Background. Recently, the percentage of older adults in developing countries has increased significantly. Objective. This study examined patterns and factors associated with primary health care services utilization in the past 1, 6, and 12 months. Method. A cross-sectional study design was used to collect data from 190 older adults in the Irbid governorate of Jordan. Results. Primary health care services were used by less than half of the participants in the past 1 month, by 68.4% in the past 6 months, and by 73.8% in the past 12 months. Primary health care (PHC) services use was associated with age, education level, tobacco use, chronic illnesses, perceived general health status today, a physical component summary score, employment, and perceived general health status in the past 6 and 12 months. The primary predictor of PHC services use at 1, 6, and 12 months was chronic illnesses (OR = 13.32), (OR = 19.63), and (OR = 17.91), respectively. Conclusion. Although many factors were associated with PHC service utilization, the strongest predictor of PHC service utilization was chronic illnesses. Recently, the percentage of older adults in developing countries has increased significantly . Middle Some studies have explored health services utilization patterns across several countries –8. A stuPatterns in health services utilization by older adults have been found to be associated with many factors –13. For A study conducted in Ghana reported that health care utilization was associated with participants' age group, occupation, medical history of chronic conditions, cognitive impairment in past 30 days, and self-reported health status. In addition, significant predictors were age group, medical history of chronic condition, cognitive impairment, self-perceived health, and difficulty with picking up items in the past 30 days . In KoreAlthough there were some studies that studied health status of older adults in Jordan –19, a coA cross-sectional study design was used to examine PHC services utilization patterns and to identify factors associated with and predictive of health care utilization/nonutilization in the past 1, 6, and 12 months. The study was conducted in the catchment areas associated with three comprehensive PHC centers which provide services from 8 a.m. to 4 p.m., and most of these health centers serve large numbers of people and offer many preventive and curative health care services situated in areas within the Irbid governorate of north Jordan .N that is at least 10 times K, where K is the number of predictors in the model. Using their criteria, the minimum sample size needed for this study was 190 participants.Adequate sample size needed for binary logistic regression  was deten = 79), center (n = 85), and north (n = 26)) of the Irbid governorate in Jordan (Irbid is the third largest inhibited city). These primary health care centers serve around 69,088 inhabitants.A proportional convenience sample of 190 older adults, aged 50 and older, participated in this study from three areas  . Perceivr, Phi coefficient (ϕ), and Cramer's V and were used depending on the level of measurement for each variable. Three binary logistic regression models were developed for utilization/nonutilization of PHC services during the past 1, 6, and 12 months. The probability (P < 0.05) was taken as minimum level of significance.Descriptive statistics were used to describe study variables. Chi-square associations with categorical variables and Pearson correlations were conducted to establish associations between independent and dependent variables and to identify which variables would enter logistic regression model. Correlation tests included were Spearman's rho, Point Biserial Permission for conducting study was obtained from the University Of Jordan School Of Nursing and the Jordan Ministry of Health Ethical Committee. Permission to use the ECQA instrument was obtained from the instrument developer and permission to use the Algerian (Arabic) SF-12v2 was obtained from Quality Metric Incorporated (License agreement CT130430/OP011094). All participants were notified that the data collected would be treated with anonymity and confidentiality. In addition, personal informed verbal consent was obtained from all participants.In the past 1, 6, and 12 months, 41 percent of participants used medical services in the past 1 month. In the past 6 months, 68.4 percent of participants had used medical services, and in the past 12 months, 73.8 percent of participants had used medical services.Participants who used significantly more PHC services in the past month were those who (a) had no formal education or had a primary school education, (b) were nonsmokers, (c) had a chronic illness, (d) had perceptions of poorer health status, and (e) had symptoms or poor physical health (lower SF-12v2 PCS score) see .Factors significantly associated with increased use of PHC services were (a) increasing age, (b) being unemployed or retired, (c) having no formal education or only a primary school education, (d) being nonsmokers, (e) having a chronic illness, (f) those having poor self-rated general health status today, and (g) those having a poor self-rated general health status in the past 6 months see .Significantly greater use of PHC services was associated with (a) increasing age, (b) being unemployed or retired, (c) having no formal education or only a primary school education, (d) being nonsmokers, (e) having a chronic illness, (f) those having poor self-rated general health status today, and (g) those having poor self-rated general health status in the past 12 months see .The variable associated with PHC services utilization in the past 1-, 6-, and 12-month period was chronic illness , , and , respectively see .For older adults in three catchment areas of the Irbid governorate of Jordan, data on medical services utilization in the past 1, 6, and 12 months were not consistent. The findings showed that rate of medical services use by older adults in the past one month was less than 50%, which was consistent with a study in Hong Kong . In the In the present study, older age was not associated with PHC service utilization in the past 1 month, similar to that found in Spain ; howeverChronic illness was significantly associated with PHC service utilization in the past 1, 6, and 12 months, consistent with studies in Thailand and Spain , 11. CogFurther studies need to be conducted to confirm that perceived general health status today correlates with PHC service utilization of older adults. Also, those older adults who perceived that they were in poor health, in particular, poorer physical health (PCS score) in the past 1 month, tended to use more PHC services in the past 1 month. This result disagrees with previous studies , 13. OldAge was not a significant predictor of PHC services utilization in the past 1, 6, and 12 months, in contrast to a study in Ghana , and eduChronic illness was the strongest significant predictor of PHC services utilization in the past 1, 6, and 12 months. Multivariate analysis indicated that older adults who had chronic illnesses were 13.3, 19.6, and 17.9 times more likely to use PHC services than those who did not have chronic illnesses in the past 1, 6, and 12 months, respectively. This finding is consistent with another study [Perceived general health today was not a significant predictor of PHC services utilization in the past 1, 6, and 12 months. Likewise, the PCS score was not a significant predictor of PHC services utilization in the past 1 month. This finding diverges from a prior study . One jusHealth care services use, history of chronic illnesses, and perceived health status of participants were self-reported. However, several attempts were made to reduce the possibility of recall bias: (a) the study used different recall periods , (b) PHC services use and history of chronic illnesses were measured as a dichotomous variable, which is easier to recall, and (c) the study participants were community dwelling older adults with a relatively low frequency of cognitive impairment (about 3.2% had evidence of cognitive impairment based on the ECAQ) . Also, tAbout three-fourths of older adults in this study reported having at least one chronic illness. Hence, there was an unusually high rate of chronic illness among the participants in this study. In terms of utilization patterns, the findings reflected high utilization rates of PHC services among older adults over a one-year period. The present study identified several predisposing and need factors that were associated with PHC service utilization. However, the strongest predictor of PHC service utilization was a medical history of chronic illnesses.The results of this study add to the body of knowledge in geriatric nursing regarding older adults' health seeking behavior which will help in developing an effective nursing care programs to promote well-being in Jordanian older adults. Good health care planning requires an understanding of PHC service use, and the approach used in this study could be replicated throughout Jordan. As such, it would enable the Ministry of Health in Jordan to gather relevant data necessary to provide appropriate health care services for older adults that are both efficient and cost-effective. Moreover, such data could help planning for healthier lifestyles throughout the life course and to build comprehensive and preventative health care programs in the PHC centers that will promote better health for older adults, instead of the current focus on cure and/or medication prescriptions.


Claudin 1 is a small transmembrane protein responsible for maintaining the barrier function that exists between epithelial cells. A tight junction protein that regulates the paracellular transport of small ions across adjacent cells, claudin 1 maintains cellular polarity and plays a major role in cell-cell communication and epithelial cell homeostasis. Long considered to be a putative tumor suppressor in human breast cancer, new studies suggest a role much more complex. While most invasive breast cancers exhibit a down regulation or absence of claudin 1, some aggressive subtypes that exhibit high claudin 1 levels have now been described. Furthermore, a causal role for claudin 1 in breast cancer progression has recently been demonstrated in some breast cancer cell lines. In this review we highlight new insights into the role of claudin 1 in breast cancer, including its involvement in collective migration and epithelial mesenchymal transition (EMT). Breast cancer is the most common cancer in women and the second leading cause of cancer death in women worldwide . A diffiMetastasis of breast cancer to secondary sites in bone, liver, brain and lungs is largely incurable and is the principle cause of death in breast cancer patients ,10,11,12To achieve metastatic potential, a few cancer cells initially acquire the ability to evade and survive the homeostatic mechanisms of the organism. These cells are selected from the genetically heterogeneous lineages of cancer cells within the microenvironment of the tumor’s origin and continue to proliferate . The conAs epithelial cells convert from the non-invasive to the invasive phenotype, cells become anchorage independent and exhibit enhanced motility as well as increased aggressiveness. Epithelial mesenchymal transition (EMT) is thought to be a major mechanism for this conversion ,18. DuriTJs form the closest contacts between adjacent cells along the apical border of the epithelial cell membrane . They arC-terminus interacts with many signaling pathways and tight junction proteins through a PDZ domain. Through this domain, claudins interact with multiple TJ proteins [The claudins are a family of integral membrane proteins (24 identified to date), central to the formation of TJs ,35. By rproteins ,43. The proteins ,47,48,49C-terminal of the claudin 1 protein [Claudin 1, the first claudin family member identified, forms the backbone of the TJ strand ,50. Knoc protein ,51. In c protein ,53,54. F protein , and mor protein , thus id protein ,58,59.A dysregulation of claudin 1 in cancer has been well documented, and both a loss and gain has been associated with several cancers including oral, lung, prostate and gastric carcinoma as well as breast cancer ,63,64,65Interestingly, aberrant expression of claudin 1 has been observed not only in different cancers but in different histological subtypes of the same cancer. The over expression of claudin 1 in type II seropapillary endometroid carcinoma can distinguish it from type I which exhibits low claudin 1 expression .et al. [in vitro studies showed that re-expression of claudin 1 alone was sufficient to induce apoptosis in a human breast cancer cell line, MDA-MB361 [Although not definitively proven, claudin 1 has long been postulated to be a tumor suppressor in breast cancer, as it was observed to be frequently down regulated or absent during disease progression. To lend further support to this hypothesis, a correlation between down regulation of claudin 1 and disease recurrence was also reported in a study involving 83 patients (57 patients with non-recurrent breast cancer and 26 patients with recurrent disease) . More reet al. , showed DA-MB361 , and a lDA-MB361 . ClaudinDA-MB361 .There have been many speculations about the mechanisms responsible for claudin 1 loss in ER+ breast cancers. A search for mutations in the promoter or coding region of the claudin 1 gene has been futile as an explanation for the down regulation of claudin 1 in breast cancer . Recent et al. [et al. [in vivo. These authors showed that Stanniocalcin 2 (STC2) promoted breast cancer metastasis in mice through its interaction with claudin 1, which directly led to an alteration of the expression of the EMT molecules including Zeb1, Slug, and Twist [Claudin 1 involvement in EMT has also been well documented in several cancers ,32. Clauet al.  suggesteet al. ,89. It het al. ,92,93,94et al.  and repr [et al.  further nd Twist .The matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases which can degrade and remodel the extracellular matrix (ECM) and its constitutive proteins, during both normal processes (such as morphogenesis and tissue repair), and pathological processes (such as the facilitation of cell migration and invasion) during EMT . HoweverIn breast cancer claudin 1 co-localized and directly interacted with membrane type MMP-1 and pro-MMP-2 and mediated its activation . MMP-2 cThis suggests a dual role for claudin 1 in its interactions with the ECM. Claudin 1 may form complexes with ECM proteins to stabilize cell-matrix interactions, and/or assist in the degradation of the ECM through MMPs during cell invasion.in vitro and in vivo studies reveal an emerging role for claudin 1 in breast cancer. The concept that there are two main patterns by which cancer cells migrate, collective migration or single cell migration, has been described several years ago, but the mechanisms are not well understood. Single cell migration, as the name implies, refers to the detachment of single epithelial cells from the tumor, and, facilitated by the acquisition of mesenchymal characteristics, then proceed to navigate through the ECM to metastasize to distal sites [Both al sites ,99. Collal sites . This coal sites ,102,103.al sites .et al. [et al. [Claudin 1 plays a key role in facilitating the collective migration of tumor cells. Work by Giampieri et al.  showed t [et al. , showed et al. [Despite the fact that many laboratories, including our own, suggest that claudin 1 is a tumor suppressor in invasive breast cancer, our studies also suggest that this is not the case for all breast cancers . We haveet al. , also reet al. . Howeveret al. ,  family receptors (protein tyrosine kinases), and its ligands can induce bidirectional signaling in epithelial cells via cell-cell contact [ contact . Claudin contact , a trans contact . The ext contact . Interes contact , which i contact . UnderstThe ESCRT machinery: It has been shown that the intracellular accumulation of claudin 1 can occur as a result of defective interactions with the Endosomal Sorting Complex Required for Transport (ESCRT) machinery, a collection of cytosolic proteins required for proper protein transport and maintenance of epithelial cell polarity [polarity . ESCRT apolarity . Thus, Epolarity . Moreovepolarity , two of polarity . Such obCD9: The tetraspanin CD9 has recently been identified as an interacting partner of claudin 1. Claudin 1 is one of a few known interacting partners of CD9. Tetraspanins are transmembrane proteins that typically reside on cell surface and do not generally function as ligands or receptors [eceptors . Rather,eceptors ,124,125.eceptors .In breast cancer, the interaction of claudin 1 with CD9 has been shown to coincide with its subcellular co-localization in breast cancer. This has now been demonstrated in multiple cell lines, including the human breast cancer cell lines MDA-MB231  and MCF7  . InteresEpCAM: Epithelial cell adhesion molecule (EpCAM), also known as CD326, is a transmembrane surface glycoprotein found expressed in epithelia and some invasive carcinomas [rcinomas . EpCAM ircinomas . EpCAM ercinomas . As a reet al. [EpCAM has recently been shown to play an important role in regulating the composition and function of TJs through specific interaction with claudin 1. Wu et al.  recentlyThe void in our knowledge about the ever-emerging breast cancer molecular subtypes presents an insurmountable challenge to finding effective strategies for managing this disease. In the healthy mammary gland, TJ permeability can be reproduced readily in response to a number of physiological stimuli , and thuThe loss of cell-cell adhesion is a crucial step in EMT, and thus strategies to overcome the altered expression of TJ proteins in cancerous tissues are attractive as they could eventually lead to the development of effective therapeutic management for treating and possibly preventing human cancers.Claudin 1 is of particular interest in the quest to identify new targets for breast cancer therapy as, (1) increasingly it has been shown to have direct involvement in the migration and proliferation rates in several cancers, including breast ,47,112 aAs well, the pattern of expression of claudin 1 in different molecular subtypes indicates that claudin 1 may be a useful biomarker for the detection, diagnosis and treatment of breast cancer and may also serve as a tool for predicting disease progression. ,73. It h


The association between heavy metals exposure and respiratory diseases or allergic sensitization showing high serum immunoglobulin E (IgE) has been suggested. However, previous findings have been inconsistent and the mechanisms responsible remain unclear. We evaluated heavy metal exposure and its association with coughing, itchy eyes in chronic cough patients with different IgE levels. Ninety outpatients in Kanazawa University Hospital were recruited between January–June 2011. Subjects whose total IgE measured by radioimmunosorbent test were asked to record their daily symptoms. We collected daily total suspended particles (TSP) from which concentrations of calcium (Ca), cadmium (Cd), chromium (Cr), iron (Fe), manganese (Mn), nickel (Ni), and lead (Pb) were determined then divided into high and low level groups. Generalized estimating equations were applied to compute the relationship between concentrations of these metals and symptoms. All metals at high levels were significantly associated with itchy eyes compared with low levels, with exception of Ca, the six others were significant in patients with IgE < 250 IU/mL. Cd, Fe, Mn had association with coughing : 1.13 , 1.22 , and 1.13 , respectively), this relationship remained significant for Cd (OR (95% CI): 1.14 ) and Mn (OR (95% CI): 1.15 ) in patients with lower IgE. Our findings demonstrate the relationship between aerial heavy metals and itchy eyes, coughing in chronic cough patients, suggesting these symptoms may be due to a non-IgE mediated mechanism. Due to economic and industrial growth, air pollution such as particulate matter (PM) and gaseous emissions have become an important environmental issue throughout the world. PM is a heterogeneous mixture of biologically active components in the air, and heavy metals and polycyclic aromatic hydrocarbons have been identified as the predominate toxic constituents ,2,3. Air10 was associated with allergic sensitization at the birth address as well as the current address and may also increase the risk of asthma and allergy in schoolchildren [Epidemiological and experimental studies have provided evidence for the adverse effects of PM exposure, particularly heavy metals, on respiratory diseases and allergic sensitization. For example, exposure to ambient PM has been identified as a high risk factor for increasing the incidence of atopic diseases and allergic sensitization in children . Heavy tchildren . Increaschildren . HoweverChronic cough is the fifth most common condition encountered by primary care physicians . In addiImmunoglobulin E (IgE) plays an important role in inflammation. It has interdependent and independent roles in the complex immune responses that manifest clinically as allergic disorders . InhaledInformation provided by longitudinal studies on the relationship between heavy metal exposure and respiratory diseases is still limited. The involvement of IgE mechanisms in the symptoms of patients with chronic airway inflammation remains unclear. Therefore, we conducted this study to investigate the relationship between heavy metal concentrations and the appearance of symptoms including itchy eyes and coughing in chronic cough patients with different total serum IgE levels.Ninety-nine patients diagnosed with at least one of the following: Bronchial asthma, atopic cough, and cough-variant asthma, were recruited at Kanazawa University Hospital between 4 January 2011 and 30 June 2011. All subjects gave their informed consent for inclusion before they participated in the study. The study was conducted in accordance with the principles of the Declaration of Helsinki of the World Medical Association, and the protocol was approved by the Medical Ethics Committee of Kanazawa University (Project Identification Code: 981). We used Japan Asthma Prevention and Management Guidelines 2011 to diagnose asthma . The criAmong the 76 patients with recorded itchy eye symptoms, 59 had a total serum IgE level less than 250 IU/mL, while that of 17 patients was more than or equal to 250 IU/mL . Forty-tPatients recorded their symptoms in an allergy diary every morning, afternoon, evening, and night from the beginning to the last day of the study. Patients started recording the frequency of coughing from 4 January 2011, while the level of itchy eyes was started on 1 February 2011. We made every effort to avoid missing data due to patients forgetting to record their symptoms in their diaries by asking them to bring their diaries with them when they came to the hospital to check their symptoms or have consultations. The average frequency of coughing and level of itchy eyes was calculated for each patient. The presence of symptoms was defined based on whether the frequency of coughing or level of itchy eyes of each patient on each day was higher than the average.At the initiation of the study, total serum IgE levels were measured from a blood sample taken from the peripheral blood vessels of all patients using a radioimmunosorbent test. Patients were divided into two groups with total serum IgE < 250 IU/mL and total serum IgE ≥ 250 IU/mL based on criteria used at Kanazawa University Hospital .Hydrochloric acid and nitric acid were purchased from Kanto Chemical Co., Inc.  for atomic absorption spectrometry. A borosilicate glass fiber filter coated with fluorocarbon ) was used. Total suspended particulates (TSP) were collected on the borosilicate glass fiber filter coated with fluorocarbon using a high-volume air sampler  at a flow rate of 1000 L, located at Kanazawa University . After 24 h of collecting ambient dust (start at 13:30 to 13:30 on the next day), the collected filter was removed and replaced with a blank filter. The collected filter was kept for 16 h–48 h in a desiccator in order to remove water and then stored at −30 °C before the extraction and analysis of heavy metals. The method of measurement used has been described previously . A quart2)), and pollen count (cedar and cypress pollen) were used as potential confounders. Data were statistically analyzed using the SPSS software program for MS Windows, version 19.0 . The significance of differences was set at p < 0.05 for all analyses.In order to assess the effects of heavy metals on the appearance of symptoms over the 6-month follow-up, we applied the generalized estimating equation (GEE) model to adjust the relationship between changes in the concentrations of heavy metals and symptoms in the same patient as well as to determine the effects of these metals on the symptoms of patients with different total serum IgE levels. We categorized the concentration of each metal into two levels (high and low levels) using the median of each one. Since patients recorded the frequency of coughing and level of itchy eyes at different periods, we used the median concentration of heavy metals in these periods. The odds ratios of high levels of each metal with 95% confidence intervals were calculated relative to the low levels. The characteristics of patients , meteorological factors , air pollutants (sulfur dioxide (SO3). The mean Fe concentration was more than 2-fold higher than that of Ca (mean 238.53 ng/m3), and at least 13-fold higher than that of Mn (mean 37.54 ng/m3) and the others. The average concentrations of metals from high to low were Fe > Ca > Mn > Pb > Ni > Cr > Cd  for adjustments, we found that all metals at high levels correlated with itchy eyes with ORs for Mn, Fe, Cd, Ni, Ca, Pb, and Cr of 1.60 , 1.56 , 1.27 , 1.23 , 1.22 , 1.20 , and 1.17 , respectively , 1.77 , 1.49 , 1.34 , 1.29 , and 1.25 , respectively. However, after using covariates : 1.22 ), Mn (OR (95% CI): 1.13, ), and Cd (OR (95% CI): 1.13 ) , 1.17 , 1.15 , and 1.12 , respectively. After adjustments for age, BMI, gender, temperature, humidity, precipitation, SO, 1.24)) .p-value of 0.027), this relationship did not account for the difference observed in the effects of Fe on coughing in patients with high total IgE levels . No correlations were observed between heavy metal concentrations and symptoms in patients with total serum IgE ≥ 250 IU/mL , 1.61 , 1.30 , 1.23 , 1.21 , and 1.18 , respectively . In anot50 IU/mL .To the best of our knowledge, this is the first epidemiological study to provide evidence for a relationship between airborne heavy metals and coughing or itchy eyes in chronic cough patients with different total serum IgE levels. The results of the present study indicate that patients with lower IgE levels are more likely to be affected by heavy metals and also that the relationship between heavy metal exposure and airway inflammation may involve a non-IgE mediated mechanism.Some environmental studies in large industrial cities in Asia such as Beijing, Guangzhou, Chengdu (China), and Seoul (Korea) reported markedly higher concentrations of PM and heavy metals than those of the present study ,33,34. D2.5 may be responsible for more severe inflammation [10 in the Netherlands with the highest average concentration, a correlation was not observed after adjusting for asthma symptoms and rhinitis but only for allergic sensitization at birth [We clearly showed that all of the metals examined correlated with the presence of itchy eyes after adjusting with covariates. Furthermore, Cd, Cr, Fe, and Mn also correlated with coughing, and this relationship remained significant for Cd, Fe, and Mn after adjusting with possible confounders. A previous study on New York children for 24 months did not find a relationship between coughing and Fe in single models . Other iammation , and shoammation . Childreammation . Althougat birth . Exposurat birth . Mn has at birth . Ocular at birth . Our resat birth , and alsat birth . The detat birth . These fat birth ,52, and at birth . Furtherat birth . The finat birth . HoweverWe clearly demonstrated that correlations existed between six out of the seven heavy metals (except for Ca) at high concentrations and the presence of itchy eyes in patients with total serum IgE levels less than 250 IU/mL. Furthermore, Cd and Mn positively correlated with coughing in patients with total serum IgE levels less than 250 IU/mL. The findings of a current study also showed that none of the metals tested correlated with the appearance of itchy eyes and coughing in patients with total serum IgE levels more than or equal to 250 IU/mL. Several possible mechanisms have been proposed for IgE-dependent allergic inflammation pathology ,56. A st2), and pollen (cedar pollen and cypress pollen) are the important strengths of our study. However, there were some limitations. First, we only obtained data on metal concentrations in TSP at Kanazawa University-Takaramachi Campus; therefore it is possible that data at the observation did not always reflective the concentration in the study area and individual exposures. Second, the findings for metals may be applicable to traffic-related constituents especially largely relate to non-exhaust emission of traffic [The present longitudinal cohort study with a detailed heavy metal concentration exposure assessment for 6 continuous months with patients recording their symptoms in a diary during the study period in addition to the adjustment of the effects of metals on each patient with possible confounders including patient characteristics , weather , air pollution (SO traffic ; however traffic ,39,54,71The results of the present study provide evidence for the effects of aerial heavy metals on itchy eyes and coughing in patients with chronic cough, particularly due to different total serum IgE levels. The present results suggest correlations between not only the non-exhaust emissions of traffic and general health, but also asthma and rhinitis. The relationship observed between heavy metal concentrations and the symptoms of our patients with total serum IgE levels less than 250 IU/mL is an important result and indicates the relevance of the impact of heavy metals on airway inflammation through a non-IgE-mediated mechanism.


Lef1, Lhx2, Lhx9, Lhx5, Lmo3, Lmo4, and Prox1), in combination with topological position, to identify the medial pallial derivatives, define its major subdivisions, and compare them between mouse and chicken. In both species, the medial pallium is defined as a pallial sector adjacent to the cortical hem and roof plate/choroid tela, showing moderate to strong ventricular zone expression of Lef1, Lhx2, and Lhx9, but not Lhx5. Based on this, the hippocampal formation , the medial entorhinal cortex, and part of the amygdalo-hippocampal transition area of mouse appeared to derive from the medial pallium. In the chicken, based on the same position and gene expression profile, we propose that the hippocampus (including the V-shaped area), the parahippocampal area , the entorhinal cortex, and the amygdalo-hippocampal transition area are medial pallial derivatives. Moreover, the combinatorial expression of Lef1, Prox1, Lmo4, and Lmo3 allowed the identification of dentate gyrus/CA3-like, CA1/subicular-like, and medial entorhinal-like comparable sectors in mouse and chicken, and point to the existence of mostly conserved molecular networks involved in hippocampal complex development. Notably, while the mouse medial entorhinal cortex derives from the medial pallium , the lateral entorhinal cortex  appears to derive from a distinct dorsolateral caudal pallial sector.We carried out a study of the expression patterns of seven developmental regulatory genes ( The hippocampal formation is a cortical structure of the telencephalic hemispheres that is essential for spatial navigation and memory formation  comprises three cytoarchitectonically distinct subdivisions, which from lateral to medial are: the subiculum, the hippocampus proper  and the dentate gyrus  and LIM-only (Lmo3 and Lmo4) genes, and Prox1 in the developing medial pallium of mouse and chicken. Although the expression of all of these genes was previously studied in the developing dorsomedial pallium of mouse  and in several anamniotes , and were subsequently processed as floating sections.Mouse embryos (Swiss) from embryonic day 11.5 (E11.5) until birth and chicken embryos from embryonic day 6  until 2 days after hatching (P2) were used in the present study. All animals were treated according to the regulations and laws of the European Union (86/609/EEC) and the Spanish Government  for care and handling of animals in research. The protocols used were approved by the Committee for handling and care of research animals of the University of Lleida. The mouse embryos were obtained from pregnant females, and were processed and fixed as previously described  or Geneservice Limited ], or obtained from other laboratories, as indicated below. The purchased clones were obtained from the BBSRC ChickEST Database ;cLhx2 .cWnt8b  protocols for the genes mentioned above. Before hybridization, the sections were abundantly washed in PBS containing 0.1% Tween-20 (PBT 1X), prehybridized in hybridization buffer (HB) for 2 h at 58°C, and then hybridized in HB containing the riboprobe overnight at 58°C . The hybridization buffer contained 50% of deionized formamide, 1.3X standard saline citrate , 5 mM ethylene-diamine-tetraacetic acid , 1 mg/ml of yeast tRNA (Sigma-Aldrich), 0.2% Tween-20, 100 μg/ml of heparin (Sigma-Aldrich), completed with water . Following hybridization, the sections were washed with a mix 1:1 of MABT 1X  and HB at 58°C during 20 min and washed abundantly at room temperature with MABT 1X (about 2 h). Following this, the sections were blocked with a solution containing blocking reagent (Roche), MABT 1X and sheep serum (Sigma) for 4 h at room temperature, then incubated in an antibody against digoxigenin  overnight at 4°C, later washed with MABT 1X and finally revealed with BM purple (Roche Diagnostics). Sections were then mounted on glycerol gelatine (Sigma).Some series of chicken embryonic brain sections (E8–E12) were processed for immunohistochemistry, following a procedure previously described . Following this, the sections were washed and incubated in the avidin-biotin complex  for 1 h at room temperature. Finally, the immunolabeling was revealed by 0.05% diaminobenzidine  in 0.05 M Tris buffer (pH 7.6), containing 0.03% HDigital photographs were taken on a Leica microscope (DMR HC) equipped with a Zeiss Axiovision digital camera. Digital images were adjusted for brightness/contrast using Adobe PhotoShop and figures were mounted and labeled using Macromedia FreeHand 10.Finally, the nomenclature used in the present study for the chicken telencephalon generally followed that proposed by Reiner et al. , except Lef1, Lhx2, Lhx9, Lmo3, and Lmo4 in the mouse embryonic medial pallium (summarized in Table 5), and data on the expression of cLef1, cLhx2, cLhx9, cProx1, cLmo3, and cLmo4 in the chicken embryonic medial pallium . The figures are organized according to both the species and the age, showing first those for the mouse and then those for the chicken, and within each species showing first those of early embryonic stages, followed by intermediate stages and finally those for late stages. For comparative purposes we also included published data on Prox1 in mouse in Table Lhx5 at early developmental stages in mouse and chicken  covering the forebrain during development , in the developing choroid plexus, and in forebrain blood vessels .Herein we present data on the expression of n Tables , Lef1 wa Figures , as wellLef1 was strongly and distinctly expressed in the ventricular zone (vz) of the medial pallium (MP) and the pallial septum (PSe) , although it also showed generally weak expression in the vz of other pallial sectors. By E13.5 in mouse , Lef1 became primarily restricted to the vz of the medial pallium and pallial septum ,  Figures  and E8 im Tables , 2A.3) and E8-E9 in chicken , the medial pallial sector was characterized by strong or moderate vz expression of Lef1 , Lhx2 , and Lhx9 , but not Lhx5 . This feature allowed the distinction of the medial pallium from other progenitor pallial sectors, such as: the pallial septum (PSe), expressing Lef1 , Lhx2 , Lhx9 , and also Lhx5  in the vz; the dorsal pallium (DP), expressing strongly Lhx2 in the vz , but not Lef1 , but no vz expression of Lef1  nor Lhx5   and, for this reason, was previously suggested to be part of the ventral pallium . In addition, in contrast to the medial pallium, the DLP did not express Lef1 ; Table Lhx9 , and Emx1  are explained in detail below, first for the mouse  and then for the chicken .In addition to the expression in the vz, most derivatives of the medial pallium of mouse and chicken also showed moderate to strong expression of Lef1, Lhx2, and Lhx9 in the vz and in postmitotic cells migrating into the mantle  and the adjacent part of the dorsal pallium  , the dentate gyrus , the CA fields , the subiculum , at least part of the amygdalo-hippocampal transition area  of mouse was distinguished by its moderate to strong expression of )] Table . Based o Figures , Lhx2 , dentate gyrus (DG), CA fields, subiculum (S), medial entorhinal cortex (MEnt); Figure Lhx9 remained moderate to strong in most of them but started to be downregulated in the developing CA1 field and, especially, in the developing subiculum , making it different from the MEnt  showed gene expression patterns highly similar to those in other medial pallial derivatives, the lateral entorhinal cortex  differed in the expression of  Figures , Lhx9 , where it remained moderate to strong  and CA1, and moderate expression in the indusium griseum (IG) and medial entorhinal cortex (MEnt), but its expression was very weak in CA3 and absent in the dentate gyrus (DG) , except the indusium griseum and the dentate gyrus, which showed only weak expression  Figure . Lmo4 exn Figure . On the Lhx2 , dorsolateral-caudal (DLP), lateral (LP), ventral (VP), ventrolateral-caudal (VLP)] could additionally be distinguished by a region-specific combinatorial expression of cLhx2, cLhx9, cLmo3, and cLmo4 in the vz, subventricular zone  of chicken embryos could be distinguished by its moderate to strong expression of  Figures , 7A, cLh Figures , 7D, and Figures , 7B,C in Figures . The rescLef1  in the APH, with an increasing gradient toward caudolateral levels , although only in part of it because it was downregulated in the vz of APHl, APHcl, and entorhinal cortex (Ent)  and the different APH subdivisions, except the APHcl and the Ent where cLef1 expression was only weak , which showed very strong expression  and the lateral pallium  , which was still visible at P2 . By E18, cLmo3 expression was weak in DG and Ent, but moderate in parts of most APH subdivisions, except APHl, where it was strong , together with analysis of topological position and published data on these or other genes, to identify the medial pallial derivatives and define its major subdivisions in mouse and chicken, and to compare such subdivisions between both species. In both mouse and chicken, the medial pallium is defined as a pallial sector adjacent to the cortical hem , the dorsal pallium , the dorsolateral caudal pallium (which does not express Lef1 nor Lhx9 in the vz), and the ventral/ventrolateral-caudal pallia (which do not express Lef1 in the vz). Based on this gene combination, often also present in the mantle, we propose that the indusium griseum, the hippocampal formation , the medial entorhinal cortex, and part of the amygdalo-hippocampal transition area of mouse are medial pallial derivatives. It is likely that the presubiculum and parasubiculum also derive from the medial pallium , the APH .The genes cLhx2 and cLhx9: Abellán et al., Lhx9 in the zebra finch: Chen et al., cLef1: Gupta et al., Lef1 during development (present data). However, while the APHcl/CDL derives from the medial pallium , the cingulate cortex and other parts of the neocortex derive from the dorsal pallium (showing lack of expression of Lhx9 at the vz), which disfavors the homology of these two structures. Our data support that APHcl/CDL is really a medial pallial derivative and, as such, part of the avian hippocampal complex; therefore, we recommend to call it simply APHcl and to abandon the term CDL, which is confusing because it is also employed by some authors to refer to the DLP . As discussed later, an ectopic migrated part of APHr appears to reach intermediate and caudal hippocampal formation levels, where it appears to correspond to the so-called parvocellular region of Atoji and Wild , is expressed in the developing hippocampal formation of chicken (E8–E9), but not in the arcopallium. After E10, Lef1 also starts to be expressed in parts of the mantle of the chicken dorsal pallium  and lateral pallium . It is important to remember that most developmental regulatory genes are expressed in more than one region; for example, this is so for Emx1, Emx2, Pax6, Lhx2, Lhx9, and ER81, expressed in several pallial subdivisions, but some also in the subpallium and outside the telencephalon, and even outside the nervous system , although these authors included the whole V-shaped area and did not mention the ventral hippocampus. Our data show that the dorsalmost part of V-shaped area , but Lef1 is downregulated, similarly to the findings in mouse  or by novel expression of Prox1 in one of the parts (the DG). To know what was the ancestral situation in amniotes, it is mandatory to study Lef1 and Prox1 in different reptiles, including lizards and other Squamates, which are currently considered a sister group of Archosauria  and, as such, excellent models for understanding the basal condition in sauropsids  also show Lmo3 expression in an area- and layer-specific way. In the mouse, the strongest expression of Lmo4 occurs in the CA1, while the strongest expression of Lmo3 is seen in the subiculum. In the chicken, the strongest Lmo4 expression is seen in APHm and APHi [roughly corresponding to the dorsomedial APH sector (DM) of Atoji and Wild, Lmo3 expression is seen in APHl [corresponding to the dorsolateral APH sector (DL) of Atoji and Wild, During early (chicken) and/or intermediate (mouse and chicken) development, in addition to Lef1 expression and radial glial fiber disposition, the APHr vz appears to be located at very rostral APH levels  appears to extend from this origin, and progressively occupies more superficial, dorsolateral and caudal positions. At intermediate and caudal levels, the Lef1 expression domain related to APHre lies at the surface of APHm  from the medial pallium neuroepithelium   from the medial pallium, and the LEnt  from the dorsolateral caudal pallium Figure . In partLmo3, Lmo4, and Cdh10, very weak expression of Lhx9, and no expression of Lhx2 and Lef1; in contrast, the avian entorhinal cortex shows moderate to strong expression of Lmo4, Lhx9, Lhx2, and Lef1, while its cortical plate is nearly free of Lmo3 and Cdh10 . Based on its embryonic origin, the so-called avian entorhinal cortex may be comparable to mammalian MEnt. As noted above, MEnt also receives a minor direct input from the olfactory bulb at least in some mammals  is or is not comparable to the dorsolateral caudal pallial field that produces LEnt in mammals, even if they occupy similar topological positions and share some similar molecular features (for example, in general weak or moderate expression of 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.


Xanthophyllomyces dendrorhous, a promising bioresource for the carotenoid astaxanthin. Using genomic and transcriptomic data, the codon usage and codon context biases of X. dendrorhous open reading frames (ORFs) were analyzed to determine their expression levels, GC% and sequence lengths. X. dendrorhous totiviral ORFs were also included in these analyses.Synonymous codons are used differentially in organisms from the three domains of life, a phenomenon referred to as codon usage bias. In addition, codon pair bias, particularly in the 3’ codon context, has also been described in several organisms and is associated with the accuracy and rate of translation. An improved understanding of both types of bias is important for the optimization of heterologous protein expression, particularly in biotechnologically important organisms, such as the yeast X. dendrorhous ORFs were identified through comparison with sequences in multiple databases, and the intron-exon structures of these sequences were determined. Although there were important expression variations among the ORFs under the studied conditions (different phases of growth and available carbon sources), most of these sequences were highly expressed under at least one of the analyzed conditions. Independent of the culture conditions, the highly expressed genes showed a strong bias in both codon usage and the 3’ context, with a minor association with the GC% and no relationship to the sequence length. The codon usage and codon-pair bias of the totiviral ORFs were highly variable with no similarities to the host ORFs.A total of 1,695 X. dendrorhous, which is more evident for ORFs that are expressed at the highest levels under the studied conditions. However, there is no direct relation between the totiviral ORF biases and the host ORFs.There is a direct relation between the level of gene expression and codon usage and 3′ context bias in The online version of this article (doi:10.1186/s12864-015-1493-5) contains supplementary material, which is available to authorized users. With the exception of methionine and tryptophan, amino acids are encoded by two to six synonymous codons according to the standard genetic code, and degenerate codons are used at different frequencies, a phenomenon known as codon usage bias (CUB) . SeveralD. melanogaster [in vivo study suggested that the codon context primarily influences the speed at which proteins are synthesized in E. coli [The existence of CUB has been described in metazoans , D. melanogaster , bacterinogaster ,13, insenogaster , archaeanogaster  and virunogaster -18. It hnogaster ,20. Prevnogaster ,22. Highnogaster -27. Thusnogaster ,29. In anogaster -33. One  E. coli . Preferr E. coli .Xanthophyllomyces dendrorhous is relevant to biotechnology, as this microorganism synthesizes the carotenoid astaxanthin. This pigment has strong antioxidant properties beneficial for human health, including potential benefits for the treatment of degenerative diseases [X. dendrorhous is a promising source of natural astaxanthin, natural production in wild-type strains is not sufficient to be economically competitive against the chemical synthesis of this pigment. Therefore, considerable effort has been made to improve the production of carotenoids in X. dendrorhous, including culture optimization, classical random mutagenesis and metabolic engineering approaches . Unfortiewed in , limitinD 67–385 ; unlike X. dendrorhous has been previously described, the analysis was performed using only ten ribosomal genes [X. dendrorhous genes using genomic and transcriptomic data obtained from the yeast cultured with two different carbon sources (glucose and succinate) during two different phases of growth . The level of gene expression was included as a parameter in these analyses for the comparison of codon usage and codon context biases among highly and lowly expressed genes, and the gene expression was also compared against totivirus genes resident in this yeast.Although the codon usage of al genes . HoweverX. dendrorhous strain UCD 67–385 was grown in minimal media supplemented with glucose or succinate as the sole carbon source, and the cells were collected at the early exponential (~18 h) and initial stationary (~72 h) phases of growth, generating a total of four different conditions . Total RNA was purified from the yeast pellets, and the quality of samples was assessed and sequenced using the Illumina GAII and HiSeq platforms. Open reading frames (ORFs) of at least 300 bp in length were predicted using transcriptome contigs, and subsequently these sequences were mapped to five genomic scaffolds of 1.1 to 2.4 Mbp in length . Only the mapped ORFs identified under the four conditions were analyzed, and ORFs showing 100% identity with genome sequences, including a well-defined exon-intron structure, were selected and compared with the database using the Blast2GO server [−85) , with RPKM values ranging from i) 1–30, ii) 31–70, iii) 71–100, iv) 101–999 and v) ≥1,000. The ribosomal ORFs commonly used as references for highly expressed genes were grouped separately (R grouping). However, as an ORF can be poorly expressed under one condition but highly expressed under another, the ORFs were also classified using the same RPKM value ranges but only considering the Highest RPKM Value observed among the four conditions (HV grouping). In addition, the ORFs were also classified according to the Average RPKM Value among the four conditions (AV grouping). The analysis of relative synonymous codon usage was performed for each group within a classification using the CodonW program/server/software , and thIn addition, the ORFs were classified according to sequence length and GC% to analyze the CUB. When the ORFs were grouped according to sequence length, all groups showed similar codon usage, and only the shorter sequences, ranging from 300 to 499 bp, showed some differences with the larger ORFs . Among the four conditions, lower expression levels were observed for the 18-h cultures containing succinate as the sole carbon source, and the expression of a majority of the genes increased from 18 to 72 h of culture in both carbon sources. As several factors influence the CUB, in the present study, we examined the X. dendrorhous CUB according to the gene expression level, sequence length and GC% of the ORF. Clearly defined ORFs were classified according to these parameters, and the 3′ codon context was also analyzed.In the present study, based on genomic and transcriptomic data, 1,695 ORFs were selected from the In the first analyses, the ORFs were classified into groups according to the expression levels observed under each of the four conditions, independently exhibiting CUB differences among the groups without a clear relationship between the CUB and the expression level. The highest or average expression levels among the four conditions were used to group the ORFs according to their expression level. In these cases, a direct relationship between the CUB and the expression level was observed, and the highly expressed genes showed a major bias, with the exception of the Asp, Cys and His codons. A comparison of the CUB among all groups, based on the expression levels, revealed that the codon usage was similar among genes with similar levels of expression. Although this finding seems rather obvious, the gene expression varied under different conditions; therefore, to classify a gene as lowly or highly expressed based on only one culture condition and state of growth could lead to errors in gene classification and analysis.X. dendrorhous strain CBS 6938 was described using ten ribosomal genes [X. dendrorhous was not observed; however, in the present study, we observed that although these codons are not the preferred codons for each amino acid or for a stop codon, these codons are indeed used in X. dendrorhous. In addition, there are more than ten-fold differences in the RSCU values determined for codons GCA (Ala), AGA (Arg), CAA (Gln), GAA (Glu), CAU (His), AGC (Ser) and GUA  between the previous and present studies.Previously, the CUB for the al genes . HoweverX. dendrorhous cultured in glucose and succinate as the sole carbon sources [X. dendrorhous was also consistent with that of other organisms in which highly expressed genes generally show a higher synonymous codon usage bias attributed to selection for efficient translation [The results obtained from transcriptomic analyses are consistent with the results of previous expression studies. For example, the expression of the genes encoding astaxanthin synthase and phytoene-beta carotene synthase was quantified using RT-qPCR in  sources , and simnslation ,25,27,47nslation ,49, mighX. dendrorhous were consistent with the described patterns, i.e., CCUAAG, GAGUCC and AACCGA, and the most preferred codon pairs were GCGCTC, GUUCCC, ACUCCU, UCUUCU and UCUUCC .Information regarding the CUB is important in the field of heterologous gene expression to achieve the efficient production of recombinant proteins, for example, enzymes relevant in the biotechnology industry . In receX. dendrorhous strain UCD 67–385 [In general, viruses do not encode tRNAs, and the synthesis of viral proteins is dependent on the host translational machinery. Thus, several virus sequences have adapted to the host codon usage, including viruses that infect humans and other mammals, particularly for highly expressed genes ,53. Two D 67–385 ; thus, wX. dendrorhous ORFs are highly expressed, particularly during the stationary phase of growth using succinate or glucose as the sole carbon source, and the majority of the ORFs showed considerable variations in expression under the conditions studied. The codon usage bias and the 3′ codon context bias showed a clear direct relation with the expression levels and GC% of the ORFs, but not the sequence length. However, no similarities among the totiviral and host ORFs were observed for either codon usage or 3′ codon context biases.In general, the identified X. dendrorhous strain UCD 67–385 (ATCC 24230) was used for next-generation whole genome and transcriptome sequencing and analysis. The strain was cultured at 22°C with constant agitation in YM medium  for DNA extraction or in Vogel minimal medium (MMv) supplemented with 2% glucose or 2% succinate for RNA extraction.The wild-type The yeast RNA was purified from the early exponential (18 h) and initial stationary (72 h) phases of growth from cultures grown in MMv medium supplemented with 2% glucose or 2% succinate. After 18 h of culture in MMv medium supplemented with 2% glucose, 1% glucose remained in the medium (confirmed using the DNS method ).X. dendrorhous DNA was isolated from protoplasts as previously described [escribed , resultiTotal RNA was extracted from the cell pellets via mechanical rupture with 0.5 mm glass beads (BioSpec) by vortexing for 10 min, followed by the addition of Tri-Reagent (Ambion). The lysate was incubated for 10 min at room temperature, and subsequently 200 μl of chloroform per ml of Tri-Reagent was added, mixed, and centrifuged for 5 min at 4,000 x g. The aqueous phase was recovered, and two consecutive extractions with acidic phenol: chloroform (1: 1) were performed. The RNA was precipitated with two volumes of isopropanol for 10 min at room temperature, and the RNA was washed with 75% ethanol and suspended in RNase-free water. RNA samples at a 260/280 ratio >1.9, measured using a V-630 UV–vis Spectrophotometer, were used for next-generation sequencing.X. dendrorhous strain UCD 67– 385 was sequenced using the Illumina GAII Sequencing System at Amplicon Express Inc.  and the Illumina HiSeq2000 System at Macrogen Inc. . Read assembly and genome and transcriptome analyses were performed using the CLC Genomics Workbench 5. We estimated that the current collection of genomic scaffolds and contigs should cover approximately 95% of the haploid genome of the yeast. For Illumina GAII genome sequencing, a 250-350-bp paired-end library and a 2,500-3,500-bp mate pair library were constructed and sequenced. In addition, 48 primer pairs across 48 gaps were designed for bulk gap closure by sequencing the PCR products with 96 primers. For Illumina HiSeq2000 genome sequencing, a 100-bp paired-end library was constructed and sequenced. The RNA samples from the 72-h culture were sequenced using a Illumina GAII, including a 250-350-bp paired-end library, and the RNA samples from the 18-h culture were sequenced using Illumina HiSeq2000, including a 100-bp paired-end library.The genome of −3; ii) the blast hits of each sequence were mapped using the Gene Ontology Consortium ; iii) the GO functional annotation was completed using a cutoff value of 10−6; and iv) functional annotation was performed using InterPro annotations. The expression level of each ORF was calculated under each condition as reads per kilobase per million mapped reads (RPKM), as previously described [Using the transcriptome data obtained under each condition, the open reading frames (ORFs) of at least 300 bp in length were predicted using the standard genetic code and the software Geneious® 8.0.2. ORFs that were present in yeast cultured under the four conditions were selected and mapped to five genomic scaffolds of 1,116,253; 1,334,503; 1,461,881; 1,770,274; and 2,396,803 bp. The mapped ORF sequences showing 100% identity with genome sequences, including a correct exon-intron structure, were selected, compared with the database and annotated using the Blast2GO  server: escribed . The resX. dendrorhous ORF names, sequences, RPKM values and the viral ORF sequences used in this work are included in the Additional file The


Angiomatoid fibrous histiocytoma (AFH) is a rare, low-grade malignant soft-tissue tumor most commonly occurring in the extremities of children and young adults and has a low potential of local recurrence and metastasis. Here, we present a case of recurrent subcutaneous perianal AFH. After an initial diagnosis as a sebaceous cyst, we were able to use immunohistochemical findings to correctly identify the mass as an AFH. The patient was effectively treated after 3 surgical resections. This case emphasizes the need to correctly diagnose soft-tissue tumors using a variety of diagnostic modalities to ensure that the patient receives proper treatment. Because of the potential for misdiagnosis, molecular diagnostics are increasingly being used to detect gene fusions characteristic of AFH.2 About two-thirds of cases occur at sites with abundant lymph nodes, such as the antecubital fossa, popliteal fossa, axilla, and inguinal and supraclavicular areas.3 AFH is often associated with systemic symptoms such as anemia, fever, weight loss, enhanced cytokine production, polyclonal gammopathy, and a Castleman disease-like lymphadenopathy.4 Local recurrence is more likely in deeply situated tumors and those with infiltrative margins.5 Surgical resection is the most common management and can effectively control local recurrences and metastases. Only a few cases have been reported demonstrating AFH arising in sites other than the deep dermis and subcutaneous tissue of the extremities, trunk, head, and neck.6 Xiang et al7 reported a case of AFH arising from the retroperitoneum.Angiomatoid fibrous histiocytoma (AFH) is a rarely metastasizing soft-tissue tumor of low-grade malignant potential that mostly affects children and young adults.8 we also review the latest literature on the etiology, diagnosis, and treatment of AFH.Herein, we present a case of a 35-year-old woman suffering from AFH presenting as a recurrent perianal subcutaneous mass, who underwent 4 surgeries. To the best of our knowledge, this is the first reported case of AFH presenting in this manner. Because the rarity of AFH often leads to the misdiagnosis of either a reactive lesion or a benign or malignant tumor,A 35-year-old woman was admitted to our hospital complaining of a perianal subcutaneous mass. Two years ago, we treated her for a similar mass that we surgically resected . Both masses were similar in tenderness, texture, and mobility. The only difference of the current mass was in its location, on the left side of her anus and exposed to air. During this visit, the mass was the size of a peanut. Approximately 1 month later, however, it grew to approximately 4 × 2.5 cm  angiography showed no abnormalities of the abdominal arteries and revealed an abnormal soft tissue mass surrounding the anus. Magnetic resonance imaging (MRI) of her pelvic cavity revealed small multifocal areas on the left, right, and back side of the anus Figure . These sGiven this diagnosis, we performed a retroperitoneal tumor resection and transverse colostomy and removed a firm hemorrhagic mass measuring 8.0 × 4.0 × 3.5 cm  Classification of Tumours of Soft Tissue and Bone, AFH is described as an intermediate soft-tissue tumor that is often locally aggressive.6 To the best of our knowledge, our case is the first report to describe a recurrent perianal subcutaneous AFH with no usual systemic symptoms such as anemia, fever, and so on.Although AFH is most commonly associated with the extremities, there have been a number of reported cases of AFH arising at additional sites such as the mediastinum, bone, intrapulmonal tissue, intracranial tissue, retroperitoneum, lung, vulva, and ovary.12 On CT images, these confusable heterogeneous soft-tissue mass in the extremities are usually isodense to muscle with high attenuating areas corresponding with hemorrhaging. MRI is superior to CT in depicting fluid–fluid levels within the cystic component of the mass, indicative of intralesional hemorrhage. AFH lesions are often homogeneously hypointense (isointense to muscle) on the T1WI and heterogeneously hyperintense on the T2WI.1 In our case, the mass on CT is slightly hypodense to muscle and its MRI fit with the reported findings.Despite previous studies, it is still hard to differentiate AFH from malignant fibrous histiocytoma, sarcoma, or other tumors with imaging alone.13AFHs have a broad range of histological features. Typical AFHs are characterized by 3 microscopic findings: solid arrays or nests of histiocyte-like cells, hemorrhagic cyst-like spaces, and aggregates of chronic inflammatory cells. Multifocal recent and old hemorrhages are a striking feature of this tumor. These spaces resemble vascular spaces, but they are not lined by endothelium. Inflammatory cells present include lymphocytes and plasma cells. A thick pseudocapsule and occasional germinal centers give this tumor a resemblance of a lymph node.14 Bohman et al4 reported a series of cases with perineurioma-like areas, with eosinophils in the associated inflammatory infiltrate, and with numerous eosinophils. To clarify the cellular differentiation features of AFH, Hasegawa et al15 once examined 4 cases of AFH by clinicopathologic, immunohistochemical, and ultrastructural analyses. Seventy-five percent examined by electron microscopy had a mixture of fibrohistiocytic, myofibroblastic, and undifferentiated cells containing cytoplasmic processes and dense core granules. The authors pointed that AFH has a variety of immunophenotypes, including histiocytic, myofibroblastic, epithelial, and neural and may occasionally have predominantly round cell morphology.15In addition to features that have been previously described such as nuclear pleomorphism, increased mitotic activity, atypical mitotic figures, reticular, myxoid patterns, lymphoplasmacytic infiltration, pseudovascular spaces filled with blood, round cell morphology, and spindle cell patterns.2 Cytogenetic analysis of our case demonstrated positivity for desmin, CD68, and CD99.The immunohistochemical features sometimes present a unique immunophenotype. For instance, approximately 50% to 60% of AFH lesions coexpress desmin, EMA, CD68, and CD99 but are generally negative for keratins, S-100 protein, CD34, and follicular dendritic cell markers (CD21 and CD35).16 followed by EWSR1-ATF1 from t,17 and the least common FUS-ATF1 from t.18 Thway et al expanded the clinicopathological spectrum of AFH by characterizing 13 AFH cases by fluorescence in situ hybridization (FISH) and reverse transcription polymerase chain reaction (RT-PCR). Besides the EWSR1 gene rearrangement detected in 4 cases by FISH, EWSR1-CREB1 fusion was confirmed in 9 cases, including a schwannoma-like AFH, and a EWSR1-ATF1 fusion was detected in a myoepithelioma-like AFH.16 These studies suggest that combinatorial FISH and RT-PCR will likely be diagnostically powerful tools to identify AFH by evaluating EWSR1-CREEB1, EWSR1-ATF1, or FUS-ATF1 fusions.2Because the role of immunohistochemistry in establishing the diagnosis of AFH is still limited, additional markers will be important to distinguish this tumor. Recently, the molecular pathogenesis of AFH has become clarified by the discovery of fusion genes resulting from various chromosomal translocations, including the most prevalent EWSR1-CREB1 derived from t,19 In the currently reported case, the initial diagnosis was as a sebaceous cyst because of the resemblance in appearance. The imaging studies we obtained were not diagnostic, but a pathological review of the immunohistochemistry confirmed our AFH diagnosis.It is important to correctly diagnosis AFH patients to avoid overtreatment when misinterpreted as a more aggressive tumor such as malignant fibrous histiocytoma, nodular Kaposi sarcoma, Ewing sarcoma, and rhabdomyosarcoma. Considering its potential for recurrence and metastasis, it is also important to avoid undertreatment by misdiagnosing it as a sebaceous cyst, perianal abscess, aneurysmal benign fibrous histiocytoma, or inflammatory pseudotumor.We report an uncommon case of AFH located around the anus without systematic symptoms. This patient suffered a frequent recurring AFH mass and underwent 3 surgical resections. AFH is a rare disease that is often initially misdiagnosed. It is a challenge to avoid overtreatment or undertreatment. Our case emphasizes the need to correctly diagnose soft-tissue tumors using a variety of diagnostic modalities to ensure that the patient receives proper treatment.


Serum, feces, and various tissues were collected from the mice after RNA consumption and analyzed for several miRNAs. Exogenous plant miRNAs were present in the sera, feces, and tissues of animals and these exogenous plant miRNAs were primarily acquired orally. MiR-172, the most highly enriched exogenous plant miRNA in B. oleracea, was found in the stomach, intestine, serum, and feces of mice that were fed plant RNA extracts including miR-172. The amount of miR-172 that survived passage through the GI tract varied among individuals, with a maximum of 4.5% recovered at the stomach of one individual, and had a range of 0.05–4.5% in different organs. Furthermore, miR-172 was detected in the blood, spleen, liver, and kidney of mice.MicroRNAs (miRNAs), a class of small RNAs, are important molecules that influence several developmental processes and regulate RNA interference (RNAi), and are abundant in animals, plants, and plant tissues that are traditionally consumed in the diet. The survival of plant small RNAs from the diet in animals, however, remains unclear, and the persistence of miRNAs from dietary plants in the animal gastrointestinal (GI) tract is still under debate. In this study, ICR mice were fed plant total RNAs in quantities of 10–50  MicroRNAs (miRNAs) are a class of small RNAs (19–25 nucleotides (nt) long) which have posttranscriptional activity in animals and plants  tract, and organs.In this report, we investigated the survival of exogenous RNA in the digestive system, blood, and several organs of mice after being fed RNAs extracted from Although the functional effect of those plant-specific miRNAs in mammals is under debate, first their ability to survive the mammalian GT tract and enter blood and organs needs to be verified. A plant specific miRNA, miR-172, was identified in the feces by polymerase chain reaction (PCR) and validated by TaqMan® assay. These results show that exogenous plant miRNA can survive in the murine GI tract, enter peripheral blood, and continue to other organs.B. oleracea (see below) in DEPC-treated water in quantities of 10 to 50 μg in a total volume of 500 μL. The solution was administered from a pipette tip directly into the oral cavity. In some of the feeding experiments, the same amount of RNAs solution was added to the food offered as diet to the animals. As a control, prior to feeding, a fecal sample from each experimental animal was examined to confirm the absence of miRNA sequences with homology to miR-172 (data not shown).All animal experiments were performed in accordance with permission from the ethics committee of Southeast University, China. All animal experiments were performed using ICR strain mice on a 12-h light/dark cycle in a pathogen-free animal feeding facility at Southeast University. Male and female ICR mice between the ages of 2–4 months were gavage fed total RNAs extracted from B. oleracea, Brassica rapa, rice, corn, Arabidopsis thaliana, and Solanum lycopersicum using Trizol®  according to the manufacturer's protocol. The concentration of total RNA was quantified by the absorbance at 260 and 280 nm. The total RNA was electrophoresed in 1.6%/Tris-Boric acid-EDTA buffer agarose gel to visualize small RNAs. A stem-loop reverse transcription polymerase chain reaction (RT-PCR) assay  was incubated at 37°C with freshly drawn serum or fecal suspension in DEPC-treated water from control mice. At various times after starting incubation , RNA samples were prepared, purified (see below), and analyzed for the presence of small RNA sequences by electrophoresis.To determine the degradation of plant small RNA in vitro, total RNA  after feeding the exogenous RNA solution to mice, fecal samples were collected. Mice were then anesthetized with ether, the abdominal fur was thoroughly washed with ethanol, and one set of surgical instruments was used to open the peritoneal and thoracic cavities by cutting the diaphragm. Finally, the still palpitating heart was punctured with a disposable 0.9-mm gauge needle, and blood (about 0.5–1 mL) was drawn with a syringe. For facilitation, in some of the experiments, blood was taken from the fundus of eyes in the mice. Thereafter, the contents of the stomach, intestines, liver, spleen, and kidneys were removed for analyzing. Control analyses on abdominal fur washing fluids performed by PCR methods revealed no evidence for detectable contamination.The contents of the stomach, intestines, and feces were dispersed in 0.5 ml DEPC-treated PBS, respectively. The suspensions were incubated at 37°C for 15 min, and subsequently nucleic acids were extracted according to the methods described above. These extracts were subsequently treated with 10 U of DNase per mL at 37°C for 30 min. Prior to use, the DNase was heated to 85°C for 10 min to destroy contaminating DNase.Total RNA was extracted using Trizol® from the liver, spleen, kidney, and blood according to the standard protocol. In some of the experiments, the blood cells were pelleted by low-speed centrifugation, and RNA was extracted separately from the cell pellet and serum by the SDS-proteinase K-phenol/chloroform procedure. Next, DNase treatment was performed to eliminate residual DNA contamination. Finally, after a second acid phenol:chloroform extraction, the RNA was resuspended in DEPC-treated water.μL total volume) was incubated with 1 μL of a cocktail containing 10 mmol/L of each of the antisense primers listed in the table To measure the maximal length of miRNA fragments resistant to GI tract degradation, gene-specific primers and reverse transcriptase were used to convert the pre-miRNA and mature miRNA from stomach and fecal samples to cDNA , 200 nmol/L of each primer, and water to a total volume of 20 μL. The reactions were amplified for 15 sec at 95°C and 1 min at 60°C for 40 cycles. The thermal denaturation protocol was run at the end of the PCR cycles to determine the number of products that were present in the reaction. Reactions were typically run in duplicate. The cycle number at which the reaction crossed an arbitrarily placed threshold (CT) was determined for each gene and the relative amount of each miRNA.Real-time quantitative PCR was performed using standard protocols on an Applied Biosystems 7500 Sequence Detection System . Briefly, 1.25 μg of RNA were treated with DNase and followed a similar protocol to that described above in the SYBR Green assay.To verify the SYBR Green results and to determine whether plant miR-172 can survive the mouse GI tract, a TaqMan® assay was conducted. TaqMan® probe is the “gold standard” in miRNA detection at present, which can distinguish a one nucleotide difference in sequence and provides a reliable means to assess the survival of miR-172 in the GI tract. The RNA samples from blood, fecal suspensions, and contents of the stomach, intestines, and organs of animals previously fed with 50, 30, or 10 To determine the percentage of originally administered plant RNA that was present after digestion, the TaqMan probe assay was used. Standard curves were constructed for mature miR-172 using a standard sample and quantifying 10-fold dilutions by real-time PCR. After collecting and quantifying the miR-172 present in the various tissues and samples, percentages of the originally administered RNA were determined.μg total RNA per gram of mature plant sample, with 371.6 μg being the average total RNA yield per gram of mature seeds or plant tissue from seven independent RNA extractions (range 214.2–905.3 μg per gram of sample) (B. rapa) than in mature seeds   . After eB. oleracea, B. rapa, A. thaliana, and S. lycopersicum as detected by semiquantitative RT-PCR analysis with 30 cycles  showed high levels of expression in Despite the lack of a low-molecular weight RNA marker, we can infer that the bright bands on the agarose gel are small RNA fragments in the range 20∼100 bp that survived degradation after different times of incubation  up to 36 h in the contents of the intestine Fig. B, up to In addition, from the experiments carried out, there was no evidence that differences in the sex of the animals or their age within the 2- to 4-month range affected the outcome of any of the experiments reported .RNA is an unstable molecule that degrades easily under normal environmental conditions . Despite the presence of mature miR-172 in fecal samples, pre-miR-172a and pre-miR-172b were not detected in mice feces 2 h after feeding. It is apparent that pre-miR-172 decomposed in 2 h, while miR-172 could survive between 2 and 72 h after feeding RNA. At later times after feeding, pre-miR-172 could no longer be detected in the mice. There was no evidence for long-term survival of pre-miRNA in the intestines of the mice.Next, miRNA survival in the GI was measured in mice fed exogenous plant RNA, as well as investigating the presence of miR-172 sequences in liver, spleen, kidney, whole blood, and serum of the animals fed with plant RNA. Between 2 h and 72 h after feeding 50 μg or 10 μg of RNA were fed to mice , the relatively large amount of exogenous miRNA detected in test samples reassures us that exogenous plant miRNAs did survive in the GI tract. Moreover, the transient survival of small RNA in the GI tract, feces, blood, and organs have been proved by TaqMan probe methods, and is a method generally accepted.μg. These values must be related to the body weight of mice (between 15 and 25 g), and thus constitute an approximate fraction of 2/106 of their body weight. For humans of average weights between 50 and 70 kg, one would thus have to postulate an intake of about 50–140 mg of RNA with the daily food uptake to parallel the situation simulated in these mice experiments. Based on the estimate of ∼500 μg of total RNA yielded per gram mature seeds, this would require about 280 g of food consumed, a reasonable human exposure.The amount of foreign RNA administered to mice was between 10 and 50 The stability of exogenous RNA in organisms and whether it can enter cells and elicit certain functional control in the recipient cells remains unclear. Zhang et al.  reportedμg of RNA to mice, and the result suggests that there is a good correlation between miR-824 and miR-172 (Fig. In this context, many questions remain to be investigated, none of which will be technically easy to control. Our data are consistent with the notion that a steady flow of exogenous genetic material persists in the GI tract, and a very small percent can enter the circulation and distribute in different organs (Zhang et al. 172 Fig. . The preWhile our results do not support general and consistent uptake of dietary plant miRNAs, additional studies are needed to establish whether or not plant or animal xeno miRs are transferred across the GI in sufficient quantity to regulate endogenous gene expression, and these experiments have been initiated (Witwer et al. This study shows that foreign plant miRNA ingested by mammals can survive through the GI system, and enter the bloodstream and various organs of mice. Experiments involving foreign miRNA entering into established mammalian, and probably other eukaryotic, genomes have to take into account the possibility that these foreign small RNA may have important consequences, with unexpected alterations of mammalian genome and expression patterns. It is important to note that we have not found phenotypic changes in all the mice fed with the foreign RNA.


In the present study, the successful management of severe paraquat (PQ) poisoning with multiple organ dysfunction syndrome is described. A 42-year-old female ingested >100 ml PQ (20% weight/volume) in an attempted suicide. After 22 h the patient was admitted to hospital with serious liver, kidney and lung damage. Comprehensive therapy that maximized poison elimination was administered, along with appropriate glucocorticoids and medication for anticoagulation and protection of the liver and kidney. The patient was successfully treated and recovered after 40 days. However, pulmonary damage was aggravated when the glucocorticoid treatment was stopped after 2 months; the lungs recovered again following systematic therapy. Subsequent to a 8-month follow-up, the patient was able to look after herself in her daily life. To the best of our knowledge, successful treatment following severe PQ poisoning is rare. Paraquat , an effective herbicide that has favorable environmental characteristics, as well as being cost-effective, was first synthesized in 1882 and has been used as an herbicide since 1955 . PQ has China is an agricultural country and PQ is used extensively. Since the first reported case of PQ poisoning, studies have focused on the mechanism and effects of combination therapies and various agents ,8. FurthA 42-year-old female, weighing ~60 kg, ingested >100 ml PQ (20% weight/volume) in an attempted suicide at 12:50 p.m. on September 13, 2012. The patient experienced nausea, vomiting and discomfort immediately following PQ ingestion and was sent to a county hospital 20 min later. A gastric lavage was immediately performed and kaolin (30 g) and mannitol infusion (250 ml) was administered within 1 h following ingestion. Hemoperfusion therapy was prescribed for 2 h. The dose for methylprednisolone was 1,000 mg/day for the first day. Due to the severity of the condition, relatives brought the patient to the Department of Poisoning and Occupational Diseases, Qilu Hospital of Shandong University at 10:55 a.m. on December 14, 2012. On admission, the patient’s vital signs were normal, with a body temperature 36.6°C, pulse 89 beats/min, respiratory rate 19 breaths/min, blood pressure 125/80 mmHg and blood oxygen saturation 98%. Physical examination was normal, with the exception of painful erosions in the oral cavity. Based on the typical poison-associated symptoms in the mouth and the gastrointestinal tract, computed tomography (CT) manifestations, significantly abnormal results in the main laboratory test results and the information provided by the patient and her relatives on admission, the patient was diagnosed with severe acute PQ poisoning.Cordyceps and depside salt from Salvia miltiorrhiza) were administered to the patient. This was termed the Qilu therapeutic schedule. The dose for glucocorticoids was 800 mg/day for the first 4 days and then reduced to 40–80 mg/day. The patient’s condition was markedly improved following the comprehensive therapy. Low doses of prednisone were maintained after 3–6 weeks. After 2 months the patient stopped taking glucocorticoids; however, the pulmonary damage became aggravated and recovered again subsequent to treatment with glucocorticoid and traditional Chinese medicines.Following admission to Qilu Hospital, the patient was subjected to complete gastrointestinal decontamination using activated carbon, Smecta and mannitol in order to thoroughly remove the pesticide residue from the gastrointestinal tract. Furthermore, other treatments such as an intravenous drip of high doses of glucocorticoids, anticoagulants, anti-free radical, liver- and kidney-protecting and water and electrolyte balance-maintaining agents, and treatment with Chinese herbs (including Xuebijing, Following multi-target comprehensive therapy, the dynamic changes in the lung CT scans are shown in PQ is a non-selective herbicide that has been widely used in agriculture since the 1960s . Althouget al (In the present case study, a patient with PQ poisoning complicated with MODS was successfully treated using multi-target comprehensive therapy. The patient ingested a high dose of PQ, resulting in severe liver and kidney damage. Gastrointestinal procedures were performed to prevent further absorption of PQ residue, accompanied by glucocorticoid treatment to reduce cell absorption, promote the elimination of PQ and control lung inflammation lesions. The mechanism of PQ toxicity has been previously investigated by Dinis-Oliveira et al . In the


Culex camposi Dyar, Culex coronator Dyar and Knab, Culex covagarciai Forattini, Culex usquatus Dyar, Culex usquatissimus Dyar, and Culex ousqua Dyar). Culex coronator has been incriminated as a potential vector of West Nile Virus (WNV), Saint Louis Encephalitis Virus (SLEV), and Venezuelan Equine Encephalitis Virus (VEEV). The complete mitochondrial genome of Cx. coronator, Cx. usquatus, Cx.usquatissimus, and Cx. camposi was sequenced, annotated, and analyzed to provide genetic information about these species.The Coronator Group currently encompasses six morphologically similar species  and the AT-rich control region. Comparative analyses of the 37 genes demonstrated the mitochondrial genomes to be composed of variable and conserved genes. Despite the small size, the ATP8, ATP6 plus NADH5 protein-encoding genes were polymorphic, whereas tRNAs and rRNAs were conserved. The control region contained some poly-T stretch. The Bayesian phylogenetic tree corroborated that both the Coronator Group and the Culex pipens complex are monophyletic taxa.The mitochondrial genomes of Cx. coronator, Cx. usquatus, Cx. usquatissimus and Cx. camposi share the same gene composition and arrangement features that match to those reported for most Culicidae species. They are composed of the same 37 genes and the AT-rich control region, which contains poly-T stretches that may be involved in the functional role of the mitochondrial genome. Taken together, results of the dN/dS ratios, the sliding window analyses and the Bayesian phylogenetic analyses suggest that ATP6, ATP8 and NADH5 are promising genes to be employed in phylogenetic studies involving species of the Coronator Group, and probably other species groups of the subgenus Culex. Bayesian topology corroborated the morphological hypothesis of the Coronator Group as monophyletic lineage within the subgenus Culex.The mitochondrial genomes of The online version of this article (doi:10.1186/s12864-015-1951-0) contains supplementary material, which is available to authorized users. Culex camposi Dyar, Culex coronator Dyar and Knab, Culex covagarciai Forattini, Culex ousqua Dyar, Culex usquatus Dyar and Culex usquatissimus Dyar are members of the Coronator Group of the subgenus Culex , Cx. pipiens , Aedes albopicutus [NC_006817] and Anopheles darlingi [NC_014275]. The latter two species were employed as outgroups. Multiple alignments were generated in Clustal X [Phylogenetic analysis was carried out using Bayesian inference from the concatenated sequences of all protein coding genes of the five newly sequenced gi NC_0145. The lalustal X  to ensurlustal X , under Alustal X  and the Cx. coronator, Cx. usquatus, Cx. usquatissimus and Cx. camposi contain 37 genes, which have been found in other metazoan and Culicidae species, including 13 protein-encoding genes, 22 tRNAs and 2 rRNA genes ,( rrnL and rrnS) and the AT-rich control region [Culex quinquefasciatus : 15,573 bp in Cx. usquatus and Cx. usquatissimus from Acre state (Cx. usquatissimus AC); 15,574 bp in Cx. usquatissimus from Rondônia state (Cx. usquatissimus RO); and 15,576 bp in Cx. coronator , whereas the GC-skew was −0.15 for all species, showing that adenine and cytosine are in the highest proportion compared to thymine and guanine, respectively, in the majority strand. Those findings are in agreement with other studies of species of the genera Anopheles and Drosophila [In all species, nucleotide composition was biased toward overall AT content. This AT-bias has been reported in the mitochondrial genomes of other metazoan species –37. In C species  and othe species , the val species . Accordi species , the higosophila .Table 3OCOX1 gene in which no canonical ATN was found, the majority of the PCGs of Culex species possessed the start codon ATN  as a start codon for Drosophila melanogaster. The author hypothesized that if an ATA, a recognized start codon, overlaps a stop codon TAA in the frame −1, the ATA of the tetranucleotide ATAA could function as a highly conserved 0 frame and as an opening frame. Notwithstanding, in the mitochondrial genome of Culex species, the closest ATAA tetranucleotide is located within the trnL2 gene, overlapping 36 base pairs of the gene. Consequently, it is plausible to suppose that the hexanucleotide ATTTAA, which usually flanks the beginning of the COX1 in mosquitoes, is involved in the initiation of the translation signal of this gene, as it was proposed for Tetrodontophora bielanensis [Except for the  insects –35 and t insects . In 1983 insects  proposedlanensis .ATP6, ATP8, COX3, CytB, NAD2, NAD3, NAD4, NAD4L, NAD5, NAD6) have complete TAA stop codons, except for COX1 and COX2 genes, which terminate with an incomplete codon T. This phenomenon has been extensively reported in other mitochondrial genomes, especially in COX genes, and so far it has been related to posttranscriptional polyadenylation, during which residual adenines are added in order to originate TAA terminators [Eleven genes  concentrated the majority of the polymorphisms; however COX1,only presented synonymous substitutions, whereas NAD2, NAD5, NAD4, CytB, and NAD1 showed 3, 2, 1, 3, and 2 non-synonymous substitutions, respectively. Additionally, despite the small length, ATP6 and ATP8 were polymorphic genes in Culex species, with 23 (2 non-synonymous) and 4 variable (2 non-synonymous) sites. Additionally, the dN/dS ratios  showed that the codons with adenine and thymine in the third position were overused when compared to other synonymous codons Table , which ctrnR gene to 72 bp in trnV gene  arm by a DHU-loop [trnS1 has been reported for Collembola species [The set of 22 tRNA genes in all species varied in length from 64 bp in  % Table . The ant insects –35 ; and the other between trnS2 and NAD1 genes with 19 bp in all samples. Although those spacers account for only 0.3 % of the whole mitochondrial genomes, they are composed almost exclusively of adenine and thymine, with an AT content that is higher than that observed in the AT-control region, reaching 94.5 % in Cx. usquatissimus from Acre . Those differences in length were caused by several indels of one to three nucleotides distributed throughout the AT regions of the mitochondrial genomes. Considering both indels and nucleotide substitutions, 34 variable sites were observed in Culex species. Similar to nuclear noncoding DNA, the AT-control region is subjected to a high level of polymorphisms that lead to variation in size and base composition. However, in species of the Anopheles gambiae complex, there is evidence that this region change more slowly than the 3rd codon position of PCGs [Anopheles, there are conservative poly-T regions of 15 to 18 bp [Culex species studied, we observed 4 conservative T-stretch regions of 6 to 15 bp. This was likely caused because this region contains several regulatory elements, including the origin of replication and transcription of the major strand of the mitochondrial genome [The size variation, ranging from 719 bp in  of PCGs . Additiol genome  and, conCulex species to verify regions with high nucleotide divergence and thus, identified potential markers that can be employed for future studies focusing on Culex species. Results of the sliding window analysis showed low nucleotide diversity (Pi), ranging from 0.0 to 0.035  among all mitochondrial genes. The COX1 presented low variation, mainly in its 5′ proximal half. This region is largely employed in studies focusing in Culicidae species, and it is considered as the universal barcode for species identification [COX1 is a conserved gene [Mitochondrial DNA is largely employed in phylogeny and evolutionary studies because it has a high mutation rate, maternal inheritance, high number of copies, and intraspecific polymorphisms . Herein,fication . Notwithved gene –53. ConsrrnL and rrnS demonstrated to be the most conserved genes within the mitochondrial genomes of species of the Coronator Group, showing low nucleotide diversity. Although rrnL usually shows high variability among some groups, i.e., Taeniidae [rrnL was 0.02. Per se, the rrnL and rrnS genes seem not to be good markers for taxonomy and evolutionary studies focusing on species of the Coronator Group.The aeniidae , Nematodaeniidae , and Octaeniidae , herein,Cx. coronator and the problematic identification of females based on morphological characters, the development of molecular markers can facilitate species identification as well as establish phylogenetic relationships within the group and other closely related species. Taken together, results of the sliding window analysis and the dN/dS ratios indicate that the NADH5, ATP8 and ATP6 genes represent potential markers for future investigations regarding species of the Coronator Group, primarily because they retain high variations among PCGs with an elevated proportion of non-synonymous substitutions. Notwithstanding, the low levels of diversity shown herein, demonstrates that the utility of sequences of the mitochondrial gene needs further evaluation in future studies employing a larger sample size and other species of the subgenus Culex as well as other Culex (Culex) species.Considering both the epidemiological importance of Cx. pipiens complex. Within the clade formed by species of the Coronator Group, the placement of Cx. coronator as sister of the lineage leading to Cx. camposi plus Cx. usquatus plus Cx. usquatissimus was strongly supported (100 % Bayesian posterior probability). Additionally, divergence among species from this group under Maximum Composite Likelihood (MCL) was low (Table Cx. usquatus and Cx. camposi to 1.4 % between Cx. coronator and Cx. usquatissimus (from Acre and Rondônia). The low nucleotide divergence observed among the mitochondrial genes of species of the Coronator Group supports the close phylogenetic relationships observed in previous studies [Culex. However, if more species were included in the study, likely the phylogenetic signal of the mitochondrial genes would be changed. For instance, studies based on morphological phylogeny [COX1 barcode sequence data of several species of the genus Culex [Cx. surinamensis and Cx. maxi. Those results suggest that Cx. surinamensis and Cx. maxi may share their ancestor with current species included in Coronator Group and as such, they should be included in the group. However, further studies need to be developed to address this hypothesis.Best-fit models selected by the Akaike Information Criterion are showed in Additional file ow Table , ranging studies –5. As hy studies , the highylogeny  and COX1ATP6, ATP8 and NADH5 for reconstructing phylogenetic relationships within the Coronator Group, a Bayesian analysis was performed only with these genes (Fig. Cx. pipiens complex species, results of Bayesian analysis recovered a polytomy between Cx. quinquefasciatus and Cx. pipiens. Those results are important to show how evolutionary behavior of the genes are different among groups of species, even within the same subgenus, and how carefully researchers need to be when selecting a molecular marker.In order to address the effectiveness of the Cx. usquatus, Cx. usquatissimus, Cx. coronator, and Cx. camposi share the same gene order and gene content of other Culicidae species with the mitochondrial genome reported to date. The overall length and the AT content of the four Culex species are within the range observed in species of the Anopheles albitarsis complex, Cx. quinquefasciatus, and Ae. aegypti. Except for trnR and trnS1, the tRNAs were folded into the typical cloverleaf-like structure. Similar to other metazoan and species of the family Culicidae, the control region presented some conservative poly T- stretches, which seems to be involved in the functional roles of mitochondrial genome. Results of the Bayesian phylogeny corroborated the monophyly of the Coronator Group. Nevertheless, new insights on the evolution of the group can be addressed using a larger species sampling, including morphologically similar species and species of other Culex subgenera, for example, Lutzia, Phenacomyia and Phytothelmatomyia species. Probably because of the functional roles played in the respiratory chain by the mitochondrial genome, PCGs showed high purifying selection and low nucleotide divergence among the species investigated here.The complete mitochondrial genomes of