Further exploration of UVR-induced and Slug-dependent pathways of cytokine induction in melanocytes may reveal novel targets for melanoma therapy.Despite extensive investigation, the precise contribution of the ultraviolet radiation (UVR) component of sunlight to melanoma etiology remains unclear. UVR induces keratinocytes to secrete proinflammatory and immunomodulatory mediators that promote inflammation and skin tumor development; expression of the slug transcription factor in keratinocytes is required for maximal production of these mediators. In the present studies we examined the possibility that UVR-exposed melanocytes also produce proinflammatory mediators and that Slug is important in this process. Microarray studies revealed that both UVR exposure and Slug overexpression altered transcription of a variety of proinflammatory mediators by normal human melanocytes; some of these mediators are also known to stimulate melanocyte growth and migration. There was little overlap in the spectra of cytokines produced by the two stimuli. However IL-20 was similarly induced by both stimuli and the NF Although the incidence of melanoma is increasing faster than that of any other neoplasm and the tumor has been studied intensively, the cause of melanoma remains incompletely understood. It is clear from epidemiologic and clinical observations that the ultraviolet (UVR) component of sunlight is an important etiologic factor; however, the precise mechanisms by which UVR drives melanomagenesis are unknown –4. Mutat An important mechanism for tumor promotion is the creation of a tumor microenvironment that fosters tumor cell proliferation and migration while suppressing an effective immune response to the tumor cells. It has been shown that many tumor cell types and their precursors secrete a wide variety of proinflammatory and immunomodulatory substances , 8. For  Based on our findings in keratinocytes, we examined the possibility that UVR also induces expression of proinflammatory and immunomodulatory mediators by melanocytes. We also investigated a possible role for Slug in modulating UVR induction of these mediators.Normal human melanocytes of neonatal origin were obtained from the American Type Tissue Collection (ATCC) and maintained in Dermal Basal Medium supplemented with Melanocyte Growth Kit (ATCC).2 UVR and harvested 24 hours later. UVR was obtained from a bank of Philips TL20 W/12 RS UVB lamps (American Ultraviolet Company). These lights emit wavelengths between 280 and 400 nm, with a peak at 313 nm. The lamps emit approximately 60% UVB wavelengths and 40% UVA wavelengths, with less than 3% of the radiation in the UVC range [ Melanocytes were grown to confluence then exposed to 300 J/mVC range .μL of adenoviral stock  mixed with one mL of medium was added to each well of a six-well plate. Cells were incubated with the adenovirus for 24 hours before the medium was changed. Cells were harvested 24 hours later, and mRNA was isolated utilizing the RNeasy kit (Qiagen).Melanocytes were grown to 75% confluence then incubated with adenoviral Slug (AdSlug) or a control adenovirus  . For traFor mRNA analysis cDNA was generated utilizing the Applied Biosystems High Capacity cDNA RT kit. Following reverse transcription, RT-QPCR was performed utilizing applied biosystems assays on demand for Snai2 and 18S together with TaqMan Universal Master Mix. Amplifications were carried out on an applied biosystem 7900HT real-time PCR analyzer, utilizing the standard curve method for quantification .μg of protein was loaded on a 4–20% tris-HCL gel. Following SDS-PAGE, protein was transferred to PVDF membrane and probed with antibodies against Slug  or GAPDH (Abcam). The secondary antibody utilized was anti-rabbit IgG HRP . Primary antibodies were used at 1 : 1000 dilutions, while the secondary antibody concentration was 1 : 5000. Blots were incubated overnight at 4°C with primary antibody and two hours at room temperature with secondary antibodies. Prior to exposure to film, membranes were incubated in Super Signal West ECL (Thermo Fisher Scientific). For quantification, densitometry was performed. Whole cell lysates in RIPA buffer plus HALT protease inhibitor cocktail (Thermo Scientific) were cleared by centrifuging at 10,000 rpm for 10 minutes at 4°C. A total of 40 For cytokine profiling, the PAHS-3803 Human Inflammatory Response and Autoimmunity Cytokine Array (SA Biosciences) was employed. This array quantifies the expression of 370 key genes involved in immune response and inflammation, including genes encoding inflammatory cytokines, chemokines, and their receptors as well as genes involved in cytokine synthesis and metabolism and in cytokine-cytokine receptor interactions. RNA was analyzed to check RNA quality using the Agilent RNA 6000 Nano kit. Only RNA with a RIN value less than 7.5 was employed. For each sample, 1000 ng of RNA was used to synthesize the first strand of cDNA using the high-capacity cDNA RT Kit from Applied Biosystems (ABI), following standard ABI Protocol. The amount of cDNA added to each of the 96-well plates was standardized. The cDNA was mixed with iTAQ SYBR Green Supermix (Bio-Rad), and each plate was run using the ABI Prism 7900 HT instrument using the following protocol: 10 minutes at 95°C for 10 minutes; 40 cycles of 0.15 seconds at 95°C and 60 seconds at 60°C; 15 seconds at 95°C; 15 seconds at 95°C and 15 seconds at 95°C. Analysis of each plate was performed using the SDS 2.3 program from ABI using the same threshold and baseline for all biological samples. Two independent sets of samples were analyzed.http://www.ingenuity.com/).Genes with expression levels altered more than two-fold in both replicate arrays were identified. Ingenuity Pathway Analysis software (Ingenuity Systems) was used to construct networks that functionally relate genes with markedly altered expression due to UVR exposure or Slug overexpression. These networks are based on algorithms that reflect the connectivity among different genes. A score is generated that indicates the relevance of any given network to the input genes (2 UVR showed a marked induction of Slug mRNA expression at 24 hours after exposure. However, Slug protein induction was much less robust, averaging a little less than two-fold at 24 hours. Thus, as in keratinocytes, Slug expression is UVR inducible [As shown in nducible .Two independent comparisons of inflammatory mediator expression in UVR-exposed versus unexposed and in Slug overexpressing versus control normal human melanocytes revealed a number of genes with expression consistently altered two-fold or more.κB transcription factor and the NFκB activator RIPK2 was unexpected, as UVR activates the NFκB pathway in many melanoma cells, and these cells constitutively express both NFκB and an alternative activator NIK [ Nine genes showed increased expression and six genes showed decreased expression in UVR-exposed versus unexposed melanocytes . Genes wator NIK . The rolator NIK . The relκB pathway [AdSlug-transfected melanocytes show markedly enhanced Slug expression . Compare pathway ; however The only gene with expression similarly increased by both UVR and Slug overexpression was IL-20, a member of the IL-10 family of cytokines. IL-20 plays a poorly understood role in cutaneous inflammation. It is highly expressed by keratinocytes in inflammatory conditions of the skin-like psoriasis and atopic dermatitis; however, the role of the cytokine in keratinocyte biology and immune regulation remains controversial , 34. ExpP value, which indicates the probability of an association between genes in the dataset and a given canonical pathway, was ≤10−30 for both gene sets. However, because the analysis was based on a limited number of genes preselected for their involvement in inflammation and autoimmunity, this degree of association is perhaps not remarkable. The canonical networks identified accounted for 11/16 genes in the UVR dataset and 13/16 in the Slug dataset. Many of the gene interactions in both datasets were centered around NFκB. The NFκB pathway is constitutively activated in malignant melanoma and is believed to be responsible for persistent expression of chemokines by melanoma cells [κB activation may occur via MAPK pathways [We investigated interactions among the genes with altered expression in UVR-exposed and in Slug-overexpressing melanocytes using Ingenuity Pathway Analysis Figures  and 3. Fma cells . For Slupathways , 36.β, TNFα, IL-6, IL-8, CCL2, CCl3, and CCL5 [UVR makes important contributions to skin carcinogenesis. UVR serves as a complete carcinogen for nonmelanoma skin cancer: it both induces critical mutations in keratinocytes and promotes tumor expansion . One of and CCL5 , 39, andand CCL5 . Our previous studies have shown that the Slug transcription factor is an important mediator of UVR-induced inflammation in the skin. Slug knockout mice are highly resistant to UVR-induced skin inflammation because UVR-exposed Slug knockout keratinocytes release fewer proinflammatory mediators than wild type keratinocytes . The rolκB, a transcription factor known to play an extremely important role in melanoma cell production of chemokines [κB and Slug expression are strongly induced by growth factors like EGF, which signals through the MAPK pathway [ The spectrum of proinflammatory genes with altered transcription in UVR-exposed versus Slug-overexpressing melanocytes showed little overlap. In fact, the only proinflammatory gene similarly altered in the two conditions was IL-20, which plays a poorly defined, although potentially critical role in cutaneous inflammation . Despite pathway . Another Tumor-derived proinflammatory and immunomodulatory molecules, like those stimulated by UVR exposure and Slug expression, clearly influence tumor growth by modulating the inflammatory and immunologic milieu of the tumor as well as by directly stimulating tumor cell proliferation, migration, and invasion . This maBoth UVR exposure and Slug overexpression induce alterations in the production of proinflammatory and immunomodulatory mediators by normal human melanocytes. These melanocyte-derived cytokines and chemokines may have important effects on the inflammatory milieu, proliferation, and motility of melanocytes and the melanoma cells to which they give rise. Further study will be required to determine the extent to which Slug mediates the effect of UVR on the induction of inflammatory mediators in melanocytes. Additional studies of cytokine and chemokine induction in melanocytes are warranted, in view of the potential therapeutic targets that may be revealed.


We sought to determine if functional measures of cardiovascular reserve, in particular the anaerobic threshold , echocardiography and arterial applanation tonometry were performed.2AT) was the most significant predictor, independently  and in the multivariate logistic regression analysis . The area under the receiver-operating-characteristic curve was 0.93, based on a risk prediction model that incorporated VO2AT, body mass index and desensitization status. Neither echocardiographic nor measures of aortic compliance were significantly associated with CCU admission.There were 70 participants . 14 patients (20%) required escalation of care from the ward to CCU following transplantation. Reduced anaerobic threshold (VO2AT has the potential to predict perioperative morbidity in kidney transplant recipients.To our knowledge, this is the first prospective observational study to demonstrate the usefulness of CPET as a preoperative risk stratification tool for patients undergoing kidney transplantation. The study suggests that VO A successful kidney transplant reduces the cardiovascular mortality risk when compared with maintenance dialysis therapy and relies on survival beyond the perioperative period The main novelty of cardiopulmonary exercise testing (CPET) lies in its ability objectively to measure cardiovascular functional reserve, in contrast to other tools frequently used to assess cardiac function such as echocardiography or cardiac magnetic resonance (CMR) which focus mainly on left ventricular (LV) morphology 2AT) provide a reliable predictor of perioperative complications and mortality following major intra-abdominal surgery Several studies have demonstrated that non-invasive CPET derived measures of cardiovascular reserve, especially the anaerobic threshold (VOThe study protocol was approved by the Black Country Research Ethics Committee (REC reference number: 09/H1202/113).All patients aged 18 or over undergoing kidney transplantation at the University Hospitals Coventry and Warwickshire NHS Trust (United Kingdom) were invited to participate within 4 weeks prior to the surgery between April 2010 and June 2012. These patients had previously undergone standard kidney transplant work-up according to our center’s protocol that included clinical evaluation, chest X-ray, electrocardiogram and echocardiography. Each prospective transplant recipient was evaluated by a transplant physician and a transplant surgeon. Thallium myocardial perfusion imaging, exercise tolerance test, coronary angiography, pulmonary function test and imaging of the iliac vessels were performed selectively as determined by the clinical assessment during the work-up period. All transplant recipients were reviewed by the anesthetist 6–36 hours prior to surgery.For the purpose of this study, all recruited patients underwent the additional CPET, arterial applanation tonometry and a study-specified echocardiography within 4 weeks prior to transplantation. CPET data were not used to determine the inclusion or exclusion of prospective kidney transplant recipients. All assessments at study entry were carried out on the first non-dialysis day for living donor kidney transplant recipients who were hemodialysis dependent and prior to first antibody removal therapy with double filtration plasmapheresis (desensitization) for those who had donor kidney directed antibodies The main outcome of interest was unplanned admission to CCU due to post-operative requirement for invasive or intensive central hemodynamic monitoring allowing for accurate infusion of volume expanders or circulatory support with vasoactive drugs. Indications for this were persisting hemodynamic instability alone or hemodynamic instability in patient with sepsis. Hemodynamic instability was defined as persistently low systolic arterial blood pressure (≤85 mmHg) despite adequate blood volume expansion targeted to a central venous pressure of 10–12 mmHg and as determined by clinicians in the ward. In addition, an observation based modified early warning scoring system (MEWS) (1) A previous coronary artery disease event , (2) cerebrovascular disease (CVD: transient ischemic attack or stroke), (3) diabetes , (4) dyslipidemia (use of lipid-lowering therapy), (5) hypertension (use of antihypertensives), and (6) smoking status (ever or never). Serial 12-lead electrocardiograms were performed at study entry, during hospital admission for surgery  and at clinic visit, 60 days following transplantation. All kidney transplant recipients who were admitted to CCU were identified. Length of stay in the CCU and hospital were recorded.Baseline clinical and demographic data were recorded including weight, height, body mass index (BMI), duration of CKD and dialysis vintage. Assessment of traditional cardiovascular risk factors included a history of any of the following: 1) and full vital capacity (FVC) of the lungs were measured. Exercise testing to maximal exhaustion using cycle ergometer ramping protocol, incorporating an individualized work rate was performed. Minute ventilation (VE), oxygen consumption (VO2), carbon dioxide production (VCO2) and other associated parameters were acquired via breath-by-breath analysis . The metabolic cart was recalibrated for consecutive patients. A 12-lead electrocardiogram was recorded continuously throughout testing and during recovery. Test was terminated at maximal exhaustion, accompanied by attainment of a respiratory exchange ratio  of a value ≥1.15. VO2peak was measured as the highest VO2 achieved during the final 30 sec of peak exercise. VO2AT was determined by the V-slope method in conjunction with analyses of the ventilatory equivalents (VE/VO2 and VE/VCO2) and end-tidal gas tensions (PETO2 and PETCO2) plots Prior to exercise testing, forced expiratory volume in 1 sec  was used to evaluate arterial compliance. Aortic pulse wave-form, augmentation pressure, and augmentation index were derived by tonometric applanation of the radial artery. Using similar technique, carotid-femoral pulse wave velocity (PWV) was determined from the time taken for the arterial pulse to propagate from the carotid to the femoral artery. A minimum of 3 readings were obtained for all parameters.2AT ‘cut-off’ values to predict CCU admission, ordinary and cross validated receiver operating characteristic (ROC) curves and area under curve (AUC) estimates were calculated. The optimal cut-off is estimated based on the Youden index criterion Results are expressed as mean, median or frequencies with corresponding confidence interval (CI) estimates depending on the distribution and type of the variable. Logistic regression was utilized to calculate odds ratios (ORs) with 95% CI limits for risk factors of CCU admission. Multiple (binary) logistic regression models were fitted to identify a prediction model for CCU admission. For this, risk factors which were significant in the univariate modeling (plus known confounders) were considered for the model allowing multiple predictor variables. Three different effect selection methods  were used for the multiple logistic regression modeling to evaluate the consistency of results. A p-value <0.05 was required for effect inclusion in the multivariate modeling and an effect elimination criterion of p<0.10 was chosen. To evaluate the accuracy of different VOEighty-one patients were screened for inclusion into the study and seventy individuals were included after the exclusions of eleven . Analysis of unplanned CCU admitted patients included the fourteen patients who fulfilled the study-defined indications for CCU admission. One patient who was admitted to CCU electively from theatre for a non-study indication (telemetry monitoring due to prior history of ischemic cardiomyopathy) was grouped as ‘non-CCU’.Baseline characteristics of seventy CKD patients are shown in Admission to CCU within 24 hours following transplantation was documented in twelve patients whilst two patients had escalation of care to CCU more than a week following transplantation (day 10 and day 12 respectively). Hemodynamic instability  accounted for all CCU admissions including two patients who also had sepsis (Gram negative bacteremia). 71.4% of CCU admitted patients received intravenous vasopressors whilst four patients had intra-arterial blood pressure and central venous pressure measurements to guide volume expansion without the eventual need for vasoactive drugs. There were no ischemic or arrhythmic ECG changes during the peri- and post-operative period; and at 60 days follow-up in all patients. The average duration of stay for those admitted to CCU was 3 days and this group had a longer hospital stay following transplantation  versus those that were not admitted to CCU . No major cardiovascular events  were recorded at 60 days post transplantation.). Univariate logistic regression modeling did not yield a significant association between preoperative LVEF, LV mass, LV filling pressure (diastolic compliance) or LA volume with the risk of CCU admission , oxygen pulse  and maximum work rate  (2AT was significantly lower in the CCU admitted compared to non CCU patients (p<0.001)  Table 3.2AT and CCU admission. Therefore, and because of its high reproducibility 2max, oxygen pulse and maximal work rate . 2AT, BMI and desensitization status; model-building strategies forward selection, backwards elimination and stepwise variable selection resulted in the same model. The Hosmer-Lemeshow test is not significant, indicating that there is no evidence for lack of fit in the final multiple logistic regression model . The area under the ROC curve is 0.93, indicating a very high probability of correctly classifying a randomly chosen pair consisting of a patient who requires CCU admission and a patient who does not +(−0.334×BMI)+(−2.342× Desensitization status)]. The measured values are entered for the risk variables VO2AT and BMI, whilst desensitization status is coded as either 1 (yes) or 0 (no). The predicted risk (probability) of CCU admission following kidney transplantation is calculated as odds/1+ odds.The odds of admission to CCU following kidney transplantation is calculated as 2AT univariately as a predictor yields an optimal cut-off of VO2AT = 11.31 ml/min/kg for distinguishing between the CCU admitted and non-admitted populations (Youden index J = 0.68). This translates into a sensitivity of 92.9% and a specificity of 75.0% at this cut-off point.Considering the variable VO2AT, measured preoperatively and CCU admission following kidney transplantation. In the preoperative assessment setting of major intra-abdominal surgery , three physiological CPET variables have been shown effectively to identify high risk patients: VO2AT 2peak 2) 2peak and VO2AT, with the additional measures of oxygen pulse and maximal work rate as markers of high perioperative risks  carries the strongest risk predicting strength independently  and remains significant after adjusting for age and gender  who underwent surgery had an VO2AT of less than 11 ml/min/kg in this study compared with previous studies, such as 29% in the one by Older and colleagues 2AT in other major intra-abdominal studies may not be transferable to kidney transplantation. The study also shows no associations between the static measures of cardiac function (echocardiography) or aortic compliance and perioperative risk among CKD patients. Additionally, there were no events of cardiac ischemia during the perioperative period to 60 days following surgery. The relevance of these is in keeping with the findings of a recent preoperative (kidney transplant) cardiac assessment study that demonstrated not only the low incidence of cardiac ischemia perioperatively among high cardiac risk cohort but also the lack of prognostic abilities of tools such as myocardial scintigraphy, dobutamine stress echocardiography or coronary angiogram (with subsequent revascularization if positive stress test) in reducing perioperative cardiac events In contrast with other studies In antibody incompatible transplantations, patients receive more cumulative immunosuppression treatment, including plasmapharesis and anti-thymocyte globulin ). This could be due to the fact that PWV provides information on the stiffness of the local vessels being studied rather than the systemic arterial stiffness PWV was found to be insignificant in predicting perioperative morbidity in this study Table 42 are suspended from transplantation. However, it is important to note that this study has shown that lower BMI is associated with significant perioperative morbidity after adjusting for important covariates as shown in 2peak in hemodialysis patients Many nephrologists and transplant surgeons have concerns about the higher rates of postoperative complications in overweight patients with their possible effects on survival outcomes and hence patients with BMI greater than 35 kg/m2AT is demonstrated in the risk-prediction model that included both the BMI and desensitization status  with a subsequent reduction in the perioperative risk associated with kidney transplantation or if the measure of VO2AT could improve the transplant work-up process and increase the inclusion of patients into the transplant programme, who are otherwise excluded based on current clinical criterions.Even though the design of this study was prospective, the limitation was the relatively small sample size. The study was also not powered for mortality. However, there were no deaths despite the lowest measured VO2AT, have the potential to predict perioperative morbidity in kidney transplant recipients. A further study of larger sample is required to validate the findings.This is the first prospective study to demonstrate the usefulness of CPET as a preoperative risk stratification tool for patients undergoing kidney transplantation. The study suggests that objective measures of functional cardiovascular reserve, in particular the VO


Obesity and obesity co-morbidities are associated with a low grade inflammation and elevated serum levels of acute phase proteins, including serum amyloid A (SAA). In the non-acute phase in humans, adipocytes are major producers of SAA but the function of adipocyte-derived SAA is unknown. To clarify the role of adipocyte-derived SAA, a transgenic mouse model expressing human SAA1 (hSAA) in adipocytes was established. hSAA expression was analysed using real-time PCR analysis. Male animals were challenged with a high fat (HF) diet. Plasma samples were subjected to fast protein liquid chromatography (FPLC) separation. hSAA, cholesterol and triglyceride content were measured in plasma and in FPLC fractions. Real-time PCR analysis confirmed an adipose tissue-specific hSAA gene expression. Moreover, the hSAA gene expression was not influenced by HF diet. However, hSAA plasma levels in HF fed animals  were increased compared to those in normal chow fed animals , and plasma levels in the two groups were in the same ranges as in obese and lean human subjects, respectively. In FPLC separated plasma samples, the concentration of hSAA peaked in high-density lipoprotein (HDL) containing fractions. In addition, cholesterol distribution over the different lipoprotein subfractions as assessed by FPLC analysis was similar within the two experimental groups. The established transgenic mouse model demonstrates that adipose tissue produced hSAA enters the circulation, resulting in elevated plasma levels of hSAA. This new model will enable further studies of metabolic effects of adipose tissue-derived SAA. Obesity, and especially central obesity, is a major risk factor for cardiovascular disease in vitro has been shown to correlate with SAA gene expression In the acute phase response, SAA is produced by the liver and its serum levels can rise thousand-fold, but the function of SAA is poorly understood. Extrahepatic production of SAA has been found in vitroIn vitro, a higher amount of SAA on the HDL particles increases the binding of HDL to proteoglycans Previous studies in humans have shown that SAA may have various effects including promoting proinflammatory cytokine production The SAA protein is transcribed from the two homologous genes SAA1/SAA2 in humans, and from Saa1/Saa2 in mice. However, there are also species differences in the SAA gene family. In mice, Saa3 is extrahepatically expressed, while SAA3 in humans is a pseudo gene. Hence, in mice, the Saa1/Saa2 and Saa3 genes are expressed in adipose tissue whereas in humans only the SAA1/SAA2 genes are transcribed. It has also been shown that, only the expression of Saa3 is increased after HF diet in mice The mature protein encoded by SAA1/SAA2 is highly conserved between mouse and human All manipulations involving mice were performed using protocols approved by the local Ethics Committee for Animal Studies at the Administrative Court of Appeals in Gothenburg, Sweden, approval numbers 201-2006 and 281-2008.5′-CCAGGGAGAACCAAAGTTGA and 5′-CCCGAGCATGGAAGTATTTG, respectively. The endogenous β-globin gene was co-amplified as an internal control.Transgenic founder mice were generated on a C57BL/6J background. The cDNA from human SAA1 (hSAA) was cloned into the pCR 2.1 vector  and verified by sequencing. A SmaI-EcoRV digested fragment containing hSAA1 was ligated into Sma1 linearized pBluescript SK II+ vector  downstream of the fatty acid binding protein 4 (aP2) promoter (kindly provided by Dr. Bruce Spiegelman) Subsequent breeding was done against C57BL/6J mice  to generate heterozygous SAA-transgenic mice. Male animals of F2 or later generations were used in experiments. Animals were weaned at 3 weeks of age and housed 3–6 per cage in a temperature-controlled (25°C) facility with a 12-h light-dark cycle with free access to chow and water. From the age of 13 weeks age-matched wild type and hSAA transgenic animals were fed either a normal chow (NC) or a pelleted high fat (HF) diet . At the end of the experiment mice were sacrificed after a 4-hour fast. Using heart puncture blood was collected in potassium EDTA tubes  from Isofluran  inhalation anesthetised animals. Brown adipose tissue (BAT), epididymal adipose tissue (eWAT), retroperitoneal adipose tissue (rWAT), gastrocnemius muscle, heart, liver, kidneys and spleen, were dissected, weighed, snap frozen in liquid nitrogen and stored at −80°C until RNA extraction.Total body weight was recorded at 11 weeks of age and weekly during the HF or corresponding NC feeding period. Analysis of total body fat was performed by dual energy X-ray absorptiometry (DEXA) using the Lunar PIXImus II Densitometer with version 2.10.041 software  after 17 weeks of HF or NC diet. During DEXA analysis, mice were anesthetized with Isofluran.RNA from the dissected tissues was prepared using the RNeasy lipid mini kit with TissueLyser homogenization  and the RNA was then reverse transcribed using the High Capacity cDNA RT kit . Real-time PCR analysis was performed using TaqMan Gene Expression Master Mix and cDNA corresponding to 10 ng of RNA per reaction with an ABI Prism 7900HT (Applied Biosystems). The following TaqMan gene expression assays were used; Hs00761940_s1 (human SAA), Mm00441203_m1 (mouse Saa3), and Mm00445878_m1 (mouse Fabp4). The Mm00725448_s1 assay was used for detection of the endogenous reference transcript ribosomal protein, large, P0 . A standard curve was analysed for each assay with a serial dilution of cDNA synthesized from pooled RNA. Samples and standards were analysed in triplicate.After 16 weeks of HF or NC diet, a 4-hour fast preceded blood sampling from the tail vein. Basal glucose and insulin were measured using Accu-Check glucometer  and Ultrasensitive mouse insulin enzyme-linked immunosorbent assay (ELISA) Kit , respectively. Plasma pools , from blood collected by heart puncture, were subjected to fast protein liquid chromatography (FPLC) gel filtration on a Superose 6 (10/300GL) column (GE Pharmacia AKTA explorer) Data is reported as mean ± standard deviation (SD) unless indicated otherwise. Statistical analysis was performed using SPSS . Differences between groups were assessed with the Mann-Whitney U-test. Differences in growth curves were assessed with repeated measures analysis of variance (ANOVA). Nonparametric correlations were performed using Spearman rank correlation test. A p-value less than 0.05 was regarded statistically significant.A construct containing the mouse aP2 promoter/enhancer region was used to generate a mouse model with adipose tissue specific expression of hSAA . Three tAt 11 weeks of age, the body weights of hSAA male mice  were slightly lower compared to wild type male mice . Groups of animals were challenged with a HF diet resembling a western diet. Even though there was an initial difference in body weight between hSAA and wild type animals, there was no difference in weight gain between hSAA and wild type mice fed NC or when challenged with HF diet .Total body fat content was measured with DEXA after 17 weeks of either NC or HF diet. Total body fat was similar in hSAA and wild type male mice on the same diet (data not shown). In line with this finding, there were no major discrepancies in adipose tissue depot sizes . At the Transgenic hSAA animals fed HF diet had reduced absolute weight of kidneys and heart . However, when relating to body weight, these significances were lost. The weight of kidneys and heart from animals fed normal chow was similar between wild type and hSAA animals. Furthermore, hSAA animals on HF, but not on NC, had significantly reduced spleen weight compared to wild type mice both in absolute mass and in proportion to body weight (p = 0.001 for both tests). The weights of gastrocnemius muscle, liver or brain were similar in wild type and hSAA animals fed NC or a HF diet (data not shown).An ELISA specific for human SAA was used to analyse hSAA concentrations in plasma from hSAA transgenic animals. We found that the hSAA protein was present in plasma from both NC (n = 7) and HF (n = 10) fed hSAA animals, while hSAA was undetectable in plasma from wild type animals (n = 8). Furthermore, significantly increased levels of hSAA were found in plasma from HF fed compared to NC fed hSAA animals . When the results were analysed separately for animals given NC and HF diets, the correlation remained significant  in animals challenged with HF diet only. Plasma mSAA levels correlated with body weight (r = 0.79 p = 1.8*10−7), and total amount of body fat  were subjected to FPLC gel filtration. In response to the 18 week long HF diet, lipoprotein cholesterol content in FPLC fractions increased in a similar way in both wild type and hSAA animals compared to NC fed animals . TriglycWe here report the establishment and initial characterization of a mouse model with transgenic expression of human acute phase SAA in adipocytes under the control of the aP2 enhancer/promoter. In hSAA transgenic animals, the hSAA protein was present in plasma, demonstrating that adipose tissue-derived SAA is released into the circulation in this model. Furthermore, plasma levels of hSAA in the transgenic mice were similar to those observed in humans and the levels were associated with the amount of total adipose tissue, resembling the association between body fat and serum SAA levels in human subjects. We found no indication of an increased expression of mouse SAA genes in the hSAA transgenic mice. On the contrary hSAA mice subjected to HF diet displayed reduced expression of the mouse Saa3 gene in adipose tissue. Furthermore, plasma levels of mSAA were similar in hSAA and wild type mice and the levels were highly correlated with body fat content as is the case in man The hSAA transgenic mice displayed no visible phenotype. Although the hSAA mice had slightly lower body weight compared to wild type mice at 11 weeks of age, growth during the 18 subsequent weeks on NC or HF diet did not differ between transgenic and wild type animals. Therefore, we can not rule out that the initially observed difference in weight between wild type and hSAA mice was a spurious finding. hSAA mice challenged with HF diet had reduced spleen sizes, which may indicate an effect on the immune system. Total body fat mass was similar between hSAA and wild type mice, but the epididymal depot mass was decreased after HF diet in wild type animals compared to NC fed mice, and compared to hSAA transgenic mice. This decrease resulted in a significant difference between wild type and transgenic mice. However, if these findings are related to the hSAA over-expression requires further analysis.The aP2 promoter/enhancer has previously been used in several projects to induce adipocyte specific over-expression Previous studies have shown that SAA serum levels are elevated in obese human subjects and reduced in response to weight loss Importantly, in the established model, plasma hSAA levels in transgenic mice fed HF and NC were in the same ranges as in obese and lean human subjects in vivoet al. demonstrated that SAA promotes vascular proteoglycan synthesis in vitro and most importantly, that a short-term adenoviral-induced hSAA production was sufficient to obtain pro-atherogenic proteoglycan synthesis in vivoSAA may affect the development of cardiovascular disease through many different mechanisms. For instance, SAA is involved in cholesterol transport, including cholesterol efflux (reviewed in Elevated CRP concentration in the blood is a well recognized risk factor for future cardiovascular disease, although some studies argue against a causative role for CRP In conclusion, we have established a transgenic mouse model with adipose tissue-specific expression of hSAA under the control of the aP2 promoter/enhancer. The increased adipose tissue-derived hSAA plasma levels in HF fed hSAA mice, correlated with amount of adipose tissue and, in plasma, hSAA concentration peaked in HDL containing fractions. Thus, the established model is similar to the situation found in human obesity and will provide a novel tool for studies of the direct metabolic and vascular effects of adipose tissue-derived hSAA.


Haemaphysalis longicornis has an intracellular (HlFER1) and a secretory (HlFER2) ferritin, and both are crucial in successful blood feeding and reproduction. Ferritin gene silencing by RNA interference caused reduced feeding capacity, low body weight and high mortality after blood meal, decreased fecundity and morphological abnormalities in the midgut cells. Similar findings were also previously reported after silencing of ferritin genes in another hard tick, Ixodes ricinus. Here we demonstrated the role of ferritin in protecting the hard ticks from oxidative stress. Evaluation of oxidative stress in Hlfer-silenced ticks was performed after blood feeding or injection of ferric ammonium citrate (FAC) through detection of the lipid peroxidation product, malondialdehyde (MDA) and protein oxidation product, protein carbonyl. FAC injection in Hlfer-silenced ticks resulted in high mortality. Higher levels of MDA and protein carbonyl were detected in Hlfer-silenced ticks compared to Luciferase-injected (control) ticks both after blood feeding and FAC injection. Ferric iron accumulation demonstrated by increased staining on native HlFER was observed from 72 h after iron injection in both the whole tick and the midgut. Furthermore, weak iron staining was observed after Hlfer knockdown. Taken together, these results show that tick ferritins are crucial antioxidant molecules that protect the hard tick from iron-mediated oxidative stress during blood feeding.Ticks are obligate hematophagous parasites that have successfully developed counteractive means against their hosts' immune and hemostatic mechanisms, but their ability to cope with potentially toxic molecules in the blood remains unclear. Iron is important in various physiological processes but can be toxic to living cells when in excess. We previously reported that the hard tick  Fe2+ is potentially toxic due to its ability to catalyze the formation of reactive oxygen species (ROS) through Fenton reaction Iron is an essential element required for various physiological processes in most living organisms. Iron metabolism involves a continuous redox cycling between the ferrous -mediated silencing of H. longicornis ticks were used throughout this study. Ticks have been maintained by feeding on the ears of Japanese white rabbits  for several generations at the Laboratory of Emerging Infectious Diseases, Joint Faculty of Veterinary Medicine, Kagoshima University, Kagoshima, Japan Parthenogenetic (Okayama strain) adult female Hlfer in unfed adult female ticks was induced by injection of double-stranded RNA (dsRNA) prepared as previously described Hlfer1 or Hlfer2 dsRNA through the fourth coxae using an IM 300 Microinjector . Control ticks were injected with the same amount of firefly Luciferase (Luc) dsRNA. To confirm silencing, total RNA was extracted from whole ticks 4 days post-injection of dsRNA for RT-PCR analysis. Ticks injected with dsRNA were held in a humidity chamber kept in a 25°C incubator for 18 h before infestation to rabbits or for 4 days before injection with ferric ammonium citrate (FAC).The silencing of Hlfer1and Hlfer2-silenced ticks and two ticks each for Luc-injected group, were also prepared for the TBARS assay. The remaining ticks were used for immunoblot detection of oxidative stress biomarkers, described in the succeeding sections. All ticks were stored in −80°C until use.For rabbit infestation, a total of 50 ticks per dsRNA injected group were attached in separate ears of rabbits, individually covered with an ear bag. Attached ticks were allowed to feed until they naturally dropped off. From the total number of engorged ticks, 30 ticks from each group were used for the thiobarbituric acid reactive species (TBARS) assay in the whole ticks. Five pooled midgut samples, comprising of three ticks each for Hlfer1 or Hlfer2 had a negative effect on tick survival after blood feeding Hlfer-silenced ticks after exposure to iron, 100 µM FAC was injected in the same manner as dsRNA injection. Likewise, sterilized high-purity water was injected to dsRNA-injected ticks for additional control. Thirty ticks for each group were used for this experiment. After injection of FAC, ticks were held as mentioned above and monitored for mortality every 12 h for 11 days. The survival experiment was repeated three times to confirm the reproducibility of results. Otherwise, unfed adult ticks not injected with dsRNA were injected with 50 or 100 µM FAC or sterilized high-purity water for control to evaluate mRNA and protein expression and iron staining in response to iron treatment.We previously found that the silencing of either Blood-fed or FAC-injected whole ticks were homogenized in phosphate-buffered saline (PBS). Midguts and salivary glands were also collected and homogenized in Tris-buffered saline (TBS) with a protease inhibitor . Hemolymph was collected from the amputated legs of immobilized ticks. Hemocytes were separated by centrifugation. Protein from whole ticks and organs was extracted as previously described To investigate the protein expression, protein samples were separated in 12% SDS-polyacrylamide gel electrophoresis (PAGE) and subjected to Western blot analysis as described previously 4[Fe(CN)6] and 10% HCl) at room temperature for 48 h as previously reported To stain native HlFER for ferric iron, protein extracts were separated in 6% native PAGE. Protein concentration was adjusted after determination of protein concentration using a Micro BCA Assay kit  or based on control immunoblotting with β-tubulin as described above An indirect immunofluorescent antibody test (IFAT) was performed as previously described Oxidative stress was evaluated by detecting oxidative stress biomarkers including malondialdehyde (MDA) and protein carbonyl (PC). MDA was demonstrated through immunoblotting using the Oxiselect Malondialdehyde Immunoblot Kit  following the manufacturer's recommendation. Engorged whole ticks were homogenized individually, whereas midguts and unfed adult ticks injected FAC were pooled. Protein was adjusted based on tubulin profile. Bands were viewed using Clarity Western ECL Substrate  and the MDA level relative to tubulin was calculated after band densitometry analysis. TBARS assay was also performed to quantify MDA Hlfer knockdown −1 cm−1 and based on protein concentration.The ferrozine assay for measuring non-heme iron was adapted to determine the amount of ferrous iron in whole ticks injected with FAC after t-test or the Mann-Whitney U test was performed, depending on data distribution. For the survival experiment, the Mantel-Cox log-rank test was performed using GraphPad Prism software. In all statistical analyses, significant difference between groups is defined by P<0.05.For band densitometry analysis, Student's Hlfer-silenced ticks after blood feeding Hlfer-silenced adult female ticks to iron by injecting 100 µM FAC into the hemocoel. Silencing was confirmed by RT-PCR analysis (data not shown). After FAC injection, ticks were kept at 25°C and survival was monitored every 12 h. No mortality was observed in the control group injected with Luc dsRNA . Eleven days after FAC injection, the Hlfer2 dsRNA-injected group showed the lowest survival rate. As an additional negative control, high-purity sterilized water was similarly injected after RNAi but this did not result in high mortality as in the case of FAC injection , a known product of lipid peroxidation Iron is known to catalyze the formation of ROS in living cells, thus promoting oxidative stress. We previously found that Hlfer-silenced ticks have significantly higher (P<0.05) levels of these oxidative stress biomarkers than the control group after blood feeding or FAC injection. Band densitometry analysis was performed to calculate the relative MDA or PC content of the samples based on tubulin. Hlfer1-silenced ticks showed the highest levels of MDA and PC, including engorged whole ticks and midguts and unfed ticks injected with FAC.Immunodetection using specific antibodies against MDA  and PC  showed tHlfer-silenced ticks after blood feeding was further evaluated using the TBARS assay, the most common technique employed in studying lipid peroxidation and oxidative damage Hlfer-silenced groups as compared to the Luc-injected control group, either in whole ticks or in midguts  ferrous iron level 72 h after FAC injection than the control group  or with injection of water in unfed adult female ticks did not result in any mortality; however, mortality increased with each day after FAC injection. The group injected with Hlfer2 dsRNA showed a more rapid increase in mortality and a lower survival rate at the end of the observation period compared to both Hlfer1-silenced and Luc-injected control groups. The injection of FAC introduced high levels of free iron in the hemocoel. Only the secretory HlFER2 is present in the tick's hemolymph. After the knockdown of Hlfer2, excessive ferrous iron, as we have demonstrated through the ferrozine assay, could have caused oxidative damage in the ticks that eventually lead to mortality. Conversely, the absence of HlFER1 after its knockdown could have led to high levels of intracellular ferrous iron.Aside from the effects of Hlfer-silenced ticks is related to ferritin function, we performed additional experiments after injecting FAC into normal unfed adult ticks, including RT-PCR, Western blotting and IFAT. In contrast to reports of ferritin up-regulation on mosquitoes following artificial feeding and in vitro exposure of cells to iron Macrobrachium rosenbergiiBombus ignitusHlfer did not change in response to iron injection. Meanwhile, Western blot analyses showed an increasing protein level in whole ticks, particularly of HlFER1, in a time-dependent manner after FAC injection. In agreement with our previous conclusion, these results demonstrated the translational regulation of HlFER1 through the binding of the iron-responsive element (IRE) to the iron-regulatory protein (IRP) To confirm that the mortality after FAC injection in Drosophila melanogasterAnopheles gambiaeInterestingly, Western blot analysis of different organs showed that FAC injection stimulated the expression of both HlFER1 and HlFER2 in the midgut but not in the salivary glands or ovary. IFAT also showed the extensive fluorescence of digestive cells for HlFER1 after FAC injection, extending from the basal lamina to the cells close to the lumen, whereas HlFER2 was strong particularly along the basal lamina in the midgut. In contrast, no fluorescence was found in the salivary glands. These results suggest that the iron in the hemolymph may cross the basal lamina of the midgut for storage in HlFER1 of digestive cells. In mammals, circulating iron bound to transferrin can enter the basolateral membrane of enterocytes through transferrin receptor 1 Hlfer1-silencing but not after the silencing of Hlfer2. We previously found that HlFER1 was still expressed after Hlfer2 silencing, particularly in the midgut Hlfer2-silenced ticks implicates HlFER1.The Prussian blue staining for ferric iron in native HlFER after native PAGE was useful in the assessment of ferric iron accumulation. We found increased staining after FAC injection, which may reflect the increased HlFER level and iron uptake of HlFER molecules at these time points. The increased ferric iron accumulation, together with the increased levels of both HlFERs in the midgut we mentioned earlier, supports our previous conclusion that the midgut is the primary organ for iron metabolism, most likely being the first organ exposed to large amounts of iron during blood feeding. Interestingly, ferric iron staining was weakened after Hlfer resulted in oxidative stress in ticks exposed to high levels of iron, either from blood meal or FAC injection. Similar to our previous study, Hlfer-silenced ticks infested on rabbits failed to engorge, weighing less than half of the Luc-injected ticks' engorged body weight, meaning they ingested a lower amount of blood. Oxidative stress was confirmed by the detection of malondialdehyde and protein carbonyl, which are products of lipid peroxidation and protein oxidation, respectively, and observation of higher levels in Hlfer1- and Hlfer2-silenced ticks than in Luc-injected ticks after blood feeding or injection of FAC. The TBARS assay was also employed to assess lipid peroxidation after blood feeding and similarly, it showed that Hlfer-silenced ticks had a higher degree of lipid peroxidation compared to the control. We also attempted to perform TBARS assay on unfed Hlfer-silenced ticks injected with FAC but due to the low sensitivity of this test Iron is known to promote the formation of reactive oxygen species that can result in damage to macromolecules, including DNA, proteins and lipids—the condition collectively termed oxidative stress Hlfer-silenced ticks during blood feeding, including altered shape, disrupted microvilli and cell membrane and vacuolated cytoplasm Hlfer-silenced ticks had high levels of MDA and protein carbonyl. Lipid peroxidation leads to alterations of biological membranes and gives rise to several products that are known to induce diverse biological effects Hlfer1-silenced ticks had the highest level of either oxidative stress biomarker, which corresponds to our previous observation of more severe abnormalities in the digestive cells of Hlfer1-silenced ticks. Moreover, we also previously reported a decrease in hematin production after Hlfer1 silencing, indicative of impaired digestive activity. Oxidative stress can also alter physiological processes including heme detoxification in the midgut We previously found abnormalities in the digestive cell morphology in Dermacentor variabilis, these antioxidant enzymes were found in the midgut at day 6 of blood feeding, corresponding to the rapid feeding stage Several antioxidant enzymes that counteract reactive oxygen species, such as superoxide dismutase, glutathione S-transferase and thioredoxin, have been identified in hard ticks H. longicornis resulted to increased levels of oxidative stress biomarkers after a blood meal or injection of iron. Our results provide evidence for the first time that two kinds of ferritin act as antioxidant molecules in a hard tick that prevent oxidative stress during blood feeding, thus ensuring tick survival. This paper provides a clearer explanation on the crucial importance of ferritin in the ticks that we reported in our previous paper on H. longicornisI. ricinusIn summary, the silencing of two ferritin genes in the hard tick Figure S1Survival rate of Hlfer-silenced ticks after injection of sterilized high-purity water. Four days after injection of Hlfer1, Hlfer2, or Luciferase dsRNA, sterilized high-purity was injected, and mortality was monitored. Low mortality was observed from all the three groups. n = 25 ticks per group. Bars represent standard error.(TIF)Click here for additional data file.Figure S2Transcription profile of H. longicornis adult ticks injected with FAC. Unfed adult ticks were injected with 50 µM or 100 µM FAC. Sterilized high-purity water was injected into the control group (0 µM). Total RNA was extracted from whole ticks at 24 h and 72 h after injection and RT-PCR analysis was performed using specific primers for Hlfer1 and Hlfer2. cDNA was adjusted based on control amplification for Hlactin. No significant difference was observed among groups.(TIF)Click here for additional data file.Figure S3Protein expression of H. longicornis ferritins in salivary glands of unfed ticks injected with different concentrations of FAC. Salivary glands were collected from ticks at 24 h and 72 h after injection of 50 µM or 100 µM FAC. Sterilized high-purity water was injected into the control group (0 µM). Western blot analysis was performed using specific primary antibodies against H. longicornis FER1 (HlFER1) or H. longicornis FER2 (HlFER2). Tubulin was used as an internal control. No significant difference was observed among groups.(TIF)Click here for additional data file.Figure S4Coomassie blue staining and Western blot analysis after native PAGE. To further confirm that the bands stained for ferric iron from tick protein samples were HlFER, we performed Coomassie blue staining and Western blot analysis using specific anti-HlFER sera. (A) Coomassie blue staining showed all the bands of high molecular weight marker (M), the commercially prepared horse holoferritin (HF) and the tick protein (T). The weak band of approximately 440 kDa in the tick protein sample was presumed to be ferritin. Western blot analyses for HlFER1 (B) and HlFER2 (C) showed a single band of approximately 440 kDa. Arrow indicates the 440 kDa band in the high molecular marker while arrowheads point to tick ferritin.(TIF)Click here for additional data file.Figure S5An IFAT examination of salivary glands 72 h after injection of different concentrations of FAC compared to control group injected with sterilized high-purity water. Frozen sections of the salivary glands were incubated with specific mouse anti-HlFER1 or anti-HlFER2 sera. Normal mouse serum was used as negative control. Anti-mouse IgG conjugated with Alexa 594 was used as secondary antibody and nuclei were visualized using DAPI. No fluorescence was observed among groups. (Bars  = 20 µm).(TIF)Click here for additional data file.


Contemporary human bones and teeth, however, are relevant in forensic DNA analyses that deal with the identification of perpetrators, missing persons, disaster victims or family relationships. They may also provide useful information towards unravelling controversies that surround famous historical individuals. Retrieving information about a deceased person’s externally visible characteristics can be informative in both types of DNA analyses. Recently, we demonstrated that human eye and hair colour can be reliably predicted from DNA using the HIrisPlex system. Here we test the feasibility of the novel HIrisPlex system at establishing eye and hair colour of deceased individuals from skeletal remains of various Twenty-one teeth between 1 and approximately 800 years of age and 5 contemporary bones were subjected to DNA extraction using standard organic protocol followed by analysis using the HIrisPlex system.Twenty-three out of 26 bone DNA extracts yielded the full 24 SNP HIrisPlex profile, therefore successfully allowing model-based eye and hair colour prediction. HIrisPlex analysis of a tooth from the Polish general Władysław Sikorski (1881 to 1943) revealed blue eye colour and blond hair colour, which was positively verified from reliable documentation. The partial profiles collected in the remaining three cases (two contemporary samples and a 14th century sample) were sufficient for eye colour prediction.Overall, we demonstrate that the HIrisPlex system is suitable, sufficiently sensitive and robust to successfully predict eye and hair colour from ancient and contemporary skeletal remains. Our findings, therefore, highlight the HIrisPlex system as a promising tool in future routine forensic casework involving skeletal remains, including ancient DNA studies, for the prediction of eye and hair colour of deceased individuals. Skeletal remains represent a unique type of biological material. Due to their unique features, bones and teeth can be resistant to degradation and, depending on the environmental storage conditions, can provide a good source of DNA suitable for genetic analysis thousands of years after an organism’s death. DNA analysis of ancient bones and teeth is used to explore various aspects of a species’ history, including archaic and modern humans. For instance, the application of next generation DNA sequencing (NGS) technologies to ancient human bones has allowed the establishment of whole genome information on Neanderthals  and DeniContemporary bones and teeth are frequently used in dedicated forensic laboratories dealing with missing person identification cases, including disaster victim identification. Although DNA extraction from skeletal remains is very complex and time consuming with limited possibilities for automation, in forensic science teeth and bones constitute the most suitable sources of DNA for individual identification from heavily decomposed bodies. Forensic DNA analysis of contemporary bones and teeth typically concerns autosomal short tandem repeats  for direct match identification with antemortem samples of the same individual, or via STR profile similarities with close putative relatives ,6. MitocOne interesting aspect of ancient and modern skeletal analysis, which has been largely overlooked thus far, is the reconstruction of the deceased person’s appearance. In ancient DNA analysis the prediction of particular externally visible characteristics (EVCs) not reflected in skeletal features, such as eye and hair colour, is the only way to get insights into how extinct species may have looked. In contemporary skeletal analysis, such as forensic cases, EVC prediction from DNA can be useful in providing leads in police investigation to reveal the identity of a deceased person if other and more direct means  are non-informative. In recent years, researchers have started seeing increased success in DNA-based EVC prediction, a field sometimes referred to as Forensic DNA Phenotyping (FDP) or DNA intelligence ,16. FromMC1R[MC1R causes light skin colour and red hair colour in modern humans [Previous ancient DNA studies have suggested that Neanderthals may have had light skin and reddish hair colour due to a particular non-synonymous variation in MC1R. Geneticn humans , albeit n humans -38. HowePreviously, we developed and forensically validated the IrisPlex system for reliable and accurate DNA prediction of blue and brown eye colour, which includes a sensitive single multiplex assay of six highly eye colour-predictive SNPs and an eye colour prediction model based on genotype and phenotype data of many thousands of individuals from various regions in Europe -26. An ith to 14th century. S25 and S26 were teeth picked up from skeletons found in the St. Andrew Church in Kraków and dated based on historical markers to come from the 14th century.The specimens subjected to DNA analysis were comprised of 21 teeth and 5 bones. Samples S1 to S3 were collected from contemporary human remains approximately one to two years after death as estimated from medico-legal and criminalistic examination. S4 was a tooth collected from modern remains of an incomplete skeleton that rested in soil; it was impossible to estimate an approximate age of the remains but anthropological examination showed they were seriously decayed. Serious degradation was also present in cases S5 to S10, which were contemporary human remains found in a cave. S5 was a fragment of a femur, S6 was a fragment of a tibia, S7 was a fragment of a humerus, S8 was a fragment of another humerus, S9 was a tooth collected from a mandible and S10 was a part of a skull; they were all found in one location in a cave situated in Dolina Będkowska Landscape Park near Kraków. S11 was a tooth collected from the exhumed remains of General Władysław Sikorski; the exact date of his death is known (1943) and thus, we know that the analyzed tooth was 69 years of age. S12 to S23 were samples collected from skulls which in 1942 were subjected to bleaching and afterwards kept in a museum in Austria until 1998. S24 was a tooth of a mysterious woman from a Benedictine crypt in Tyniec near Kraków, which according to historical evidence comes from the 12Multifarious efforts were undertaken in the laboratory in order to prevent contamination. Full protective clothing and separate working localities for extraction, amplification and SBE reaction setup were used. All DNA extractions were done in a separate room reserved for low template DNA samples. DNA extraction and PCR were performed in clean room facilities with Laminar Air Flow benches equipped with HEPA filters . All working areas and equipment were regularly UV-irradiated and cleaned with bleach. In total, 10 negative extraction controls were included. Each presented case was subjected to separate DNA extraction, except for samples S12 to S23 where three negative extraction controls were used. Negative controls were also included for each PCR amplification performed. All samples were first subjected to STR analysis which showed no traces of DNA admixture that could indicate contamination. Moreover, in order to exclude any possibility of internal contamination, STR ) and HIrisPlex profiles were determined for all 10 members of the laboratory staff. No match was found for any of the samples analysed in both STR and HIrisPlex profiles.DNA was isolated using standard organic extraction protocols. Before extraction, bone material comprising teeth or fragments of femurs was subjected to a purification and decontamination procedure. Samples were treated with 15% bleach, repeatedly shaken with 70% ethanol and distilled water, and while drying were subjected to UV irradiation. The entire tooth or bone fragments were then pulverized using FreezerMill 6750  and subjected to the extraction procedure. When available, 3 g (in case of tooth samples 1 to 1.5 g) of bone powder were incubated overnight in a water bath set at 56°C with 3 mL of buffer composed of 0.5 M EDTA  and 10% SDS  with the addition of 225 μL proteinase K (10 mg/mL)  and 120 μL 1 M DTT . The samples were then subjected to double extraction with a phenol-chloroform-isoamyl alcohol  and concentrated after extraction using Amicon Ultra 4 – 30k columns and Microcon 100  to the final volume of 60 to 70 μl in all cases. Negative extraction controls were used to check the purity of the used chemicals and consumables.Quantifiler™ Human DNA Quantification Kit , following the protocol recommended by the manufacturer.DNA concentration was assessed on a 7500 Real Time PCR system using MC1R gene, rs12913832 from HERC2, rs12203592 from IRF4, rs1042602 and rs1393350 from TYR, rs4959270 from EXOC2, rs28777 and rs16891982 from SLC45A2, rs683 from TYRP1, rs1800407 from OCA2, rs2402130 and 12896399 from SLC24A4, rs12821256 from KITLG and rs2378249 from ASIP. All samples were genotyped at least twice.Multiplex genotyping was performed as described in detail recently . The HIrEye colour inference from the genotyping results of the six SNPs (the same as used in IrisPlex) was obtained via model-based prediction using a database of thousands of Europeans and a convenient Microsoft Excel macro  published previously ,26. Hairth century) and 5 contemporary bones. All samples were subjected to multiple genotyping using the HIrisPlex assay and to model-based eye and hair colour prediction from the combined HIrisPlex genotypes using large genotype/phenotype databases described elsewhere [Using a standard organic extraction protocol, we purified genomic DNA from 21 teeth aged between 1 year and approximately 800 years  was obtained for brown eye colour  cases, remain unsolved because no STR profile match with the antemortem samples, or because no informative STR profile similarities with known putative relatives can be obtained. In all such cases, EVC information inferred from the scene sample, including skeletal remains, may be valuable for the continuing investigation. Representing typical forensic casework samples, we included three teeth collected from partially or completely skeletonized bodies of approximately one to two years post mortem age. S1 was a cadaver and S2 was a skeleton, both found in small villages in different regions of Poland in the open soil. S3 was a cadaver found inside a building located in Kędzierzyn-Koźle, a small city in southern Poland. The teeth were previously subjected to standard forensic identification using STR and mitochondrial DNA profiling. Complete STR profiles ) were obtained in each case indicating reasonably good preservation of nuclear DNA. The amelogenin gender test revealed that skeletons S1 and S3 were males and S2 a female, corroborating results of the anthropological investigation of the skeletons (data not shown). Complete HIrisPlex profiles were determined for all three samples  indicated merely 7 pg/μl of human DNA. As template DNA concentration in the HIrisPlex reaction equalling approximately 30 pg was below the genotyping accuracy threshold of approximately 60 pg as previously established [MC1R) HIrisPlex SNPs were obtained successfully. Blue eye colour was inferred with high probability (P = 0.919) and accuracy (P = 97.4), (Table MC1R genotype. Although this DNA variant (indel) is very rare in the general European population , its impact on red hair colour is very strong.Occasionally, human skeletal remains are discovered coincidentally by workers or sightseers. They may be of old or young post mortem age. Sample S4 came from the human remains found in the field near Sieradz in central Poland. Anthropological examination revealed that the bones belong to a woman of about 40 years of age at the time of death. The skeletal remains may have rested in the soil for a very long time as they appeared to be seriously decayed. Two teeth were taken from the maxilla for DNA extraction. A partial NGM STR profile was determined (seven markers were genotyped successfully) and the positive amelogenin result confirmed the female origin suggested from the anthropological investigation. In this case the level of DNA concentration was critical as ), Table . Blond hMC1R genotype was missing. The obtained data show that from all 24 HIrisPlex polymorphisms indel position 86insA in MC1R seems most affected by suboptimal DNA concentration and/or DNA degradation. The S7 profile was also inconsistent among three repeated analyses carried out in terms of rs4959270. From the HIrisPlex profile ascertained in sample S5 we could further conclude that drop-out (rs4959270C/A -> A) occurred in this position in sample S7 (observed twice among three repetitions) keeping with the hypothesis that indeed both samples derive from the same individual. Inferred eye and hair colours for S5 and S7 are blue and black/dark brown, respectively, with highly similar probabilities and accuracies agreeing with the idea that they were derived from the very same individual. Notably, strong PCR inhibition prevented determination of DNA concentration in samples S5 and S8 using Quantifiler™ Human DNA Quantification Kit . Anthropological examination indicated that the revealed 13 skeletal elements, including pelvis, femurs, humerus, ribs, vertebras, mandible comprising two teeth and an incomplete skull, may belong to 6 individuals. The same mtDNA and STR profiles were ascertained in the femur (S5) and the humerus (S7) indicating that these bones likely belong to a single female skeleton (data not shown). Overall genetic analysis showed that in fact all remains came from only five individuals, two females (S5/S7 and S9) and three males . However, only a partial STR profile was determined in S7 showing more serious degradation compared with S5. Both types of bone (femur and humerus) are usually considered a good source of DNA and thus the observed difference may be coincidental. Complete 24 SNP HIrisPlex profiles were generated for all samples except for S7 where again the ins86A P = 0.950) and high accuracy (99%) as well as blond hair colour (P = 0.852) and a light hair colour shade (P = 0.964) so that light blond can be assumed as the most likely hair colour (accuracy 69.5%). It is worth noting that in this particular case the eye/hair colour predictions could be confirmed with known data about Władysław Sikorski’s appearance. Although colour photographs of the General were unavailable and all known colour portraits were painted many years after his death and thus may not be considered most reliable, the fact that Władysław Sikorski indeed had blue eyes and blond hair, as predicted via HIrisPlex, was obtained from historical scripts [DNA analysis from skeletal remains may help to solve various controversies concerning historical figures. As an illustrative example, we analysed tooth S11 . In 1942, 14 skulls were sent to the Natural History Museum in Vienna and at that time they were exhibited as examples of “sub-human” skulls. In 1998, these skulls were returned to Poland, and the Institute of National Remembrance ordered examination of these human remains. In all 12 cases, besides mitochondrial DNA data, complete Minifiler STR profiles and partial Yfiler Y-STR profiles were obtained (data not shown) indicating some level of genomic DNA fragmentation. All 12 skulls were included in this study for teeth sampling. In all 12 cases, complete HIrisPlex SNP profiles were generated , while only male monks were expected. One of the two DNA samples was sufficiently preserved to enable analysis of other nuclear markers (data not shown) and was used here for HIrisPlex analysis. The mysterious woman was predicted to have dark blond/brown hair (accuracy of 78.5%) and brown eyes (accuracy of 90.4%), , Table . The somP = 0.899, accuracy of 95.6%), but hair colour could not be inferred because of missing genotypes at three DNA variants . The sample S26 revealed a prediction of blond hair colour (P = 0.784) together with a light hair colour shade (P = 0.918) concluding that the individual had light blond hair (accuracy of 69.5%). Eye colour prediction of S26 revealed blue eyes   was found to be seriously affected by decay, which was reflected by a very low DNA concentration (3 pg/μl) and incomplete autosomal and Y chromosome STR profiles (NGM and Yfiler). Complete mtDNA HVI and HVII profiles were generated in both teeth (data not shown). From these data it was possible to conclude that both skeletons are of male origin and are unrelated in both maternal and paternal lines. From the partial HIrisPlex profile ascertained from S25 we successfully inferred blue eye colour  and the polymorphism C/A rs4959270 (15. position in the assay). N29insA was most sensitive to suboptimal quality/quantity of template DNA. The peak height is generally lower compared to the other SNPs in the HIrisPlex assay and did not reach detection limit settled at 50 RFU five times in the course of repeated analyses. It should be also mentioned that in three cases  this polymorphism remained undetermined  . Here wee colour . A similquantity . In one th century sample (S24) collected from a skeleton that rested in relatively favourable conditions in a church. On the other hand, a partial profile was obtained from a relatively young contemporary sample (S17) that was found in open soil. However, it is difficult, as had been outlined previously [th century sample stored under expectedly similar conditions also excavated from inside a church (S25) yielded a partial HIrisPlex profile.The results obtained here provide further evidence that quality of DNA templates from bone material depends not only on the storage time but more so on environmental conditions affecting the decomposition of the remains. Low temperature and low humidity are known to prevent DNA degradation , allowineviously , to drawObviously, special care is needed when EVC prediction is performed for samples containing DNA in suboptimal quantity and/or quality. Previous preliminary sensitivity testing of the HIrisPlex assay  providedIt can be anticipated that DNA prediction of EVC will soon become more widely used in genetic studies of human remains in evolutionary, anthropological and forensic investigations. The recently introduced HIrisPlex system  providesaDNA: Ancient DNA; DTT: Dithiothreitol; DVI: Disaster victim identification; EDTA: Ethylenediaminetetraacetic acid; EVC: Externally visible characteristic; FDP: Forensic DNA phenotyping; HGDP-CEPH: Human genome diversity cell line panel Foundation Jean Dausset-CEPH Paris; indel: Insertion-deletion polymorphism; mtDNA: Mitochondrial DNA; NGS: Next generation DNA sequencing; RT-PCR: Real time polymerase chain reaction; SBE: Single-base extension; SDS: Sodium dodecyl sulfate; SNP: Single nucleotide polymorphism; STR: Short tandem repeat (polymorphism); WGS: Whole genome sequencing; Y-STR: Y chromosome STR.The authors declare that they have no competing interests.WB and MK conceived and designed the study, provided resources, interpreted results and wrote the manuscript. JDB carried out the genotyping and elaborated results. EP participated in genotyping data analysis. SW carried out the prediction of phenotypes from genotypes and helped in manuscript writing. TK and HG provided the samples and helped to draft the case description parts of the manuscript. All authors read and approved the final manuscript.


In order to accurately model the human MMA disorder we introduced this mutation onto the human methylmalonyl-CoA mutase locus of a bacterial artificial chromosome. A mouse model was developed using this construct.The mutation R403stop was found in an individual with The transgene was found to be intact in the mouse model, with 7 copies integrated at a single site in chromosome 3. The phenotype of the hemizygous mouse was unchanged until crossed against a methylmalonyl-CoA mutase knockout mouse. Pups with no endogenous mouse methylmalonyl-CoA mutase and one copy of the transgene became ill and died within 24 hours. This severe phenotype could be partially rescued by the addition of a transgene carrying two copies of the normal human methylmalonyl-CoA mutase locus. The “humanized” mice were smaller than control litter mates and had high levels of methylmalonic acid in their blood and tissues.This new transgenic MMA stop codon model mimics (at both the phenotypic and genotypic levels) the key features of the human MMA disorder. It will allow the trialing of pharmacological and, cell and gene therapies for the treatment of MMA and other human metabolic disorders caused by stop codon mutations. MUT). Within the first days of life those with the 0mut form of MMA usually exhibit severe metabolic acidosis along with poor feeding, vomiting, lethargy, hypotonia and secondary metabolic disturbances Disruption of the conversion of methylmalonyl-CoA to succinyl-CoA results in an increased level of metabolites in tissues and fluids of the body. This inherited disorder, methylmalonic aciduria (MMA), can be caused by stop mutations in the methylmalonyl-CoA mutase gene  has no mouse methylmalonyl-CoA mutase gene (Mut) and recapitulates the symptoms of the human 0mut form of MMA. This knockout mouse can be partially corrected by the normal human MUT gene  to produce a rescue mouse model  We currently have a knockout MMA mouse model Mut+/+MUTstop+/−) and its partial rescue with a normal human MUT gene .This manuscript describes the development and characterization of the first humanized MMA mouse model with a stop codon mutation , RPCI-11-463L20 (pBAC_MMA) by a two-stage homologous recombination Figure 1Three microinjection sessions resulted in two mice positive for the transgene: one male and one female. The microinjection sessions were complicated by low fertility, lysis of embryos after 12 h, and lack of pregnancies following transfers.The integrity of the transgene in the MMA stop codon mouse model was determined by PCR of the ends of the BAC and by PCR for exon 3 and exon 12 of the mutase locus. The transgene present in the male founder mouse was confirmed to be intact, however in the female was fragmented. Due to fragmentation of the BAC involving at least the mutase locus, the female mouse was not characterized further and was excluded.Metaphase FISH of fibroblast culture from the male founder transgenic mouse identified a single site of integration in chromosome 3 Figure 3The founder mouse was bred against C57BL/6 mice with transmission of the transgene to more than 12 generations. Normal litter sizes, numbers of male and female mice and Mendelian inheritance of the stop codon transgene occurred.Mut+/+MUTstop+/−) were similar to control mice (Mut gene (Mut+/+). There was also no differences in mouse growth, development and longevity suggesting the transgene has not caused insertional problems.Metabolite levels in mice which were hemizygous for the stop-codon transgene (Mut gene and one MUTstop gene (Mut+/−MUTStop+/−) had double the mRNA expression levels of mice with just one Mut gene or one MUTstop gene; however the expressed MUTstop gene would be truncated and inactive. Never the less these mice had normal metabolite and growth patterns (data not presented).Breeding the MMA stop codon mouse model against the MMA knockout mouse model Mut−/−MUTStop+/−) appeared normal at birth, however developed symptoms and died by 24 h in a similar time frame to MMA knockout pups Pups which harbor only the transgene stop codon mutase (no endogenous mouse mutase)  Mut−/−MUT2h+/−MUTstop+/−). These MMA stop codon rescue mice weighed significantly less than control mice from 4 weeks old . The sick mice were provided with a liquid mixture of ground up food pellet and baby formula and placed on a heat pad. They recovered within 2 hours. When provided this diet once per week during the first part of life these episodes were prevented. Heterozygous mice showed none of these health issues.p<0.001, n = 9). When liver weight was compared to body weight (liver weight/whole body weight) the liver size was found to be in proportion to the mouse size. The other organs were not different at this age , even though the body weight of MMA stop codon rescue mice was significantly smaller than control mice .Comparison of mouse organ weights showed that at 6 months of age the liver of MMA stop codon rescue mice was significantly smaller than control mice . When the weight of the mice was taken into account , there was no significant difference in the testis, kidney, heart, lung, spleen or brain sizes. Whilst the relative weights of MMA stop codon rescue mouse liver  and kidney  were significantly higher than control mice.At 2 years of age the liver, kidney, heart, lung, spleen and brain showed no significant difference in size between control and MMA stop codon rescue mice. However the MMA stop codon rescue mice testis were significantly smaller  levels of normal pups did not change with time; however there was an increase in mean concentration in both the MMA knockout-stop codon mouse pups and MMA stop codon rescue pups Table 1.Comparing the change in methylmalonic acid with age showed that there was a decrease with age in tissue and plasma levels Figure 5Urine methylmalonic acid concentrations in control samples did not vary greatly with age and had an average of less than 150 µmol/mmol creatinine over a 2 year period. MMA stop codon rescue pups had 1130±305 µmol/mmol creatinine (n = 19) at 3 days old, which then rose to 23400±7820 µmol/mmol creatinine (n = 25) at 6 weeks of age and remained consistently high.mut0 MMA. Mice with only the stop codon gene  died within 24 hours of birth with drastically increased urine and blood metabolite levels. This severe phenotype could be partially rescued by breeding the mice to also harbor the normal human methylmalonyl-CoA mutase locus.A new mouse model which recapitulates the metabolite levels of a patient with methylmalonic aciduria has been developed. The mouse model contains the human methylmalonyl-CoA mutase locus carrying a stop codon mutation identified from a patient with mut MMA mut MMA mutations result from a stop codon Various studies have identified over 100 pathological mutations from patients with Microinjection of the modified pBAC_MMA resulted in one transgenic mouse line being produced with an intact locus and single integration site. Surprisingly a high copy number of the transgene was determined. As it is planned to use these mice in gene correction studies this may be beneficial in providing a number of sites to target for correction.Hemizygous MMA stop codon mice were found to have a normal phenotype and unaltered life span. When this mouse model was bred against the MMA knockout mice, pups with no endogenous mouse mutase gene were produced. The MMA stopcodon knockout pups were not physically different to control pups at birth, however the high metabolite levels soon caused severe health problems leading to death, similar to the problems seen in human cases of MMA.in utero treatment strategies, generally babies with the disorder are not identified until they are born, therefore it would be more valuable to have a model which can be tested for treatment options once the are pups born. Thus we developed the MMA stop codon rescue mice.MMA stopcodon knockout pups have a very similar etiology to the MMA knockout mouse model with high levels of metabolites in blood and tissue, and limited life span. Whist these mouse models are recapitulating the human disorder, and maybe useful for testing Mut knockout gene and the normal human MUT gene to produce mice which had no endogenous mutase gene and one copy of each of the normal and stop mutation human mutase genes (‘MMA stop codon rescue mice’). In comparison to the MMA rescue mouse model this new MMA stop codon rescue mouse model has very similar phenotypic characteristics, which shows that the stop codon mutation is not producing functional full length methylmalonyl-CoA mutase protein and thus not alleviating the phenotype.MMA stop codon mice were bred against mice which harbored the MUT gene, suggesting that the human mutase in a mouse background is less efficient  than the native mouse enzyme. The health of the partially rescued mice is initially generally good, but they have episodes where they deteriorate over a couple of days, although can be treated with additional nutrition. This is similar to the MMA rescue mouse model, where the health of the mice is more stable after about 6 months of age.The MMA stop codon rescue mice, similar to the MMA rescue mouse model, is only partially rescued by the human This MMA stop codon rescue mouse model has significantly higher methylmalonic acid in the urine compared to the blood as organic acids are typically cleared from the bloodstream by the kidney. Soon after birth the amount of methylmalonic acid in the kidney also increases. The effects of high metabolite levels in the urine may cause an enlargement of the kidney in 2-year old mice. The liver was also enlarged at 2 years, whilst other organs were not affected. Human cases of MMA may show an enlarged liver as a symptom as well as long term detrimental effects on the kidney. Many of the severe symptoms of MMA are not recapitulated in this mouse model; however the key biochemical indications are present.The MMA stop rescue mouse model size and weight gain have been significantly affected, which is a concerning feature in the human disorder. One of the diagnostic characteristics of children with MMA is their failure to thrive, similar to this mouse model. Whilst there was a significant difference in weight between control and MMA stop codon rescue mice, the difference in mouse length was less distinctive. The weight difference can most likely be accounted for by the lack of abdominal fat deposits in the MMA stop codon rescue mice.in vivo testing of stop codon read through compounds for a human mutation. More importantly the mutation is introduced centrally onto a 170 kb human BAC and is therefore within the context of the flanking human sequence with appropriate human sequence regulatory elements. Due to the intermediate phenotype of the mouse it allows measurement of phenotypic correction (growth and metabolite levels).Whilst there are mouse models for missense mutations in vivo and ex vivo gene therapy strategies (e.g. gene correction or suppression of stop codon recognition) with direct applicability to human DNA sequences.This new stop codon carrying mouse model recapitulates the key features of the human MMA disorder at both the phenotypic and genotypic levels, providing an effective tool for the evaluation of All procedures involving treatment of mice were performed with ethics approval from the Murdoch Childrens Research Institute Animal Ethics Committee (Projects: A550 and A649). All surgery was performed under isoflurane or avertin anesthesia, and all efforts were made to minimise suffering.R cassette into the R403stop mutation region of pBAC_MMA exon 6.The R403stop mutation was inserted into the human mutase locus of bacterial artificial chromosome (BAC), RPCI-11-463L20 (pBAC_MMA) by two-stage homologous recombination to form pBAC_MMA*, R403stop. The first stage involved insertion of the I-Sce I/kanSce I/kanR resistance cassette was amplified by long range PCR with proof reading Taq , from the plasmid pST98-AS/KanF. The primers  used to amplify this cassette (forward TACTGCAATAGAAGCAATGGCAGCAGTATTTGGAGGGACTCAGTCTTTGCGGTCCGA ACTCTAAACTGCTGA-3′5′- and reverse TTGTGAGACATTCCATCATGTAAGAACCTCCC CAAGGATCAGCCACTTTGGGATCTGAGGTTCTTATGGCTCT-3′5′-) incorporated fifty bases of homology to the targeting mutase sequence onto the end of each primer (underlined sequence). The targeting recombination event deleted 100 bp between the homology regions and inserted the 2.1 kb cassette into exon 6 of the mutase locus (MUT exon 6 forward (GGATCCCTACAATAATATTGTCC) and reverse (CTTACCTTTAAAGCAGCAT CATA) primers external to the recombination site identified colonies with the correct recombination.The I-se locus Figure 15′-GGATCCCTACAATAATATTGTCC-3′ and exon6R 5′-CTTACCTTTAAAGCAGCAGCAT CATA-3′. In order to amplify only the mutated allele, the 255 bp exon 6 PCR product was sub-cloned into pGemT . A TaqI restriction digestion of the exon 6 PCR product from positive colonies enabled determination of a clone containing the R403stop allele. The C→T mutation in exon 6 removed the TaqI restriction site, replacing it with a TGA stop codon.The R403stop mutation was amplified from patient genomic DNA, using primers exon6F Sce I/KanR) cells. BAC DNA was extracted from the overnight culture, restriction digested (the I-Sce I site was utilized as a counter selection to linearize non-recombinants thereby enriching for correct recombinants) and electroporated into DH10B cells. pBAC_MMA*, R403stop positive colonies were sequenced and restriction digested to exclude rearrangements.The second stage of homologous recombination was carried out using a 255 bp exon 6 PCR product amplified from the sub-cloned fragment and electroporated into DY380 . Mice were developed on the C57BL/6 background.ad libitum.The male founder was bred against C57BL/6 mice producing the mouse colony (MMA stop codon mice). Mice were kept and cared for in the mouse facility at the Murdoch Childrens Research Institute. They were provided irradiated Barastoc pellets  and tap water MUT exon 6 (exon6F and exon6R primers) resulting in a 255 bp product. This product was digested with TaqI restriction enzyme. The R403stop mutation removed the restriction site, thus is not digested, whereas a normal human gene produces two bands of 124 and 131 bp.DNA was extracted from a tail tip sample of each mouse and screened. The DNA was firstly amplified using PCR primers for the human Mut gene ; Neo/Kan which amplified the knockout cassette ; and Exon 12 which amplified the human MUT gene .When breeding the mice against the other MMA mouse models, the genotyping PCR was a multiplex looking for three genes: Exon 3 which amplified the mouse The number of copies of the human mutase locus was determined relative to the mouse by PCR of a highly homologous region and comparison of relative intensities. Primers, expF and expR 5′-TTCTATAAGGACAACATTAAGGCTGGTC-3′) and hmMUT-R (5′-CAATAGCAACTCCAGCCATTCC-3′). Expression was normalized to human beta actin (forward primer 5′-AGGCACCAGGGCGTGAT-3′ and reverse primer 5′-TCGCCCACATAGGAATCCTT-3′). The hmMUT primers bind to the methylmalonyl-CoA mutase cDNA sequence prior to the stop codon mutation. The results were analyzed using the “delta-delta comparative Ct method” and presented as fold change in mRNA levels in cells treated with different compounds, relative to untreated controls.The level of mRNA expression from the human transgene relative to the endogenous mouse locus was compared. Real time RT-PCR was performed using the ABi7300 Real Time PCR system with SYBRgreen PCR Master Mix . methylmalonyl-CoA mutase cDNA expression was detected using the primers hmMUT-F , using nick translation according to the manufacturer's recommendation. The slides were mounted and examined using an Olympus BX60 epifluorescence microscope. Images were captured using Cytovision software .2H3-methylmalonic acid; Cambridge Isotope Laboratories, Andover, MA), protein precipitate removed, before the supernatant was dried and the samples butylated, then neutralized. Samples were run using a 5 µm, Hypersil ODS 2.1×100 mm C18 column . The mobile phase consisted of 20% Buffer A (9∶1 water: 0.2 ammonium formate pH 3.5) and 80% Buffer B (9∶1 methanol: 0.2 mol/L ammonium formate pH 3.5) run with a flow rate of 0.2 mL/min. The transitions 231>119 m/z and 234>122 m/z were used in the selected reaction monitoring mode for butylated methylmalonic acid and 2H3-methylmalonic acid respectively. Plasma methylmalonic acid analysis was performed in the same manner, without the initial sample preparation steps.Tissue methylmalonic acid analysis  was performed on frozen tissue samples that were weighed precisely, homogenized in water, sonicated and then centrifuged. The supernatant was then mixed with 1 mmol/L internal standard  and analyzed by direct injection electrospray MSMS according to standard methods t-test. Statistical significance was accepted at p<0.05.Data expressed as mean (± SEM). Analyses were performed using a two-sample


Lipid bodies (LBs), also known as lipid droplets, are complex organelles of all eukaryotic cells linked to a variety of biological functions as well as to the development of human diseases. In cells from the immune system, such as eosinophils, neutrophils and macrophages, LBs are rapidly formed in the cytoplasm in response to inflammatory and infectious diseases and are sites of synthesis of eicosanoid lipid mediators. However, little is known about the structural organization of these organelles. It is unclear whether leukocyte LBs contain a hydrophobic core of neutral lipids as found in lipid droplets from adipocytes and how diverse proteins, including enzymes involved in eicosanoid formation, incorporate into LBs. Here, leukocyte LB ultrastructure was studied in detail by conventional transmission electron microscopy (TEM), immunogold EM and electron tomography. By careful analysis of the two-dimensional ultrastructure of LBs from human blood eosinophils under different conditions, we identified membranous structures within LBs in both resting and activated cells. Cyclooxygenase, a membrane inserted protein that catalyzes the first step in prostaglandin synthesis, was localized throughout the internum of LBs. We used fully automated dual-axis electron tomography to study the three-dimensional architecture of LBs in high resolution. By tracking 4 nm-thick serial digital sections we found that leukocyte LBs enclose an intricate system of membranes within their “cores”. After computational reconstruction, we showed that these membranes are organized as a network of tubules which resemble the endoplasmic reticulum (ER). Our findings explain how membrane-bound proteins interact and are spatially arranged within LB “cores” and support a model for LB formation by incorporating cytoplasmic membranes of the ER, instead of the conventional view that LBs emerge from the ER leaflets. This is important to understand the functional capabilities of leukocyte LBs in health and during diverse diseases in which these organelles are functionally involved. Intracellular inclusions containing lipids are widely present in eukaryotic cells  and bacteria. In many cells, including adipocytes, steroidogenic cells and hepatocytes, these inclusions, termed lipid droplets (LDs), are extensively recognized for their roles in neutral lipid storage and metabolism , are also dynamic organelles with functions including serving as niduses for synthesis of arachidonic acid-derived inflammatory lipid mediators (eicosanoids) With the recognition that leukocyte LBs are sites for the regulated biosynthesis of inflammatory lipid mediators such as prostaglandins and leukotrienes Some of the difficulties in the study of LBs originate from their unique architecture. In contrast to vesicles and membranous organelles that have an aqueous content surrounded by a phospholipid bilayer membrane, LBs and LDs are encircled just by a monolayer of phospholipids By conventional transmission electron microscopy (TEM), LBs generally appear as homogeneous organelles. However, the use of freeze-fracture An emerging application of automated electron tomography renders feasible the observation of the three-dimensional (3D) architecture of cellular components Given the broader functional roles for LBs in cells from the immune system, here we used electron tomography as well as conventional TEM and immunogold EM to study structural aspects of LBs in human eosinophils, cells involved in a diversity of allergic and inflammatory diseases , T cell expressed, and secreted (RANTES/CCL-5), tumor necrosis factor-alpha (TNF-α), interferon-gamma (IFN-γ) or medium alone for 1 h and prepared for conventional TEM. In addition, eosinophils from patients with the hypereosinophilic syndrome (HES) were studied. Some samples were also post-fixed with reduced osmium to increase contrast and facilitate identification of very small LBs. At least 450 electron micrographs randomly taken were carefully examined at different magnifications.The numbers of LBs were quantitated in electron micrographs showing the entire cell profile and nucleus. LB numbers significantly increased in eosinophils stimulated with the 4 different cytokines compared to unstimulated controls . Greater2 (PGE2), and is associated with intracellular membranes, including the ER Next, the presence of the membrane-inserting protein cyclooxygenase (COX), also termed prostaglandin endoperoxide synthase, was investigated in naturally formed LBs from HES patients. COX expression was analyzed because this enzyme is linked to the synthesis of the inflammatory mediator prostaglandin EAlthough the analyses of thin sections showed occasionally the presence of membranes within LBs from both unstimulated ii and stReconstruct) for tracking both the LB and its membranous structures contours through the entire tomogram. By tracing these contours -mediated formation of LBs in macrophages Here, leukocyte LB formation was evaluated, for the first time, by quantitativeTEM after stimulation with different stimuli. Our data showing increased, eotaxin-1-, RANTES-, TNF-α– or INF-γ –induced LB numbers within human eosinophils  support Consistent with the role of leukocytes in inflammation, LBs formed by these cells constitute sites for the production of inflammatory lipid mediators (eicosanoids). LBs within inflammatory cells contain arachidonyl lipids, which serve as precursors for eicosanoid generation and all of the enzymes necessary for eicosanoid syntheses Our comprehensive study of the LB ultrastructure by conventional TEM and using therefore 80 nm thickness-thin sections revealed occasionally the presence of membranes within these organelles . These sz axis resolution of the tomograms is 6–8 nm and the x and y axis resolution nearly 4 nm. Compared to imaging capabilities of other microscopic techniques, this z axis resolution is approximately 20-fold better than of classical EM thin sections and approximately 100-fold better than of typical images obtained with standard confocal light microscopic images , were localized fully within LBs as demonstrated here in human eosinophils  and leukotriene Cinophils  and in pLBs have been identified as sites of eicosanoid lipid mediator formation not only in leukocytes but also in other cells  After computational alignment and reconstruction of the tilt series, followed by modeling, the 3D architecture of LBs could be appreciated. The organization of the LB internal membranes described here resembles that of the ER. Accordingly, our conventional TEM findings demonstrate portions of ER at the LB periphery and even intermingled in the lipid content. Although it is extensively recognized that lipid-rich organelles originate from the ER, it is still a matter of debate on how these organelles are formed. The prevailing model for LD biogenesis assumes that LDs are formed by accumulating neutral lipids between the cytoplasmic and luminal leaflets of ER bilayer membranes followed by the budding off of LDs surrounded by a phospholipid monolayer derived from the cytoplasmic leaflets of ER membranes  from the healthy donors were stimulated with recombinant human eotaxin-1 ; RANTES  TNF-α or INF-γ  in RPMI-1640 medium plus 0.1% ovalbumin (OVA) , or medium alone at 37°C, for 1 h. Eosinophil viability and purity were greater than 99% as determined by ethidium bromide  incorporation and cytocentrifuged smears stained with HEMA 3 stain kit , respectively.Granulocytes were isolated from the blood of different healthy and hypereosinophic donors. Eosinophils were enriched and purified by negative selection using human eosinophil enrichment cocktail  and the MACS bead procedure , as described 2 in 0.1 M sodium cacodylate buffer, (pH 7.4) for 1 h, at room temperature (RT), washed in the same buffer and centrifuged at 1500 g for 1 min en bloc in 2% uranyl acetate in 0.05 M sodium maleate buffer, pH 6.0 for 2 h at RT and washed in the same buffer as before prior to dehydration in graded ethanols and infiltration and embedding with a propylene oxide-Epon sequence . Alternatively, samples were post-fixed in 2% aqueous osmium tetroxide and 1.5% potassium ferrocyanide in 0.1 M sodium phosphate buffer, pH 6.0 (reduced osmium) before dehydration and embedding as above. After polymerization at 60°C for 16 h, thin sections were cut using a diamond knife on an ultramicrotome . Sections were mounted on uncoated 200-mesh copper grids (Ted Pella) before staining with lead citrate and viewed with a transmission electron microscope  at 60 KV. At least 450 electron micrographs randomly taken at different magnifications were analyzed.Purified eosinophils were immediately fixed in suspension (not pelleted) in a mixture of freshly prepared aldehydes  containing 2.5% CaClP<0.05).To quantify the number of LBs, a total of 295 electron micrographs showing the entire cell profile and nucleus were randomly taken from unstimulated and stimulated eosinophils and the numbers of LBs were counted. Data were compared by the Mann-Whitney “U” test  were cut and analyzed by automated EM tomography . The tomogram corresponds to (AVI)Click here for additional data file.