a GeneChip Scanner 3000 7G (Affymetrix). A CEL file was generated for each array. All microarray data from the three groups  have been deposited in Gene expression Omnibus  under GEO Accession number GSE56805.Microarray analysis was performed as previously described . In brie2. For the technological variability, we checked several quality controls including Hybridization Controls (provided by Affymetrix), Histogram, Profile Plot, Matrix Plot, 3D PCA, Pearson's correlation coefficient, and hierarchical clustering analyses following the standard protocols provided by the manufacturers. Among them, the results of Hybridization controls and Pearson's correlation coefficient were shown in Supplementary Figures S1B and S1C in Supplementary Materials available online at http://dx.doi.org/10.1155/2015/121575, respectively. Each value of Pearson's correlation coefficient is indicated as follows: 1 indicates perfect positive correlation between two samples, 0.80 to 1.0 indicates very strong correlation, and 0.60 to 0.79 indicates strong correlation. Expressed genes that showed a fluorescence intensity greater than 100 were further analyzed. Average gene expression level was calculated for three samples in each group and used for the comparison. To make the stringent criteria, several statistical analyses were performed. First, the data obtained from the differently expressed genes between the 2 groups were analyzed by one-way ANOVA and cut off with the corrected p-value (p < 0.05) according to Benjamini-Hochberg (BH) method. Furthermore, Tukey's honestly significant difference (HSD) test was used as the post hoc test, and the differently expressed genes between the 2 groups were extracted. Among 28,869 gene probes, 12,713 gene probes were left after one-way ANOVA and BH analyses . From these 12,713 gene probes, more than 2-fold differentially expressed gene probes were selected between the two paired groups.The data were analyzed with GeneSpring GX12.0 . The normalization and summarization of CEL files were performed by “Exon RMA 16” algorithm. After that, the signal values of probe sets were transformed to the value of loghttp://david.abcc.ncifcrf.gov/) [http://www.genome.jp/kegg/) pathway tools.Functional analyses were performed using the Database for Annotation, Visualization and Integrated Discovery (DAVID) v6.7 (rf.gov/) , 21. MajTo analyze the molecular profile of hOFs, we prepared three types of cells, hOFs, hDFs, and hOF-iPSCs. Three independent cell lines from different donors were chosen to obtain accurate results from each cell type. Heat map and hierarchical clustering analysis revealed that the gene expression pattern in each group was conserved, except for hDF3 (TIG114) and hOF4, for which intermediate patterns between fibroblasts and hOF-iPSCs were identified . NotablyNext, average gene expression signal values in each group were calculated and used for further comparative analyses. Figure S1C shows the scattered plot of gene profile comparison between hOFs and hDFs. Each gene expression of samples was indicated as a spot, and most of them were exhibited within 2-fold line (green line). Therefore, threshold can be set at 2-fold to find the difference of gene expression profile between hOFs and hDFs.Out of 12,713 probes, we found that 5,738 probes and 5,672 probes (45%) were more than 2-fold differently expressed in hOFs and hDFs, respectively, compared with those in hOF-iPSCs ( micro RNA302a (MIR302A), MIR302B, lin-28 homolog A (LIN28A), Nanog homeobox (NANOG), developmental pluripotency associated 4 (DPPA4), glypican 4 (GPC4), prominin 1 (PROM1), growth differentiation factor 3 (GDF3), POU5F1/OCT4, and SOX2. We also found that the reprogramming factors KLF4 and c-MYC were highly expressed in hOFs and hDFs than in hOF-iPSCs. These results were consistent with previous observations [At the beginning, we confirmed the expression levels of several embryonic stem cells (ESCs) markers and reprogramming factors that had been generally observed in iPSCs . As expeTo elucidate the characteristics of hOFs, we first compared the gene profiles of fibroblasts (hOFs or hDFs) with hOF-iPSCs in steady-state condition. For prediction of the biological function of respective gene profiles, we matched functionally related gene groups to the known pathways by pathway analysis using DAVID linked with KEGG. Genes in thirty pathways were expressed at lower levels in hOFs and hDFs than in hOF-iPSCs, suggesting that these pathways are functionally active in hOF-iPSCs . High exConversely, 46 pathways were enriched among the highly expressed genes in hOFs and hDFs compared with those in hOF-iPSCs . We foun distal-less homeobox 5 (DLX5), LIM homeobox 8 (LHX8), paired box 3 (PAX3), PAX9, and transcription factor AP-2 alpha (TFAP2A), were expressed at a remarkably high level in hOFs .Next, we performed pathway analysis to understand the intracellular events in hOFs > hDFs and hOFs < hDFs. In the group of hOFs > hDFs, eleven pathways were enriched . However, the downstream genes, cyclin D1 (CCND1), growth arrest and DNA-damage-inducible beta (GADD45B), serpin peptidase inhibitor, clade E, member 1/plasminogen activator inhibitor type 1 (SERPINE1/PAI-1), and insulin-like growth factor binding protein 3 (IGFBP3) were downregulated in hOFs. These molecules regulate cell cycle, DNA repair, antiangiogenesis, and the anti-insulin-like growth factor 1 (IGF-1) pathway [ Tenascin C (TNC), integrin, alpha 1 (ITGA1), cartilage oligomeric matrix protein (COMP), and ITGA6 were identified and seen to overlap in ECM-receptor and focal adhesion pathways.Conversely, only three pathways  were enriched in hOFs < hDFs , suggesting that the genes reflect the plasticity or undifferentiated property of multipotent hOFs by enhancement. Second, 103 genes were highly expressed in hOFs compared with those in hDFs and hiPSCs . The genes in G2 were highly expressed in hOFs but may be kept at low or absent expression levels in hDFs. Therefore, it was suggested that the genes in G2 can exhibit the specificity or differentiated property of hOFs. Third, 70 genes in hOFs had expression levels similar to hOF-iPSCs but were expressed at lower levels than in hDFs . The genes in G3 are defined as the specificity of hDFs; however, these genes could be also involved in the plasticity of hOFs by being suppressed. Twenty-two genes in hOFs were expressed at lower levels than in hDFs that showed expression levels similar to those of hOF-iPSCs , suggesting specificity or differentiated property of hOFs by suppression and, reciprocally, plasticity or undifferentiated property of hDFs.We further analyzed the individual components in the G1–G4 groups, and we categorized them into seven groups, such as ECM/secreted, membrane, receptor, enzyme, signaling, transcriptional regulator, and others . The gen transcription factor 7-like 1 /T-cell factor-3 (TCF7L1/TCF3) and transducin-like enhancer of split 1 (E (sp1) homolog, Drosophila Groucho) (TLE1) are involved in controlling ESCs status by functioning as components of wingless-type MMTV integration site family (WNT) signaling. The transcriptional regulators in G2 are involved in the early developmental regulation, and they are also recognized as markers of the cranial neural crest. The transcriptional regulators in G3 are rich in HOX genes, which are involved in determining localization and morphology.Then we observed that the transcriptional regulator accounted for 10% in both G1 and G2, 34% in G3, and 0% in G4, highlighted by pink color in  Gli-similar 1 (GLIS1), methyl-CpG-binding domain protein 3 (MBD3), retinoic acid receptor, gamma (RARG), and T-box 3 (TBX3), was detected in hOFs and hDFs , was expressed at the higher level in hOFs than in hDFs and hOF-iPSCs.Lastly, we surveyed expression levels of reprogramming regulators because these can support plasticity in fibroblasts. Compared with hOF-iPSCs, the higher expression of reprogramming enhancers, such asIn this study, we elucidated the unique characteristics of hOFs through comparative analyses of gene expression profiles among hOFs, hDFs, and hOF-iPSCs. In x) GSL antigen. Stage-specific embryonic antigen-1 (SSEA-1), a marker for both mouse ESCs and embryonic carcinoma cells (ECCs), is also included in this group, and it contains Lex and mediates homotypic adhesion related to compaction or autoaggregation [ UDP-GlcNAc:betaGal beta-1,3-N-acetylglucosaminyltransferase 5- (B3GNT5-) deficient mice, has been reported to cause preimplantation lethality [First, we noted activated GSL metabolism in both hOFs and hDFs compared with hOF-iPSCs Figures . GSLs arregation . Furtherethality  or multiethality . The gloethality , 36. in vivo [ in vitro [ UDP-Gal:betaGlcNAc beta 1,3-galactosyltransferase, polypeptide 5 (B3GALT5), which catalyzes the conversion from globotetraosylceramide (Gb4cer) to globopentaosylceramide (Gb5cer) . Because Gb3cer and other glycosphingolipids are also involved in caveolar-1 oligomerization [In our profiles, GSL-related GTs in the globo-series and ganglio-series GSL biosynthetic pathways were highly expressed in both hOFs and hDFs compared with their expression in hOF-iPSCs, whereas GTs in the lacto-series/neolacto-series GSL biosynthetic pathways were less expressed . GSL exp in vivo  and diffin vitro , 38. Gloin vitro , 38. Bas Figures , was lowrization , their aα), and cytochrome c oxidase (COX) could regulate the metabolic transition from aerobic glycolysis in mouse ESCs to anaerobic glycolysis in mouse epiblast stem cells (EpiSCs) and human ESCs [ HIF1α and COX were similar among hOFs, hDFs, and hOF-iPSCs in our profiling data (data not shown). Collectively, hOFs and hDFs appear to exhibit the bioenergetically intermediate phenotype between stem cells and terminally differentiated cells, showing the potential to select cell fate, together with membrane sensing of GSLs.Another unique “fibroblastic” feature is the underexpression of aerobic and anaerobic glycolysis-related genes in hOFs and hDFs . This finding suggested that hOFs and hDFs are bioenergetically less active than hOF-iPSCs. A recent study reported that the metabolic switching of energy metabolism is linked with cell fate decision , consistman ESCs . However in silico analyses. The glycoprotein group was highly enriched in hOFs compared with hDFs  related factor 2 , nuclea enzymes ), peroxil stress ), PPAR dneration ), prion activity ), retino protein ), and si protein ). These We performed comparative analyses between hOFs and hOF-iPSCs, which were generated from parental hOFs  to eluci TCF7L1/TCF3 and TLE1, the negative regulators of canonical WNT signaling. In human ESCs, canonical WNT signaling actively regulates pluripotency. However, to differentiate into specific cell types of mesodermal and endodermal lineages, WNT signals need to be transiently downregulated by TCF7L1/TCF3 and TLE1 [ Tcf7l1/Tcf3 regulates stage-specific WNT signaling during the reprogramming of fibroblasts into iPSCs [ TCF7L1/TCF3 in skin wound healing was reported by demonstrating that TCF7L1/TCF3 was upregulated in epithelial cells at the site of injury, accelerating wound healing in vivo through lipocalin-2 (Lcn2) induction [ glypican-3 (GPC3) [ R-spondin 1, 2  [ WNT16 [ RSPO1 has been demonstrated to commit to the specification of germ cells, and RSPO2 plays a role in craniofacial, limb, and branching development [ WNT16 is involved in the specification of hematopoietic stem cells [ HOX genes were repeatedly categorized into the underexpressed gene group of hOFs  –77, a seSPO1, 2) , 79, and [ WNT16   (lung), LIM homeobox 8 (LHX8) (nerve), microphthalmia-associated transcription factor (MITF) (melanogenesis), PAX9  [ PPARG (adipocyte) [The specificity of hOFs is mainly characterized by a high expression of cranial neural crest markers . Forkheand limb) , and PPAipocyte) , 83 are  RARG [ TBX3 [ nuclear receptor subfamily 5, group A, member 2/liver receptor homolog 1 (NR5A2/LRH-1) [ Oct transcription [Rarg and Nr5a2 combined with achaete-scute complex homolog 1 (Ascl1), POU domain, class 3, transcription factor 2 (Pou3f2/Brn2), and neurogenin 2 (Ngn2) enhanced the efficiency of transdifferentiation from MEFs to functional neurons [ TBX3 is necessary to maintain pluripotency of mouse ESCs and also to regulate differentiation, proliferation, and signaling [ TBX3 in hESC regulates proliferation and differentiation [ RARG and TBX3 in hOFs are not fully understood, it might be possible for these to regulate the plasticity of hOFs  , and dircription , 89, andcription . Recentl neurons . Conversignaling , 91, 92,ntiation . Althoug of hOFs . GLIS1, [ NR5A2/LRH-1 was underexpressed in both hOFs and hDFs. LIN28A, a miRNA and a reprogramming repressor controlling cell plasticity [ MBD3, the suppression of which can increase reprogramming efficiency [ POU5F1/OCT4 can introduce reprogramming [ in vitro and in vivo [In the other transcription factors, GLIS1,  was highasticity , 96, wasficiency , was higgramming , 99. Furgramming –103. Tragramming . Recentlgramming . Since D in vivo –105, hOF MEG3 was expressed at a quite high level compared to those of hDFs and hOF-iPSCs . Interestingly, hOFs highly expressed both paternal imprinted genes, DIRAS3 and IGF2, and maternal imprinted genes, H19 and MEG3, compared to those in hOF-iPSCs. Furthermore, the expression of DLK1 and DIO3, which are paternally expressed genes and located within the same region as MEG3, was lower than that of MEG3 in hOFs. We found a similar expression pattern within 14q32 in hOF-iPSCs. MEG8, known as Rian in mouse, is also located within 14q32 and maternally expressed, long noncoding RNAs were not on the lists of gene profiles. The expression of DIRAS3, PEG10, and IGF2 was reciprocally observed between hOFs and hOF-iPSCs. Furthermore, although H19 and IGF2 exhibit maternal and paternal expressions that are located in the same region, both genes were highly expressed in hOFs. At this moment, we do not know the biological meaning of their expression patterns. Further analyses will be required. MEG3 is also known as a tumor suppressor via p53 activation [ p53 in hOFs, MEG3 could also be involved in the specificity of hOFs by controlling p53 signaling as shown in In addition,OF-iPSCs . Becausetivation , 107. Be WNT and p53 gene network as shown in We elucidated the fibroblastic plasticity and specificity by analyzing transcriptome profiles of GSL metabolism in hOFs and hDFs. The uniqueness of hOFs is defined as partly primed cells committed to the neural crest cell lineage with plasticity and longevity controlled byThe reliability of microarray hybridization techniques were confirmed by the company-supplied hybrydization control.


Escherichia coli isolates have been increasingly reported worldwide. Animal to human dissemination, via food chain or direct contact, of these resistant bacteria has been reported. In Tunisia, little is known about avian ESBL- producing E. coli and further studies are needed. Seventeen ESBL-producing Escherichia coli isolates from poultry feces from two farms (Farm 1 and farm 2) in the North of Tunisia have been used in this study. Eleven of these isolates (from farm 1) have the same resistance profile to nalidixic acid, sulfonamides, streptomycin, tetracycline, and norfloxacine (intermediately resistant). Out of the six isolates recovered from farm 2, only one was co-resistant to tetracycline. All isolates, except one, harbored blaCTX-M-1 gene, and one strain co-harbored the blaTEM-1 gene. The genes tetA and tetB were carried, respectively, by 11 and 1 amongst the 12 tetracycline-resistant isolates. Sulfonamides resistance was encoded by sul1, sul2, and sul3 genes in 3, 17, and 5 isolates, respectively. The qnrB1 was detected in nine strains, one of which co-harbored qnrS1 gene. The search for the class 1 and 2 integrons by PCR showed that in farm 1, class 1 and 2 integrons were found in one and ten isolates, respectively. In farm 2, class 1 integron was found in only one isolate, class 2 was not detected. Only one gene cassette arrangement was demonstrated in the variable regions (VR) of the 10 int2-positive isolates: dfrA1- sat2-aadA1. The size of the VR of the class 1 integron was approximately 250 bp in one int1-positive isolate, whereas in the second isolate, no amplification was observed. All isolates of farm 1 belong to the phylogroup A (sub-group A0). However, different types of phylogroups in farm 2 were detected. Each of the phylogroups A1, B22, B23 was detected in one strain, while the D2 phylogroup was found in 3 isolates. The virulence genes iutA, fimH, and traT were detected in 3, 7, and 3 isolates, respectively. Two types of gene combination were detected: iutA+fimH+traT in 3 isolates and iutA+fimH in one isolate. The isolates recovered in farm 1 showed the same profile of PFGE macro-restriction, while isolates of farm 2 presented unrelated PFGE patterns. We conclude that these avian ESBL-producing E. coli isolates show homo- and heterogenic genetic background and that plasmids harboring ESBL genes could be involved in the dissemination of this resistance phenotype.Avian ESBL-producing  Escherichia coli is a commensal bacterium in humans and animals and considered an indicator of fecal contamination of food. Antimicrobial resistant isolates and resistance genes of E. coli can be transferred to humans through the food chain. This transfer represents a potential risk for public health .Sixty-five fecal samples were collected from healthy chickens in 2013. Of these samples, 45 came from 58-week-old chickens in a farm located at Sidi Thabet in the North of Tunisia (Farm 1) and 20 from 7-week-old chickens in a farm located at Morneg region (Farm 2). These samples were cultivated on Mac-Conkey agar containing 2 mg/L of cefotaxime and incubated overnight at 37°C. For each sample, one colony with typical E. coli ATCC25922 was used as ESBL negative and Klebsiella pneumoniae 700603 was used as ESBL positive reference strain.Antimicrobial susceptibility testing was carried out using the agar disk diffusion method on Mueller–Hinton agar plates in accordance with the Clinical and Laboratory Standards Institute guidelines , sulfonamide , and quinolones  were investigated by PCR as reported previously  (Table All primers used to characterize the resistance genotype are presented in Table s) Table .fimA, TartT, iutA, MaIX, Ibe, FyuA, BmaE, papGalleleIII, papC, colV, cdtB, papG alleleI, nfaE, SfaS, iha, iss, ire, ehxA, sxt1, sxt2, eltA, fasA, estII, aggC, esat1, cdt, ipah, hly, cnf1, and bfp) was determined by using PCR in all ESBL-positive E. coli strains  , trimethoprim-sulfamethoxazole, sulfonamides, streptomycin and tetracycline. But, they remained susceptible to imipenem, gentamicin, tobramycin and chloramphenicol. With the exception of one strain that was co-resistant to tetracycline, isolates, from farm 2, were susceptible to all non beta-lactam antibiotics (Table Eleven 24%) and 6 (30%) ESBL-producing 4% and 6 blaCTX−M−1 genes. One strain co-harbored blaTEM−1 gene. The blaSHV gene was not detected.PCR and sequencing showed that all strains, except one, harbored the dfrA1- sat2-aadA1, encoding resistance for trimethoprim, streptothricin and streptomycin, respectively. In one isolate, the VR of class 1 integron was amplified and yielded a DNA fragment of ca. 250 bp, whereas no amplification was observed in the second int1-positive isolate.In farm 1, class 1 and 2 integrons were found in one and ten isolates, respectively. In farm 2, class 1 integron was found in only one isolate, while class 2 was not detected. Amplification of the VRs of class 2 integrons showed identical DNA fragments with an approximate size of 2000 bp. All these VRs contained a unique gene cassette arrangement, being 2, and B23, each in one isolate, and D2, in 3 isolates). The virulence genes iut(A), fimH, and traT were detected in 3, 7, and 3 isolates, respectively. The other genes were not detected in our collection. Two types of gene combination were detected: iutA+fimH+traT (in 3 isolates); iutA+fimH (in one isolate).In farm 1, all isolates were found to belong to phylogroup A (sub-group A0); however, different phylogroups were detected in farm 2 , sat1 (encoding streptothricin resistance) and aadA1 (encoding streptomycin/spectinomycin resistance)  (Farm1) and 20 samples of 7-weeks-old breeder chickens in the region of Morneg (Farm 2). Seventeen samples (26.1%) contained cefotaxime-resistant isolates, 11 (24%) and 6 (30%) positives samples were observed in farms 1 and 2, respectively. The frequency of cefotaxime-resistant isolates is similar to those reported by other authors worldwide   and topoisomerases IV (parC and parE) Ib-cr have been increasingly reported in bacterial pathogens since 2000 -Ib-cr genes, while other authors reported their presence in E. coli from animal origin  of fluoroquinolones (ciprofloxacine and norfloxacine) and were generally not detected by disk diffusion method. Indeed, the resistance toward quinolones in 7 amongst the 11 nalidixic acid resistant isolates could be explained by these qnr genes but it could also certainly explained by the presence of chromosomal mutations in QRDR of GyrA and/or ParC .Tetracycline resistance was encoded by o Sköld, . Resista Hawkey, . HoweverE. coli strains according to phylogroups, it is well-known that pathogenic strains producing extra-intestinal infections (ExPEC) belong mainly to the B2 group and less to the D group. They are responsible for meningitidis, abscess, peritonitis, septicemia and urinary tract infections . In the literature, the majority of animal E. coli isolates producing ESBL belonged to phylogroup A and B1 . The low number of genes detected could be explained by fitness notion or the competitively of strains. Indeed, these avian intestinal E. coli isolates were under selective presser by antibiotics. This selective pressure enhances the expansion and maintenance of antibiotic-resistant strains rather than virulent ones.Concerning the distribution of pathogenic sul genes, qnr genes and virulence genes) might be due to independent acquisition of genes carried by plasmids or integrons.PFGE showed that the 11 isolates of the farm 1 were indistinguishable, while isolates of farm 2 were unrelated. The cloanlity of these isolates would evoke a strong power of dissemination of clonal isolates in this poultry breeding. The difference in genetic contents in isolates of farm2 (E. coli in these two farms, our study showed two typical epidemiologic characteristics of ESBL-producing bacteria. Firstly, clonal isolates are disseminated within the same farm. Secondly, “singleton” isolates occur with limited ability of spread, evoking the potential horizontal transfer of ESBL genes between different E. coli populations. Plasmids or integrons might be implicated in the mobilization of blaCTX−M−1 genes among avian isolates. Further studies should be performed in the future to track the evolution of ESBL types and their frequencies in different ecosystems.Despite the limited samples analyzed in this study, that might not reflect the real epidemiological situation of avian ESBL-producing HK, MA, RM, SH, NC, and IB: Conceiving and designing the study, collecting and interpreting the data, and writing the article. SS, RS, IJ, and SF: interpreting the data, revising the article.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.


Nucl. Acids Res. (2010) 38 (15): 4929-4945. doi:10.1093/nar/gkq200Editorial Expression of Concern The Editors express a note of concern regarding the article“A tripartite paternally methylated region within the Gpr1-Zdbf2 imprinted domain onmouse chromosome 1 identified by meDIP-on-chip “ by Hiura H, Sugawara A, Ogawa H,John RM, Miyauchi N, Miyanari Y, Horiike T, Li Y, Yaegashi N, Sasaki H, Kono T, and ArimaT, which was published in issue 15, volume 38 (2010) of Nucleic Acids Research.Supplementary Data accompanying this expression ofconcern. Having reviewed the original data, we believe that the results and conclusion ofthe article are valid but wish to alert our readership to these concerns.The Editors wish to alert the readers that questions have been raised about the validity ofsome of the figures presented in this article. The Editors note that Figures 2, 4, 6 and S3contain duplicate panels. New figures are available in the Keith Fox Barry Stoddard Senior Executive Editors Nucleic Acids Research


Myeloid sarcoma represents the proliferation of myeloblasts of acute myeloid leukemia (AML) at extramedullary sites. While extramedullary involvement in AML is uncommon in itself, isolated myeloid sarcomas, that is, myeloid sarcomas without any bone marrow involvement, are extremely rare and pose a diagnostic and therapeutic challenge. Here, we present the case of a middle-aged woman with isolated myeloid sarcoma in the stomach—an organ seldom involved by this disease. Additionally, the literature on the epidemiology, diagnosis, pathology, prognosis, and therapeutic options in myeloid sarcomas has been reviewed. This entity can involve almost any organ system, including the skin (“leukemia cutis”), lymph nodes, bone, brain, breast, cervix, and visceral organs. Here, we present the case of a middle-aged woman who was found to have isolated MS in the stomach, a very infrequent site of involvement.Acute myeloid leukemia (AML) is a cancer of the myeloid elements of the bone marrow characterized by the rapid proliferation of abnormal blasts in the bone marrow that interfere with normal hematopoiesis. Rarely, however, the disease can manifest with extramedullary organ involvement, known as myeloid sarcoma (MS) or, previously, chloroma or granulocytic sarcoma. Defined as the presence of proliferating myeloid blasts in an extramedullary site that disrupts the normal architecture of the organ in which they are found , MS is rin situ hybridization). CD10, CD128, TdT, and BCL-2 staining were variably positive, and the proliferation fraction (Ki-67) was 60%. However, morphologic bone marrow examination, IHC, karyotyping, and FISH (fluorescence in situ hybridization) using probes designed to detect rearrangements of the c-MYC (8q24), CDKN2A (9p21), and MLL (11q23) loci as well as t and t were normal. Complete blood count (CBC) revealed leukocytes of 3,000/μL, hemoglobin of 11.8 g/dL, and platelets of 201,000/μL; the blood smear showed no circulating blasts. A diagnosis of isolated gastric MS, monocytic subtype (by the French American British classification), was made based on the expression of CD34, CD117 (both blast markers), CD4, CD68, CD163, and lysozyme (suggestive of monocytic differentiation). Positron emission tomography with CT (PET/CT) showed intense and diffuse radiotracer uptake in the thickened gastric wall with a maximum standardized uptake value (SUVmax) of 14.7 that was closely associated with adjacent liver, pancreas, and spleen  and slight decrease in size of the mass. However, EGD-guided gastric biopsy revealed no malignant cells. Immunophenotyping was unremarkable as well. Given the PET/CT findings, however, it was thought that the biopsies had missed malignant sites. Surgical resection was not recommended considering the involvement of adjacent structures. Radiation therapy to a total dose of 27 Gy over 15 fractions was administered. Significant reduction of metabolic activity in the gastric region (SUVmax 6.2) was noted on a follow-up PET/CT scan. Three months later, the patient presented with abdominal pain and vomiting. CBC was normal, but CT scan showed peritoneal carcinomatosis. Fine needle aspirate of a pelvic lymph node was consistent with the previously rendered diagnosis of MS. At this point, the patient decided to forego active treatment and died a few weeks later.A 52-year-old otherwise healthy female presented with a month-long history of dyspepsia. A trial of proton-pump inhibitor therapy proved unsuccessful and she developed severe dysphagia and odynophagia over the ensuing three months. Examination was unremarkable besides epigastric tenderness. Computed tomography (CT) revealed 3-cm gastric wall thickening, most prominent in the fundus and body, extending into the gastrohepatic ligament and along the celiac artery, inseparable from the pancreatic body and tail. Esophagogastroduodenoscopy (EGD) showed a gastric fundus mass and pathology revealed infiltration of the lamina propria with large atypical cells that demonstrated nuclear pleomorphism with prominent nucleoli. Immunohistochemistry (IHC) was positive for CD4, CD33, CD34, CD43, CD45, CD68, CD117, CD163, and lysozyme. The neoplastic cells were, however, negative for CD3, CD5, CD8, CD20, CD30, CD56, CD138, PAX5, cyclin D1, MUM-1, BCL-6, pan-keratin AE1/3, TIA, myeloperoxidase, and EBER (Epstein Barr encoded RNA) by ISH , MS is initially misdiagnosed, with lymphoma being the most common incorrect diagnosis , 16. IndMLL) rearrangements (8.5%) were the commonest abnormalities [ de novo AML (101 with extramedullary involvement and 230 without) showed that t(8:21) was present in only 3.9% of extramedullary cases and 5.7% of cases without extramedullary involvement, a difference that was statistically insignificant [μL versus 9,500/μL) in cases with extramedullary involvement. The Italian study had shown CD68/KP1 to be the most commonly expressed marker (100%), followed by myeloperoxidase (83.6%), CD117 (80.4%), CD99 (54.3%), CD68/PG-M1 (51%), CD34 (43.4%), terminal-deoxynucleotidyl-transferase (31.5%), CD56 (13%), CD61/linker for activation of T cells (2.2%), CD30 (2.2%), and CD4 (1.1%). Taken together, key observations from the two studies mentioned above include the association of 11q23 abnormalities, CD56 expression, and AML with myelomonocytic/monoblastic differentiation (M4/M5a) with MS formation. A retrospective study showed that 17 out of 20 cases of MS associated with inv(16), the other CBF abnormality in AML, had abdominal involvement with intestine being the most common site (13 of 17 cases) [Previous studies had indicated that the core-binding factor (CBF) abnormality t8:21) was the most common cytogenetic abnormality associated with MS formation , 20. How was the 7 cases) . UnfortuMS has traditionally been considered a poor risk factor in AML. Whether it truly confers a poor prognosis is, however, uncertain. A retrospective study showed a nonsignificantly increased two-year event free (32% versus 18%) and overall survival  in isolated MS as compared to leukemic AML . The optIsolated MS is an extremely rare disease and isolated gastric MS even more so. Like all isolated MS cases, it poses diagnostic challenges, given its rarity. Clinical suspicion is the key to correct diagnosis. Morphologic evaluation, along with IHC, flow cytometry, cytogenetics, and molecular studies should be performed and systemic treatment for AML, with or without local therapies, pursued.


The present treatment of childhood T-cell leukemias involves the systemic administration of prokary-otic L-asparaginase (ASNase), which depletes plasma Asparagine (Asn) and inhibits protein synthesis. The mechanism of therapeutic action of ASNase is poorly understood, as are the etiologies of the side-effects incurred by treatment. Protein expression from genes bearing Asn homopolymeric coding regions (N-hCR) may be particularly susceptible to Asn level fluctuation. In mammals, N-hCR are rare, short and conserved. In humans, misfunctions of genes encoding N-hCR are associated with a cluster of disorders that mimic ASNase therapy side-effects which include impaired glycemic control, dislipidemia, pancreatitis, compromised vascular integrity, and neurological dysfunction. This paper proposes that dysregulation of Asn homeostasis, potentially even by ASNase produced by the microbiome, may contribute to several clinically important syndromes by altering expression of N-hCR bearing genes. By altering amino acid abundance and modulating ribosome translocation rates at codon repeats, the microbiomic environment may contribute to genome decoding and to shaping the proteome. We suggest that impaired translation at poly Asn codons elevates diabetes risk and severity. IRS2, encoding an insulin signal transducer, is the gene at the top of the list in Despite similar Asn codon usage, ~4%/gene, from plants to humans , mammalsin vitro (in vivo (Asparaginase (ASNase) is a component of highly effective chemotherapeutic regimens used to treat pediatric acute lymphoblastic leukemia (ALL) ,5 and soin vitro  and in v. In the abstract, reduced rates of translation anywhere along an mRNA would result directly in a reduced overall rate of target protein synthesis and, depending on protein halflife, result indirectly in decreased steady state levels of such proteins. High rates of translation may even increase the halflife of an mRNA , and eight percent with vasculature and hematopoesis. Of the remaing ~14%, many can be classified as involved with chromatin modification, DNA maintainance and repair, RNA transcription and processing or protein synthesis and turnover, some have Leucine rich repeats that can serve as pattern recognition elements. Some genes fall into multiple categories, e.g. epilepsy , aspectsepilepsy , Alzheimepilepsy –73, retiepilepsy , hippocaepilepsy , long teepilepsy , ataxia epilepsy , cardiacepilepsy , kidney epilepsy , renal depilepsy , breast epilepsy ,82, rhabepilepsy  and, in topoesis ,85. A lidiabetes .CXCR2, (EPQ18419), which has a 60N-hCR. Other genes of interest from mouse, that differ from human in N-hCR length, include MDR1 and CFTR , and TNFRSF16/BEX3A/NGFRAP1 (implicated in diabetes)  . For exaiabetes)  as well s)   . The genMPK/SIX5 ,112, GCLX5  subset of children and its mouse homolog regulates energy homeostasis and body weight (cf. KCNH43N-hCR and KCNH83N-hCR). Pancreatitis and diabetes are associated, respectively, with CFTR4N-hCR and SLC26A93N-hCR, the products of which physically and functionally interact. CFTR is an ion channel, closely related, by membership in the superfamily of ATP-binding cassette proteins, to the multidrug resistance transporter (MDR1) , very similar in location to Asn416 in the N-hCR of CFTR. Such polymorphisms in MDR1 have been proposed  between the membrane spanning domain (MSD) and the nucleotide binding domain (NBD) could, by analogy to the key MDR1 Gly412 substitution, alter translation rate at its Asn 415 to 418 region, under conditions of low Asn, to result in generation of CFTR protein folding variants (SLC26A93N-hCR)  –153. Somproposed  to affecproposed . The N-hvariants  with alt93N-hCR) –158, Saltibility , and timtibility .TRPM64NhCR and TRPM73N-hCR,4NhCR. Allelic variation of the former has been associated with elevated risk of diabetes, osteoporosis, asthma, and heart and vascular diseases  but also CFTR (Ile507 and ΔF508) (SCARB1 (Ala350)  and ischemic stroke  receptor that participates in lipid metabolism and flux of cholesterylesters   and SCAR(Ala350) . Rs5888,c stroke –171. Traylesters  into e.gylesters  and thusylesters . SCARB1 farction  and renafarction ,177 actifarction , and caufarction ; it indifarction , mitigatfarction , and afffarction  and macufarction . By inflfarction –188. A sfarction ; this suF53N-hCR104t (encoding coagulation Factor V) have been linked to coronary artery disease  associated with ischemic stroke  in diabetics  , age of onset of amyotrophic lateral sclerosis (ALS) (TTLL4 and LAMA3) (TMEM106B) (TTLL5) (TTLL5 and PHACTR1) (LRP4 and NCOA1) (TBC1D3 and TBC1D3F) (GRAIL/RNF128/isf2)   ,217, breCAM5/CEA , CYP19A1romatase , IRS2, C CLEC10A , LRP6 and TBC1D5 ], spinald LAMA3) , dementiMEM106B)  retinal  (TTLL5) , large aPHACTR1)  decreased NCOA1) , ovarianTBC1D3F)  T cell a28/isf2) –230, ast28/isf2) –233 and  -B, -C)  suggestiDZIP1, MAML2, BOC and CDON, and may present attractive targets for drug discovery via small molecules that accelerate turnover of specific proteins encoded by N-hCR bearing-genes, synergistically magnifying the impact of ASNase by altering the replacement rate and perhaps by establishing lowered steady state levels of the targeted protein. There is already a precedent for synergism of prednisolone with ASNase, which occurs by an as yet unknown mechanism. The halflife of WNT signalling complexes and the contribution of DSV to turnover of WNT coreceptors FZD and LRP6 has recently been characterized   ,244 comeisorders . Since SANK3, IRS2, SNCAIP, XIRP2, PPP1R9A and CACNA1-C. Low plasma Asn, via the 17 N-hCR-bearing-genes listed in SNCAIP; dental caries and peridontal disease as a diabetes comorbidity through TMEM178B or ANKRD17 in children (cf. LRP1B and periodontitis in adults) (LRP1B are age at menarche (cf. glaucoma), conditional erectile dysfunction in African American men, heart rate and multiple cancers. Deafness , heroin addiction vulnerability in African Americans (XIRP2 (PHACTR1 (cf. LRP6) and PPP1R9A  ; erythropoesis and quality control of mitochondria by BNIP3L; nucleic acid processing by COIL, PAPD5, THRAP3, MEX3B and C1orf86/FAAP20; and diabetes by THRAP3 (cf. CHRM3), PTPRD and IRS2.Adverse neurological outcomes have also been associated with N-hCR-bearing-genes children ,253; (cf adults) . Also afmenarche , APOE anmenarche , protectmenarche  as well Deafness ,259 is americans  and hears  ; bone dePEG10 and BNIP3L, have transcripts with long N-hCR that are encompassed within their initial two dozen codons. Both BNIP3L and PEG10 are apoptosis-related genes that are candidates for mediation of the cell death that has been observed to follow depletion of Asn either in cell culture  . Temperature sensitive mutants of Asn tRNA synthetase undergo cell cycle arrest in early S phase at the nonpermissive temperature, a phenomenon that has been posited to be consistent with the existence a protein required for cell cycle progression that is highly sensitive to the level of charged Asn-tRNA (cf. COIL above).The discussion above has focused on adverse events elicited by ASNase therapy, not the induction of tumor remission. Two N-hCR-bearing-genes,  culture  or in pend RYR2) , JAK2, K2, KCNA3 , UBE2Q2    were performed with defined templates containing Asn codons at predetermined sites. T7 RNA polymerase was used to generate transcripts that were either devoid of Asn codons or contained one, two, five or 23 Asn codons between the N- and C-terminal segments of a bipartite hybrid protein composed of two human genes with no Asn codons. The N-terminal portion was derived from TCL1A, and the C-terminal portion was derived from CKS2. The central, intragenic N-hCR was, on occasion, substituted by the programmed ribosomal frameshifting (PRF) region from PEG10 which contains an Asn (AAC) codon at the frameshifting site. The resulting in vitro transcripts were translated in rabbit reticulocyte cell free lysates with isotopically labelled 35S-methionine and the products were analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis followed by autoradiography. This template gave extremely clean IVT results without the partial products seen with other templates such as PEG10 or gaussia luciferase. It was determined, with some appropriate control experiments, that there were quantities of ASNase that could be added to the translation mix to create different ratios of partial to full length products which could reflect relative degrees of pausing at the different poly Asn regions of length zero, one, two, five and 23 codons. Free Asn could subsequently be added back to the depleted reaction mix to 'chase', to a first approximation, the short 'TCL1A' proteins into longer, hybrid, 'TCL1A/CKS2' proteins. Conditions were also established in which the relative efficiency of frameshifting at the Asn codon of the PRF site of PEG10 was affected by exogenous ASNase added to the in vitro translation reaction, but this result was far less compelling than the effect of ASNase on translocation at N-hCR.ASNase in bsiella) , is encolower Km . Studiesynthesis . To deteynthesis ,274 in vSalmonella ASNase, of in vitro translation reactions  in adults compared to children underscores the difference between the physiology of children and adults.TBC1D33N-hCR gene of hominids, and the expansion, and perhaps positive selection in humans, of eight members of this N-hCR bearing-gene family (cf. TBC1D5 with Rab7 (TBC1D3 is involved in pinocytosis with ARF6 (cf. 285). These genes could also potentially regulate insulin or amino acid release from vesicular or lysosomal storage , or in human, mouse, rat, lizard, ~frog and chicken (BNIP3L), indicating that the two isoacceptor tRNAs may indeed be differentially regulated.Most of the poly Asn codon runs reported here consist of the two isoaccepter codons AAT and AAC used in about equal frequency with a slight bias towards homopolymeric runs of AAC. In the gene ructures , RNA seqructures ]. InterePPP1R9A5Nx2-hCR, unique among the 17 genes of FAAP20 can form a highly structured α helix upon ubiquitin binding; this domain is interrupted by a 5N-hCR in certain variant isoforms. The 2N-hCR of TP53 is similarly located: adjacent to a pair of transactivation domains (TADs) that gain structure upon ligand binding   ]. The 4N member)  that may member) . A simil member) ,306, as  member) , a regio member) ,309. The member)  perhaps se model . TXNIP ose model ,312 and se model . Alteredse model . Deletiofunction ,314. Regfunction , but seefunction . hCR mayfunction  where prfunction . An altefunction .At least five different human tRNA synthetases can serve as autoantigens in inflammatory responses . Human tcf. L-hCR length polymorphisms and diabetic nephropathy in CNDP1   (RYR2 and CHRM3. RYR2 is involved with heart disease (CHRM33N-hCR is associated with response to an antidiabetic drug in African Americans (cf. CHRM24N-hCR associated with metabolic syndrome)  cf.  as well echanism , to mBEXm  . Asn lev disease  and asso disease  and ALL  disease , CHRM33Nmericans  (cf. CHRyndrome) . Another) : IL9R (ARID5B (cf. KCNA3) (IL9R shares a common γ subunit with other interleukin receptors) ]. ARID5B encodes part of a histone lysine demethylase complex : IL9R  and ARID. KCNA3) . IL9R shceptors)  IL9R has complex  and is n complex ,364–369  complex , low bir complex , diastol complex  rheumato complex , respons complex  (an anti complex , lipid b complex  and trig complex , as well complex . The contigation .We propose that the impaired translation which has been described above be termed the 'translational N-hamper effect' because there is nothing intrinsically impaired about a protein polymerization reaction in which one of the required components, activated Asn tRNA, is ratelimiting for the translocation reaction on the template mRNA. The verb of choice for slowed translocation could just as well have been cumbered movement instead of hampered movement. If the argument was first made for Gln, the Q-cumber effect could have encompassed this hypothetical phenomenon.The 'translational N-hamper effect' is a mechanism whereby protein expression is modulated by coupling fluctuations in appropriate aminoacylated-tRNA availability to ribosome translocation rates at corresponding hCR. Thus, ribosome movement could pause at hCR which would serve as punctuation marks to allow relative intracellular amino acid pool sizes to influence mRNA decoding and protein synthesis. Amino acid level fluctuation could potentially affect: mRNA halflife and accessibility to regulatory complexes, ribosome frameshifting efficiency, initiation rate and formation of stable translation complexes, and elongation rate and vestibule residence time to affect steady state levels of these proteins and of higher order structures in which they participate.Our model holds that Asn level reductions, such as those accompanying the administration of ASNase, cause impaired translation of N-hCR-bearing-genes to precipitate metabolic, vascular, immunological and neurological disorders and contends that this could result in insulin desensitization, impaired insulin release and, ultimately, diabetes. Thus the microbiome, by endogenously generating ASNase, could cotranslationally regulate a constellation of N-hCR-bearing-genes to initiate complex disease pathologies.


Both CXCL12 and CXCR4 are constitutively overexpressed in pituitary adenomas and their signalling induces cell survival and proliferation, as well as hormonal hypersecretion. In this review we focus on the physiological and pathological functions of immune-related cyto- and chemokines, mainly focusing on the CXCL12/CXCR4-7 axis, and their role in pituitary tumorigenesis. Accordingly, we discuss the potential targeting of CXCR4 as novel pharmacological approach for pituitary adenomas.Chemokines are chemotactic regulators of immune surveillance in physiological and pathological conditions such as inflammation, infection, and cancer. Several chemokines and cognate receptors are constitutively expressed in the central nervous system, not only in glial and endothelial cells but also in neurons, controlling neurogenesis, neurite outgrowth, and axonal guidance during development. In particular, the chemokine CXCL12 and its receptors, CXCR4 and CXCR7, form a functional network that controls plasticity in different brain areas, influencing neurotransmission, neuromodulation, and cell migration, and the dysregulation of this chemokinergic axis is involved in several neurodegenerative, neuroinflammatory, and malignant diseases. CXCR4 primarily mediates the transduction of proliferative signals, while CXCR7 seems to be mainly responsible for scavenging CXCL12. Importantly, the multiple intracellular signalling generated by CXCL12 interaction with its receptors influences hypothalamic modulation of neuroendocrine functions, although a direct modulation of pituitary functioning  Differeresponse  and deveresponse , 4. CKs response , 5; moreresponse , 7 whereresponse . Recentlresponse , 10. Eviresponse –13.α-subunit of glycoproteic hormones) [α-subunit is released and more rarely they are classified as true nonsecreting adenomas. Importantly, all pituitary adenomas may induce hypopituitarism and neurological symptoms (for example compression of the optic chiasma) due to mass effect [Anterior pituitary adenomas account for approximately 15% of primary intracranial tumors. They are classified by size  and on the basis of their ability to produce hormones, as secreting or functioning tumors (about 50% of adenomas) or as clinically nonfunctioning pituitary adenomas (NFPA) that do not release hormones or, more often, secrete clinically nonrelevant  or nonbioactive hormones (ormones) , 15. Alms effect , 17. CliThis review will focus on the diverse role of CXC chemokines and their receptors in normal pituitary cell functions and pituitary tumor development and progression, summarize recent progress in CXCR7 functions, and discuss the present issues and future perspectives.Human CK system includes approximately 50 peptides and 22 GPCRs. CKs are classified, according to the number and spacing of the first two cysteine residues of a conserved cysteine motif, into four groups: (1) CXC ; (2) CC (two adjacent cysteine residues: CCL1-28); (3) XC ; (4) CX3C  , 19.α, and Gβ-γ subunits), with the remarkable exception of CXCR7 which is exclusively biased towards β-arrestin-mediated signaling [CK receptors are typical GPCRs, all of them signaling through heterotrimeric G proteins , which are unable to initiate downstream conventional G-protein-dependent signaling, resulting, from a functional point of view, in the inability to induce directional cell migration . These r+ or ELR−), which affects receptor binding specificity and biological effects. Notably, ELR− CXC chemokines  are interferon γ-inducible and act as potent angiostatic factors to impair angiogenic stimuli induced by growth factors. Conversely, ELR+ CKs  are proangiogenic [− but mediates tumor-promoting angiogenesis via its receptor CXCR4 [CKs belonging to the CXC family can be further grouped according to the presence or absence of a the tripeptide motif Glu-Leu-Arg (ELR) preceding the CXC domain . Their activation dissociates the GTP-bound Gα subunit from the Gβ-γ dimer, and both these active components trigger intracellular signals, such as activation of phospholipase C (PLC)/inositol triphosphate (IP3)-Ca2+/diacyl glycerol (DAG)/protein kinase C (PKC) and inhibition of adenylyl cyclase (AC)-cAMP/protein kinase A (PKA). Moreover, these receptors control the activity of different kinases, including extracellular regulated kinases (ERK1/2), c-Jun N-terminal kinase (JNK), p38, phosphatidyl inositol 3 kinase (PI3K)-Akt, and the focal adhesion kinase (FAK).Upon ligand binding, CK receptors undergo conformational change that activates the Gα subfamily which they activate: Gαi inhibits AC but also activates tyrosine kinases of the Src family, favoring signal integration; Gαq increases PLCβ activity [2+ release from intracellular stores. Finally, Gα12 controls the activity of the small G protein RhoA, via Rho-GEF. On the other hand, CK receptor activation of Gβγ subunits results in the activation of PI3K leading, through the phosphoinositide-dependent kinase 1 and 2 (PDK1-2), to Akt phosphorylation and subsequent activation of its downstream signal proteins such as glycogen synthase kinase 3 (GSK3), mammalian target of rapamycin (mTOR), and FAK, which control migration in different types of normal and tumor cells [The distinct transductional cascades regulated by CK receptors mainly depend on the Gactivity , to cleaor cells .α subunit, followed by its reassociation with Gβ/γ in an inactive complex. Moreover, receptor desensitization, internalization, and lysosomal degradation are mediated by G protein-coupled receptor kinases (GRKs) and arrestins [After stimulatory responses, the inactivation of CK receptor signaling occurs after the hydrolysis of GTP to GDP by the intrinsic GTPase activity of Grrestins . via a CK gradient which directs leukocyte recruitment to the site of inflammation. Furthermore, CKs directly activate specialized effector lymphocytes during the different steps of immune response, for example, CXCL8 (formerly named IL8) recruits neutrophils, basophils, and eosinophils expressing its receptors, CXCR1 and CXCR2 [γ released by natural killer and T helper 1 (Th1) cells that express CXCR3, the receptor for CXCL9-L11 [CKs are constitutively secreted by leukocytes, fibroblasts, endothelial, and epithelial cells to mediate cell activation, trafficking, and homing , 30. BesXCL9-L11 , thus amCKs also play a key role in embryogenesis, organogenesis, angiogenesis, and germ cell migration, especially during neural development . The conFocusing on the CXCL12/CXCR4-R7 network, it exerts a variety of functions in CNS development as well as in mature brain. CXCL12 directs the migration of embryonic and adult stem cells in the developing central and peripheral nervous system , 36, conAs far as CNS development is concerned, the pivotal role of CXCL12/CXCR4 emerged from studies using knockout mice for either the ligand or the receptor. Both models exhibited a superimposable abnormal neuron migration in the cerebellum, dentate gyrus, and dorsal root ganglia , 42, 43.α chemoattractant, I-TAC,), which, in turn, interacts with CXCR3 [2+ mobilization and inhibition of cAMP production, since this receptor does not seem to be coupled to Giα. Based on its ability to rapidly sequester and degrade CXCL12 and thus to suppress CXCR4 activity, CXCR7 was firstly proposed to be a decoy receptor [β-arrestin-dependent pathway [β-arrestin is influenced by both CXCR4-CXCR7 dimer formation and the oligomerization state of CXCL12 [CXCR7, the second CXCL12 receptor, has a 10-fold higher binding affinity than CXCR4 but also binds CXCL11  and CXCR7  . CXCR7 rmovement . This filability .Deletion of one of the CXCL12 receptors is sufficient to generate a migration phenotype that corresponds to the CXCL12-deficient pathway and interfering with the CXCL12-scavenging activity of CXCR7 causes loss of CXCR4 function . For exa+ cell, and stem cell migration [The relevance of CXCL12 and CXCR4-R7 system in CNS ontogeny and functions is even more crucial in the view of their expression in both embryonic and adult brain stem cells, a subset of undifferentiated cells characterized by self-renewal through asymmetric division, differentiation into multiple lineages, and constant proliferation that in adults acts in tissue maintenance and repair . The roligration , 89. Theigration , 90, 91. via CXCR4.The stem cell concept applied to cancer has radically changed the research approach to tumorigenesis and treatment, since the subset of cancer cells, namely, cancer stem cells (CSCs), seems responsible for tumor initiation, metastasis, and resistance to therapy . AlthougThrough the release of growth factors , cyto/chemokines, and neuroendocrine proteins  neuronal and neuroendocrine pathways regulate fundamental functions within the CNS and its interaction with the immune system . ComplexEGF expression has been observed at all stages of pituitary development and in the adult pituitary, and the EGFR pathway contributes to pituitary physiology and tumorigenesis .β receptors were detected in pituitary cells [2+ fluxes [IL-1ry cells , and the+ fluxes –103. in vitro [ via the modulation of AC and PLC activities [IL-6 and its receptors are also expressed in the pituitary gland , 105 whein vitro . IL-6 isin vitro , 108 regin vitro , PRL, LHin vitro –112 via tivities . Interestivities  and stimtivities .IL-18 was also proposed to exert paracrine effects in pig anterior pituitary being the ligand and its receptor expressed by different subsets of GH secreting cells .Finally, interleukins' regulation of the hypothalamic-pituitary-adrenal axis also involves the modulation of vasoactive intestinal peptide- (VIP-) secreting pituitary cells to control, in a paracrine manner, PRL release . via the hypothalamic-pituitary axis or autocrine/paracrine regulation. CXCL1 is expressed in the posterior pituitary, in the paraventricular nucleus (PVN) of the hypothalamus and the median eminence [β-GFP rats) that express green fluorescent protein in S100β-positive pituitary FS cells in the anterior pituitary [β-positive cells [γ inducible protein 10 kDa, IP-10) were identified in a subpopulation of these cells. Importantly, CXCR3, the receptor for CXCL10, was shown to be expressed in corticotrophs, suggesting a possible autocrine/paracrine effect of CXCL10, released from FS cells, on ACTH-producing cells [More recently, several studies were directed on the role of CKs in pituitary. CKs can affect pituitary hormone secretioneminence . In respeminence  to reacheminence . Similareminence . The genituitary  led to tve cells , 121 andng cells .CXCL12/CXCR4 is the major regulatory axis not only connecting the immune and nervous systems, but also playing a role in neuroimmune regulation of the anterior pituitary physiological functions . in vivo [CXCL12 was detected in both rat pituitary  and hypo in vivo . The exp in vivo . Since A in vivo . AVP and in vivo  and a fu in vivo . Indeed, in vivo . Interes in vivo . CXCL12/ in vivo .In humans, a slightly different pattern of expression was found in autoptic normal pituitaries. Scattered expression of both CXCR4 and CXCL12 within the anterior lobe was detected by immunohistochemistry, revealing a nonhomogeneous positivity for both proteins throughout the tissue, including large negative areas, others showing few positive cells and rare zones with higher expression  Figure . InteresThe activity of CXCR7 in normal pituitary deserves further investigation; however its expression in pituitary adenoma tissues , 131 sug via p38 MAPK [Beside direct CXCR4-dependent activation of ERK1/2, transactivation of tyrosine kinase receptors is currently a relevant mechanism in tumor cell responses. Mainly, the transactivation of epidermal growth factor receptor (EGFR) family members mediates the mitogenic activity of different CKs in human cancer. A cross-talk between CXCL12 and EGFR and/or HER2/neu phosphorylation was demonstrated in breast and ovarian cancer cells through G protein-dependent activation of kinases of the Src family –134. Morp38 MAPK . CXCR4 sp38 MAPK , 138. Thp38 MAPK –141. Thip38 MAPK , 143. in vitro and in vivo models: CXCL12 stimulates proliferation and migration of glioblastoma cells and xenografted tumors inducing ERK1/2 and Akt phosphorylation [The concomitant expression of ligand-receptor pair in the same tumor cells, responsible of autocrine/paracrine activation, is one of the leading causes of clinical aggressive behavior in various cancer types , 145. Exrylation –149. Therylation , 151, whrylation .β-estradiol contributing to its proliferative effects and mediating, via a Src-dependent mechanism, EGFR transactivation [Similar CXCR4 tumor-promoting effects were observed in breast carcinoma , suggesttivation , 154. via an autocrine mechanism, binds to CXCR4 impairing chemotaxis towards CXCL12-producing target organs and metastatic spread [Furthermore, CXCL12 is also indirectly implicated in tumor pathogenesis, acting as chemoattractant for CXCR4-positive cells, directing tumor cell migration –157 and c spread . In addic spread , 161, inc spread , colon [c spread , brain [c spread  ovarian c spread , prostatc spread  and oralc spread  carcinomc spread ). Importc spread , CXCR4 aRaf/MEK/ERK and PI3K/Akt pathway dysregulation is a common alteration responsible of tumor initiation and progression. While the pathways are classically activated by growth factors, cross-talks and transactivation mechanisms with neuropeptide-cytokine-CK/GPCRs have been increasingly recognized. This cross-talk activates ERK1/2, which is directly responsible for cell growth and differentiation, depending on the cellular context and represents one of the major proliferative pathways in cancer , 169.The overexpression or constitutive activation of receptors for growth factors, cytokines, and CKs potentiates the activation of Ras/Raf/MEK/ERK pathway also in pituitary adenoma . MAPK phSimilarly to cell proliferation, survival mechanisms also sustain pituitary development and tumorigenesis: in particular the balance of pro- and antiapoptotic factors , physiolα, a key molecule in hypoxic pathways triggering vessel formation, was detected in pituitary tumor tissues [Recent studies, however, highlighted that, when considering the complexity of regulatory pathways involved in pituitary cell survival and proliferation, it should take into account not only apoptosis but also senescence, an alternative process acting during tumor-suppressive cell fate. Importantly, senescence is gaining biological significance also in pituitary adenomas, whose typical benign nature could result from protective antiproliferative mechanisms. Several transformation events  induce preventive cellular senescence, characterized by cell cycle exit and subsequent irreversible proliferation arrest. Thus, pituitary tumors may be more prone to activate senescence-associated pathways, maintaining their benign behavior, preventing malignant transformation, and regulating their development . Interes tissues , 190 and tissues . via CXCR4 and CXCR7, represent candidate mediators of the above described intracellular pathways, determining proliferative, antiapoptotic, and angiogenic signals, thus possibly concurring to pituitary tumor development and aggressiveness.CKs, particularly CXCL12 signaling AIP [ MEN1 [ GNAS, [ CDKN2A, or P16; FGFR2/melanoma associated antigen -MAGE-3 pathway) [Pituitary adenomas are common intracranial tumors of the adenohypophysis causing serious morbidity, due to excessive hormonal secretion, mass effects, and local invasion of surrounding structures. At present, the understanding of biological and molecular pathogenesis and mechanisms of progression of these tumors is largely incomplete . E AIP ; multiplP [ MEN1 ; guanine [ GNAS, ), and eppathway)  mutationpathway) .The improvement of this knowledge is even more helpful taking into account the peculiar properties of pituitary adenomas as compared to other malignancies: they commonly grow slowly but with local invasive behavior and occasionally develop into high aggressive tumors. This often prevents the efficacy of surgical and systemic medical treatments, the latter hampered by the lack of definite mechanisms underlying pituitary cell transformation and potential therapeutic targets.The role of CKs in pituitary tumor development has been scantily investigated. However, few studies addressed the potential role of components of this peptide family in regulating human pituitary tumorigenesis.β and inhibited by GH releasing hormone (GHRH) [CXCL8 mRNA was identified in a small percentage of anterior pituitary adenomas , 200, ale (GHRH) . Thus, aHowever, the majority of studies focused on the analysis of CXCL12 and CXCR4 expression in human neoplastic pituitary tissues and their role in adenoma cell proliferation , 203–205 in vitro basal secretion of CXCL12 by human pituitary adenoma primary cultures resulting in an autocrine constitutive stimulation of DNA synthesis [CXCR4 mRNA is expressed in almost all GH-secreting pituitary adenomas and in the great majority of NFPAs, whilst CXCL12 was identified in about 2/3 of these tumors. Notably, most CXCL12-positive cells also express CXCR4 strongly suggesting an autocrine/paracrine regulation of tumor cell proliferation , 205. Thynthesis   [Interestingly, GH4C1 rat pituitary adenoma cell line expresses CXCR4 but not CXCL12, thus it was proposed as a suitable model to characterize the molecular pathways regulated by this receptor in pituitary adenomas, without the interference of the endogenously released CK , 212. Ins (BKCa) , 212. Sis (BKCa) . in vitro and in mice xenografts [ Phidiana militaris, which reduced GH4C1 proliferation, migration, and ERK1/2 phosphorylation [To date, the role of CXCL12/CXCR4 as potential pharmacological target in acromegalic patients has been scantily investigated. It was shown that a synthetic antagonist of CXCR4, d-Arg3FC131, is able to inhibit the growth of GH3 tumor cells and trigger apoptosis bothnografts . Similarrylation .CXCR7, the second CXCL12 receptor, was reported to be expressed in the AtT20 mouse corticotroph pituitary adenoma cell line , but the in vitro. However, such generalized CXCR4 and CXCL12 overexpression in human pituitary adenomas, as compared to normal pituitary [ in vitro on a small number of primary cultures of adenoma cell derived from GHoma, NFPA, and ACTH-secreting adenomas [ in vitro a significant basal secretion of CXCL12 causing a constitutive, autocrine stimulation of DNA synthesis [ in vivo.Studies on human pituitary adenoma cells derived from postsurgical specimens are limited, mostly due to their low proliferative activityituitary , strongladenomas . To avoiadenomas  was usedynthesis . Thus, tOverall, these observations imply that CXCL12/CXCR4 axis might play an important biological role in pituitary adenoma as potential growth and angiogenic factor for pituitary cells. The increased CXCR4 activation may result from either endocrine  and/or autocrine/paracrine mechanisms . ImportaChemokines are key factors in CNS physiology and pathology, being relevant mediators in cancer development. The CXCL12/CXCR4-R7 signaling pathway plays a unique role in the regulation of a variety of cell types, including embryonic and cancer cells. In particular, deregulation of this chemokinergic system is strictly related to tumor initiation and progression, and the balance of its activity within the tumor microenvironment is highly complex phenomenon.Multiple factors cooperate to pituitary tumor pathogenesis, but, although to date not explored in depth, a pivotal role of CK and their receptors seems to be important. Several mechanisms by which CXCL12/CXCR4 modulates pituitary function and promotes adenoma cell proliferation and their target as potential therapeutic approach have been suggested. CXCL12 is overexpressed in pituitary cells where it can directly influence adenoma formation, through autocrine mechanisms resulting in constitutive CXCR4 activation. This pathway grants pituitary cells with a proliferative advantage that triggers clonal expansion of transformed cells and sustains tumor cell survival. Increasing knowledge about pituitary cell origin and development might provide significant insights into deregulated pathways in pituitary tumorigenesis.Finally, CXCR4 is an easily druggable target and the characterization of its role in pituitary adenomas could pave the way for novel pharmacological approaches, especially for those adenoma subtypes,  still waiting for efficacious drugs.


Aeribacillus pallidus strain 8m3, a thermophilic aerobic oil-oxidizing bacterium isolated from production water from the Dagang high-temperature oil field, China, is presented here. The genome is annotated to provide insights into the genomic and phenotypic diversity of the genus Aeribacillus.The draft genome sequence of  Aeribacillus contains a single species, Aeribacillus pallidus   deposited at DDBJ/EMBL/GenBank has a G+C content of 57.4%    . Represeallidus) , productallidus) , 3, hot allidus) , 5, oil-allidus) , and froallidus) . The onlThe genomic DNA of this strain was isolated from the biomass using the DNeasy blood and tissue kit , according to the manufacturer’s instructions. The TruSeq DNA sample preparation kit  was used to create the libraries for genome sequencing. Genomic DNA was sequenced using the MiSeq reagent kit version 2 (Illumina). The shotgun library was constructed with a 500-bp paired-end library.Approximately 7,344,862 paired-end reads were generated, providing 200-fold genome coverage. The resulting reads were assembled with SPAdes version 3.1.0  for autoA. pallidus strain 8m3 revealed a genome size of 3,818,610 bp, with an average G+C content of 38.86%, which was close to the G+C content of A. pallidus type strain DSM 3670. The draft genome contained 3,717 genes, 3,520 coding DNA sequences, 15 coding rRNAs , 60 tRNAs, and 5 noncoding RNAs (ncRNAs). The 16S rRNA and gyrB gene sequences revealed in the genome were identical to those determined earlier for the strain (The draft genome sequence of This genome sequence is expected to provide deep insights into the metabolic potential of this organism and its compliance to the conditions of the deep high-temperature petroleum reservoir.LWBR00000000. The version described in this paper is version LWBR01000000.This whole-genome shotgun project has been deposited at DDBJ/ENA/GenBank under the accession no.