The present data on the evaluation of platelet (PLT) parameters in Chinese Han population and Tibetans are still limited. The objective of this study was to determine the differences in common PLT indices between Han population and Tibetans in China, through a large-scale investigation of healthy people.2131 Han people from Chengdu Plain, 1099 Tibetans from Qinghai-Tibet Plateau and 956 Plateau Han migrants were included in this study. All the subjects were healthy people through the health screening. PLT indices were measured with Sysmex XE-2100 and XT-1800i blood cell automatic analyzer.P<0.01) but lower mean platelet volume (MPV), platelet distribution width (PDW) and platelet-large cell ratio (P-LCR) (P<0.01); while Plateau Han migrants had lower PLT count, MPV and P-LCR (P<0.05). When compared with Tibetans, Plateau Han migrants had lower levels of mean PLT count but higher PDW and P-LCR (P<0.05).Compared with Han people in Chendu Plain, Tibetans had higher PLT count (There are ethnic differences in PLT indices between Chinese Han population and Tibetans. Based on this finding, it would be reasonable to conduct formal prospective studies to determine the clinical significance of these differences and to explore the effects of genetic background on these indices. Chinese Tibetans have lived in a very high altitude (mean 4500 m) and hypoxic environment at the Qinghai-Tibet Plateau for thousands of years. They thus have a suite of distinctive physiological traits that enable them to tolerate the extreme environment, such as decreased arterial oxygen content Platelets (PLTs) are well known to play an important role in hemostasis and blood coagulation. Once defects happen in amount and/or function of PLTs, the risk of bleeding will increase Chronic hypoxia is demonstrated as a risk factor of multiple cardio- and cerobro-vascular disorders, such as congestive heart failure This study was approved by the Ethics Committee of Chinese Human Genome and the Ethics Committee of West China Hospital, and written informed consent was obtained from all participants.2131 Han people from Chengdu Plain, 1099 Tibetans from Qinghai-Tibet Plateau and 956 Plateau Han migrants were included in this study. All volunteers have lived in their residence for more than 3 years. Every subject underwent an interview with a physician using a life-health questionnaire and a physical examination form.Exclusion criteria included body mass index (BMI) ≥28, high blood pressure (systolic number≥140 mmHg and/or diastolic number≥90 mmHg), alcohol assumption>30/g, tobacco use>20 cigarettes, drugs intake within 2 weeks, surgery within 6 months, blood donor or transfusion within 4 months, pregnancy, less than 1 year after childbirth, hypothyroidism and hyperthyroidism, diabetes, atherosclerosis and vascular disease, cardiopathy, chronic nephropathy, hepatobiliary disease, allergic diseases, hematological disease, myopathy, autoimmune disease, burns and muscle trauma, the presence of acute and chronic infection, plasma fasting glucose>7.0 mmol/L, serum creatinine>120 µmol/L, serum creatine kinase>400 U/L, positive hepatitis B surface antigen, positive anti-hepatitis C virus antibody, positive anti- immunodeficiency virus antibody and positive urinalysis.After strict screening, a total of 4186 blood samples were obtained. Samples were collected in West China Hospital of Sichuan University and Hospital of Chengdu Office, People's autonomous Government of Tibetan from October 2011 to May 2012. Demographic features of all participants were provided in 2 anticoagulant at 8∶00–9∶00 a.m. Complete blood Counts (CBC) were all completed within 30 minutes after blood drawing.For all participants, a 2-ml fasting blood sample was drawn into a BD Vacutainer tube with EDTA-KXE-2100 blood cell automatic analyzer in West China Hospital and XT-1800i blood cell automatic analyzer in hospital of Chengdu Office, People's autonomous Government of Tibetan were all purchased from Sysmex, Japan.Before and after samples testing, three levels of controls  were performed. Clinical laboratories of two hospitals regularly participated in proficiency test (PT) of Chinese Ministry of Health, and laboratory of West China Hospital has gained accreditation from the College of American Pathologist.ANOVA and Student-Newman-Keuls were performed to detect the difference among groups. Spearman's correlation was used as a test of correlation between two continuous variables. P Values less than 0.05 were considered significant. 95% confidence interval (CI) of each parameter was expressed by calculating the 2.5th–97.5th percentile.Data were analyzed using SPSS 17.0 software . There were no significant differences in either age or gender among the three groups of subjects .P<0.01). When compared with Plateau Han migrants, mean PLT count, MPV and P-LCR of Han people in plain was significantly higher (P<0.05), while there was no obvious difference of PDW between these two groups (P>0.05). Specific data and 95% CIs see As shown in P<0.001, P<0.001, P<0.001, P<0.001, P<0.001, P<0.001, As shown in Although a various of studies allowed researchers to realize the distinctive physiological traits of Tibetans in China, the physiological changes of PLT indices in Tibetans are still unknown. Our results demonstrated that there were ethnic differences in PLT indices between healthy Chinese Tibetans and Han population. Tibetans in Plateau had higher mean PLT count but lower MPV, PDW and P-LCR as compared with either Han population in Chengdu Plain or Plateau Han migrants.Several studies have independently demonstrated that high-altitude hypoxia exposure had great impact on the generation or function of not only red blood cells (RBCs) but also platelets in the blood 9/L] was lower than that of national average level [(125∼325)×109/L] . Decrease of mean PLT number of Han people in Plain made the PLT count of Tibetans seems to be higher. But, the reasons why Han people in Chengdu Plain had lower PLT number are still unclear, and exploring the specific mechanisms on the basis of environments and genetics is our next work.However, it was interesting to find that Tibetans living in Plateau had a higher mean PLT count than that of Han people in Plain, which was not be consistent with the previous studies. Then we compared the 95% confidence interval (CI) of PLT count in this study with the reference range of PLT count in Chinese healthy adults,and results indicated that the mean PLT number of Han people living in Chengdu Plain . In our study, MPV of Tibetans was obviously lower than that of both Han people in Plain and Plateau Han migrants, which could be the reason why Tibetans did not live with high prevalence of vascular disease.PDW is an index reflecting the heterogeneity of platelets, while P-LCR is the proportion of large platelets. Generally, the more large platelets exist in blood, the higher MPV and PDW are There were also differences in the PLT indices between Han people in Plain and Plateau Han migrants in this study, although these two group subjects had the same genetic background. Results suggested the remarkable impact of environment on PLT indices.To our knowledge, this is the first report to explore the ethnic differences in PLT indices between healthy Chinese Tibetans and Han population in so large-scale. The model of PLT indices in Tibetans was higher PLT count accompanied by lower MPV, PDW, and P-LCR, which was possibly the result of their adaption to the extreme environment in Qinghai-Tibet Plateau. The PLT count of Han people living in Chengdu Plain was lower than Chinese mean level, and this phenomenon was worth further research. Genetic and environment were two main factors causing physiological variations, therefore,unique reference ranges of some routine inspection items should be established for people of different ethnic groups or in different regions.


Extragastrointestinal stromal tumors (EGISTs) are neoplasms located outside the gastrointestinal tract in sites including the omentum, mesentery and retroperitoneum. EGISTs of the transverse mesocolon are rarely noted in the literature. Herein, we describe a rare case of giant EGIST concomitant with gastric cancer in a 78-year-old male who presented with upper abdominal pain and a palpable mass. The patient underwent en bloc resection of the tumor with a distal gastrectomy, with a D2 lymphadenectomy for the gastric cancer, accompanied with resection of a segment of the transverse colon. The patient received targeted therapy  and adjuvant chemotherapy with FOLFOX (six cycles). Neither recurrence nor metastasis was observed after 24 months of follow-up. Gastrointestinal stromal tumors (GISTs) are rare neoplasms that arise from mesenchymal cells of the gastrointestinal tract. Extragastrointestinal stromal tumors (EGISTs) have no contact with the stomach and intestine, by definition, and typically occur in the omentum, mesentery or retroperitoneum ,2. HowevThe most common type of carcinoma associated with GISTs is gastric carcinoma . To the The study was approved by the Ethics Committee of Qilu Hospital, Shandong University, China. The patient’s family consented to this study.A 78-year-old male was admitted to hospital for abdominal distension and pain. The patient had no history of epigastralgia or peptic ulcers and there was no significant relevant family history. A physical examination revealed a 20x12 cm palpable mass in the middle and lower abdomen, with minimal intrinsic mobility. Carcinoembryonic antigen (CEA), cancer antigen (CA) 724 and carbohydrate antigen (CA) 19-9 levels were normal. A computed tomography (CT) scan revealed a 21x14 cm heterogeneous mass extending from the fundus to the mid-abdomen, with a number of solid and cystic components within the tumor . A gastr2 (on day 1); leucovorin IV, 200 mg/m2 (on days 1 and 2); 5-FU (fluorouracil) IV, 400 mg/m2 (on days 1 and 2) and 5-FU IV 22 hours in fusion, 600 mg/m2 (on days 1 and 2). This regimen was repeated every 2 weeks for 6 cycles. Additionally, the patient was simultaneously administered imatinib at a dose of 400 mg/day for 1 year. No evidence of tumor recurrence was identified after 24 months of follow-up.Macroscopic examination of the distal gastrectomy specimen revealed a Borrmann type II tumor in the stomach, measuring 3x3 cm. On histopathological examination, the tumor was identified to be an adenocarcinoma with poor to moderate differentiation that exhibited transmural infiltration . No nervGISTs identified outside the gastrointestinal tract as apparent primary tumors are designated as EGISTs. EGISTs may occur in the omentum, mesentery or retroperitoneum, adjacent to  the stomach and intestine. Their true origin is uncertain; however, their histological appearance and immunophenotypes are typically identical to those of classical GISTs. The EGIST in the present case was located inside the mesocolon, outside the gastrointestinal tract. EGISTs of the mesocolon have rarely been noted in the literature –11. The et al demonstrated that the c-kit and PDGFRA mutation in EGISTs was similar to that of GISTs. Therefore, from a molecular genetics perspective, EGISTs may be a unique subtype of GISTs (The clinicopathological or immunohistochemical features of EGISTs and GISTs were not observed to be markedly different. Primary EGISTs of the omentum and mesentery demonstrated clinicopathological and immunohistochemical characteristics similar to a GIST of the digestive tract described previously in the literature . This suof GISTs .The risk categories of EGISTs are contested. A tumor size >10 cm, mitotic activity >2 per 50 high-power fields, tumor necrosis, marked nuclear atypia, >10% Ki-67 protein expression and epithelioid-/mixed cell-type, have acted as significant predictors of survival . Survivaet al, 39% of patients with EGISTs had an adverse outcome, which suggested that EGISTs were aggressive and were more similar to GISTs located in the distal gastrointestinal tract (et al analyzed 9 EGISTs and revealed that the average overall survival was 26.4 months (et al demonstrated that 66.6% of mesenteric cases with a high mitotic rate exibited hepatic metastasis at 6 and 32 months, respectively (There are few data regarding the clinicopathological factors of EGISTs that predict the patient’s prognosis. In a study by Reith al tract . Barros ectively . It was Surgery is the standard treatment for non-metastatic EGISTs. A preoperative diagnosis is difficult, and the patient undergoes an operation based on the diagnosis of an abdominal mass. Where possible, en bloc resection with contiguous tissues and regional lymph nodes is conducted. It is crucial for the pathologist to examine whether the tumor is adhesive to the gastrointestinal wall or other tissues. A histological diagnosis of EGIST is often unsatisfactory. In the present case, the patient underwent an en bloc resection of the tumor and partial transverse mesocolon, accompanied with a distal gastrectomy with a D2 lymphadenectomy. Positive immunohistochemical staining for CD117 is a defining feature of EGISTs. C-kit-positive tumors are most responsive to treatment with a c-kit selective tyrosine kinase inhibitor. Due to the advent of targeted therapy, imatinib, an inhibitor of the tyrosine kinase activity of c-kit, has revolutionized the treatment of this disease. The recommended first-line treatment of advanced GIST is 400 mg/day imatinib . In certThe coexistence of EGIST with other abdominal malignancies is a rare phenomenon; we suggest that their synchronous occurrence may be a coincidence. Although there are data regarding the co-occurrence of EGISTs and other malignant tumors, the mechanism of synchronous tumorigenesis remains unclear. Surgical resection is currently the mainstay of treatment for EGISTs. The existing data on the coexistence of EGISTs with other types of gastric carcinoma are insufficient to reach a final conclusion regarding the treatment, prognosis and recurrence.In summary, the present case was a giant EGIST in the mesocolon accompanied with gastric cancer in an elderly male. We stress that, although their prevalence is very low, surgery-based multimodal treatment is the preferred strategy for compound tumors. Adjuvant chemotherapy and targeted therapy are important for high-risk EGISTs accompanied with digestive tract malignancies. Due to the limited number of cases, the existence of a common tumorigenesis factor among the different tumors cannot be identified. Further studies are required to expound the possible association.


Human cytomegalovirus (HCMV) has been shown to induce increased lipogenesis in infected cells, and this is believed to be required for proper virion envelopment. We show here that this increase is a consequence of the virus-induced redistribution of the host protein viperin to mitochondria and its capacity to interact with and block the function of the mitochondrial trifunctional protein (TFP), the enzyme that mediates fatty acid-β-oxidation. The resulting decrease in cellular ATP levels activates the enzyme AMP-activated protein kinase (AMPK), which induces expression of the glucose transporter GLUT4, resulting in increased glucose import and translocation to the nucleus of the glucose-regulated transcription factor ChREBP. This induces increased transcription of genes encoding lipogenic enzymes, increased lipid synthesis and lipid droplet accumulation, and generation of the viral envelope. Viperin-dependent lipogenesis is required for optimal production of infectious virus. We show that all of these metabolic outcomes can be replicated by direct targeting of viperin to mitochondria in the absence of HCMV infection, and that the motif responsible for Fe-S cluster binding by viperin is essential. The data indicate that viperin is the major effector underlying the ability of HCMV to regulate cellular lipid metabolism. Rsad2 gene. Human cytomegalovirus (HCMV) paradoxically induces expression of viperin independently of the interferon response, and we previously showed that a virus-encoded protein transports the induced viperin to mitochondria where it interferes with fatty acid b-oxidation, a major energy generating system of the cell. We show here that this ultimately results in enhanced lipid synthesis by the infected cell that is essential for production of infectious virus. The mechanism involves sensing the depletion in ATP levels caused by inhibition of fatty acid b-oxidation by the enzyme AMP-induced protein kinase. This induces a cascade of events that result in the increased transcription of genes encoding lipogenic enzymes and consequent lipid biogenesis that is needed by the virus for adequate membrane envelope formation. Thus HCMV uses the interferon-inducible protein viperin, known to be antiviral for other viruses, even for HCMV itself if viperin is pre-expressed in cells prior to infection, to modulate the metabolic status of the cell to facilitate its replication.Virus infection induces the production of interferons, which in turn stimulate the production of a set of proteins that often have antiviral functions. One of these interferon-inducible proteins is viperin, the product of the human  These are sterol regulatory element binding proteins (SREBPs) and carbohydrate responsive element binding protein (ChREBP), which are insulin- and glucose-responsive transcription factors, respectively de novo lipogenesis. Glucose activates ChREBP by regulating its redistribution from the cytosol to the nucleus by a phosphorylation dependent mechanism de novo lipogenesis Two major classes of transcription factors regulate Upon infection HCMV directly induces the interferon (IFN)-inducible iron-sulfur (Fe-S) cluster-binding protein, viperin Viperin interaction with the mitochondrial trifunctional protein (TFP) depletes cytoplasmic ATP Activation of AMPK induces GLUT4 expression ChREBP is a glucose-responsive transcription factor that activates the transcription of lipogenic enzymes and is highly regulated by GLUT4 in adipose tissue de novo fatty acid synthesis by inducing expression of lipogenic enzymes ChREBP translocation to the nucleus induces lipogenic enzyme transcription, and HCMV infection is known to increase de novo lipogenesis are dominantly regulated by ChREBP rather than SREBP1, and that this process is viperin-dependent. To address this we measured lipogenesis in cells expressing GLUT4- and ChREBP-specific shRNAs during HCMV infection  induction ng cells . We alsong cells . No chanHaving previously observed that HCMV-encoded vMIA is responsible for targeting viperin to the mitochondria Enhanced lipid synthesis is thought to provide the membrane necessary for proper HCMV envelope formation. To explore the role of viperin in this process we analyzed the viral particles generated in the presence and absence of viperin by quantitating cytoplasmic non-enveloped particles (capsids and tegumented capsids) and enveloped particles (double-layered particles formed by tegumentation and secondary envelopment in the secretory pathway) in control and viperin knockdown cells by electron microscopy, late in infection to allow accumulation of quantifiable virions . In norm14C-labeled acetate as a substrate  did not show the increase in mRNA levels, nor did they increase total lipid synthesis or accumulate LDs (de novo lipogenesis observed was dependent on GLUT4-mediated glucose uptake we measured lipogenic enzyme transcripts in cells expressing the chimeric proteins in the presence or absence of glucose in the medium (de novo lipogenesis. Similar results were generated using viperin knockout murine embryonic fibroblasts (MEFs) expressing the chimeric proteins  ubstrate  in humanubstrate  and S3A,late LDs . This inlate LDs . This exe medium . In the e medium . The expe medium . The datproteins . In contproteins , accumulproteins , and incproteins .Our previous work showed that viperin interaction with the mitochondrial enzyme TFP inhibits fatty acid β-oxidation Finally, to control for the specificity of the viperin shRNA effects and confirm that viperin is involved in lipogenesis in the context of viral infection, we infected cells with a recombinant HCMV (HCMV.mVIP) in which the loci US7–US16, nonessential for in vitro replication, were replaced by mouse viperin-GFP under an inducible promoter such that expression could be enhanced by the addition of doxycycline, although some breakthrough transcription occurred in the absence of doxycycline The effects of HCMV upon the metabolic status of infected cells reflect a commitment to macromolecular synthesis, including lipid biosynthesis, rather than generation of energy The cumulative data suggests that viperin-mediated inhibition of fatty acid β-oxidation reduces the generation of ATP and activates AMPK, which results in the induction of GLUT4 to increase glucose uptake. Activation of AMPK has been shown to be important for HCMV-mediated alterations in metabolism Lipid synthesis is induced by HCMV infection, and in cells lacking viperin expression this increased synthesis is not observed. Presumably because of this the increase in size and number of LDs induced by infection is also not seen in the viperin shRNA-expressing cells. These findings are precisely replicated in the same cells uninfected but expressing mitochondrially-targeted viperin. HCMV infection in the absence of viperin induction both eliminates the size increase and reduces the number of LDs, suggesting that initiating viral envelope formation consumes pre-existing lipids but without the viperin-mediated induction of lipogenesis they are not replaced. This results in a significant decrease in the production of infectious virus. Envelopment of HCMV proceeds by a complex process, involving initial envelopment in nuclear membrane that is shed in the cytoplasm. Secondary envelopment occurs by budding into the secretory pathway. Electron microscopy clearly showed that the number of enveloped particles formed in the secretory pathway was substantially reduced in the viperin knock down cells. However, the synthesis of viral proteins and the total number of viral particles, assessed by genome copy number was unaffected.Although HCMV-induced cleavage of SREBP1 is independent of viperin expression, HCMV infection does not induce expression of lipogenic enzymes and lipid synthesis in the absence of viperin. There are two genes encoding SREBPs, SREBP1 and SREBP2, and SREBP1 exists in two forms, a and c, depending on the use of alternate promoters. This renders a simple interpretation difficult, but mice lacking SREBP1c do exhibit a reduction of some, but not all lipogenic enzyme transcripts in the liver Overall, the data indicate that viperin is the key regulator of HCMV-induced modulation of lipid metabolism, and it may present a novel target for HCMV therapy. We are still left, however, with the vexed question of the normal function of viperin. Are there physiological circumstances in which a modification of lipogenesis mediated by viperin would be important, and if that is so, how does the molecule enter mitochondria to mediate this effect? Does this play a role in any of the inhibitory effects on other viruses that have been attributed to viperin? The answers to these questions await further experiments.Rsad2) knockout C57BL/6 mice Human foreskin fibroblast (HFF) cells were purchased from Yale Skin Diseases Research Center Core. Telomerase-immortalized human fibroflast (HFtelo) cells were kindly provided by Dr. T. Shenk (Princeton University). Trifunctional protein (TFP) β subunit (HADHB) deficient fibroblasts (GM20265 and GM20266) were obtained from the Coriell Institute. MRC5 fibroblasts were kindly provided by Dr. E.S. Mocarski (Emory University). Murine embryonic fibroblasts (MEFs) used in the study were isolated from viperin (HCMV strain AD169 was kindly provided by Dr. W.J. Britt (University of Alabama at Birmingham). HCMV strain RVHB5 and HCMV mutant RVHB5ΔvMIA were provided by Dr. E.S. Mocarski (Emory University) UL86) (28-4), pp65 (UL83) (28-19), pp28 (UL99) (41-18), gB (UL55) (27-78) and IE-1 (UL123) (P63-27) were gifts from Dr. W.J. Britt (University of Alabama at Birmingham). mAb to viperin (aa 263-277) (MaP.VIP) was described previously HCMV-encoded proteins were detected with monoclonal antibodies (mAb). mAbs to MCP -FMK  and AMPK inhibitor, Compound C (Sigma) were used.HFtelo cells stably expressing the indicated shRNAs were plated in duplicate and serum starved for 24 hr. Cells were infected with HCMV at a multiplicity of infection (moi) of 2. The cells were treated with DMSO or the AMPK inhibitor, Compound C (5 µM). At 2 dpi, the cells were harvested in lysis buffer  (Roche), Phosphatase inhibitor cocktail (Roche) and 1 mM DTT). The cell lysates were assayed for AMPK activity using CycLex AMPK Kinase Assay Kit . Absorbance of each sample was measured at wavelength of 450 nm using a Victor2 fluorometer (PerkinElmer).Previously generated HFtelo cells stably expressing two distinct short hairpin target sequences to viperin (Viperin shRNA1 and 2) and a negative control, a target sequence to luciferase (Luc shRNA) were used pGIPZ lentiviral plasmids with the short hairpin target sequences to GLUT4 and ChREBP were used (Open Biosystems). To increase knockdown efficiency, two lentiviral plasmids including different target sequences were combined: GLUT4 shRNA1 (clone ID: V3LHS_376729 and V3LHS_376733), GLUT4 shRNA2 (clone ID: V3LHS_376728 and V3LHS_376730), ChREBP shRNA1 (clone ID: V3LHS_334248 and V3LHS_334249) and ChREBP shRNA2 (clone ID: V3LHS_334249 and V3LHS_313472). HFtelo cells stably expressing the indicated shRNAs were generated as described previously 14C] acetate (2 µCi/ml) and incubated at 37°C for 3 hr. Labeled cells were washed three times with ice-cold PBS and lysed in 0.3 ml of Triton X-100 (0.5% in H2O). Lipids were extracted by sequentially adding 0.75 ml of methanol, 0.75 ml of chloroform, 0.75 ml of chloroform, and 0.75 ml of H2O, with vortexing. Samples were centrifuged at 4,000 rpm for 15 min; 1.2 ml of organic phase (lower phase) was recovered and counted in a scintillation counter.Lipid synthesis was measured as described The mouse wild type viperin cDNA construct of was generated by PCR amplification and then cloned into pMXs-IRES-Thy1.1. Chimeric viperin constructs were also generated in which residues 1–42 of WT viperin, WT viperin-GFP or mutant viperin  were replaced by the residues 1–34  of vMIA and then cloned into pMXs-IRES-Thy1.1 to yield MLS-viperin, MLS-viperin-GFP or MLS-viperin (DCA), respectively. For the chimeric viperin construct Tom70-MLS-viperin, residues 1–42 of WT viperin were replaced by the MLS (residues 35–68) of human Tom70 and cloned into pMXs-IRES-Thy1.1. EGFP was also fused to residues 1–34 of vMIA (MLS-GFP) or residues 35–68 of Tom70 (Tom70-MLS-GFP). Plasmids were electroporated into viperin, GLUT4 and ChREBP knockdown HFtelo cells and TFP β subunit (HADHB) deficient HFF cells using a Nucleofector kit (Lonza). At 24 hr after transfection, the transfected cells were sorted by Streptavidin Microbeads (MACS Miltenyi Biotec) according to the manufacturer's instructions. The sorted cells were used for quantitative RT-PCR to measure the mRNA levels of lipogenic enzymes.Lipid droplets (LDs) were monitored by immunofluorescence. The HCMV-infected or transfected fibroblasts were grown in 24-well tissue culture plates containing a 13-mm-diameter coverslip. The coverslips were harvested by first washing the cells with PBS and then fixing the cells for 45 min at room temperature in 3% paraformaldehyde freshly prepared in PBS. The coverslips were washed in PBS and permeabilized with 0.5% saponin in PBS for 10 min. The coverslips were then blocked with 0.2% Tween in PBS containing 10% normal goat serum for 20 min at room temperature, followed by the addition of the anti ADRP mAb, and incubated for 1 hr at room temperature. Following washing with 0.2% Tween in PBS, the coverslips were incubated with goat anti-mouse Ig secondary Ab conjugated to dye diluted in 0.2% Tween in PBS containing 2.5% normal goat serum for 1 hr at room temperature. The coverslips were washed three times, rinsed once in PBS and mounted with ProLong Gold Antifade reagent (Molecular Probes). The images were acquired with a Leica TCS SP2 confocal microscope.en bloc stained with 2% uranyl acetate, dehydrated, infiltrated, and embedded in Epon. Sixty nanometer sections were stained with lead citrate and examined using a Tecnai 12 Biotwin electron microscope.HFtelo cells stably expressing the indicated shRNAs were infected with HCMV or recombinant HCMV.mVIP at an moi of 2 and examined by electron microscopy at 7 dpi as described previously g for 3 min. Supernatants were collected and boiled in reducing sample buffer. The supernatants were separated on 8% SDS-PAGE gels and transferred to PVDF membranes (Millipore). The immunoblots were blocked in 5% skim milk, 0.05% Tween in PBS and incubated with mAb against SREBP1, probed with anti-mouse Ig horseradish-conjugated secondary antibody, followed by incubation with enhanced chemiluminescence (ECL) reagents (Pierce).Cleavage of SREBP1 was assayed by immunoblot as described HCMV proteins were detected by immunoblotting. Cell pellets were lysed in 1% Triton X-100 in TBS containing proteinase inhibitors. Supernatants of lysates were collected and mixed with reducing sample buffer. The supernatants were separated on 8% or 10% SDS-PAGE gels. The immunoblots were probed with the indicated antibodies as described above.GCTCCGGCATGTGCAA (Fwd) and AGGATCTTCATGAGGTAGT (Rev); GLUT4, GGAGCTGGTGTGGTCAACACA (Fwd) and GGAGCAGAGCCACAGTCATCA (Rev); ACL, TGTAACAGAGCCAGGAACCC (Fwd) and CTGTACCCCAGTGGCTGTTT (Rev); ACC2, GACCACAGGTGAAGCTGAGA (Fwd) and GTGTTCCCGTCCCCTCTTC (Rev); FAS, AGGCTGAGACGGAGGCCATA (Fwd) and AAAGCTCAGCTCCTGGCGGT (Rev); DGAT1, TCGCCTGCAGGATTCTTTAT (Fwd) and GCATCACCACACACCAGTTC (Rev); DGAT2, TCACCTGGCTCAATAGGTCCA (Fwd) and CCAGCAATCAGTGCAGAATATG (Rev); ChREBPα, AGTGCTTGAGCCTGGCCTAC (Fwd) and TTGTTCAGGCGGATCTTGTC (Rev); ChREBPβ, AGCGGATTCCAGGTGAGG (Fwd) and TTGTTCAGGCGGATCTTGTC (Rev). The Q-PCR was performed in triplicate for each sample. Quantitation was performed by the comparative Ct (2–[delta][delta]Ct) method. The Ct value for each sample was normalized by the value for β actin gene. Three independent experiments were analyzed statistically for differences in the mean values, and P values are indicated in the figures.Cells were collected and total RNA extracted using the RNeasy Mini kit (Qiagen). cDNA synthesis was performed with 1–2 µg RNA using AffinityScript Multi Temperature cDNA synthesis kit according to the manufacturer's instructions (Stratagen). The cDNA obtained from cells was quantified by real-time quantitative PCR (Q-PCR) using SYBR Green (Applied Biosystems) on Stratagene Mx3000P QPCR system. The following primers were used; β actin, UL55 gene were used; ACGTGAAGGAATCGCCAGGA (Fwd) and AGTTCCAGTACCCTGAAGTC (Rev).The viral genome copy number was also quantified by real-time PCR as described previously HFFs or HFtelo cells stably expressing the indicated shRNA were infected with HCMV or recombinant HCMV (HCMV.mVIP) at an moi of 0.2 for 2 hr, washed, and cultured in normal medium or medium supplemented with doxycycline. Supernatants and cells were harvested at 6 dpi, and virus yield was measured by a fluorescence-based virus infectivity assay t test using GraphPad Prism 4 software. P values less than 0.05 were considered significant.Results from all studies were compared with unpaired two-tailed Student's Figure S1A) Viperin knockdown efficiency was analyzed by immunoblot using anti-viperin antibody (MaP.VIP). Grp94 served as a protein loading control. (B) Glycolysis. At 1 dpi, the cells were transferred to glucose and glutamine-free medium, incubated at 37° for 1 hr and glycolysis assessed by measuring the extracellular acidification rate (ECAR), using an XF Extracellular Flux Analyzer (Seahorse Bioscience). Data are presented as mean ± SEM of duplicate samples and are representative of two individual experiments.Viperin expression is necessary for HCMV-induced modulation of metabolism. HFtelo cells stably expressing no shRNA, control luciferase (Luc) shRNA or viperin shRNAs were infected with HCMV at an moi of 2 for the indicated days. ((TIF)Click here for additional data file.Figure S2de novo GLUT4 and ChREBP-mediated lipogenesis during HCMV infection. (A) Whole cell extracts were prepared from the infected cells. SREBP1 was detected by immunoblot using a specific antibody (IgG-2A4). Actin served as a protein-loading control. SREBP1-P, SREBP1 precursor; SREBP1-M, cleaved mature form of SREBP1. (B) HFtelo cells stably expressing two distinct GLUT4 shRNAs or ChREBP shRNAs were infected with HCMV at m.o.i. of 2 for 3 day. The knockdown efficiency was analyzed by RT-PCR. Actin served as a loading control. (C) GLUT4, ChREBP and lipogenic enzyme mRNA levels in HFtelo cells expressing luciferase (Luc), GLUT4 or ChREBP shRNAs after HCMV infection at an moi of 2 for 1 or 3 days. Total RNA was isolated, and the mRNA levels were measured by quantitative RT-PCR and normalized to β actin mRNA. Data are presented as means ± SEM of triplicate samples and are representative of two individual experiments. GLUT4, glucose transporter 4; FAS, fatty acid synthase; DGAT1, diacylglycerol acyltransferases 1. (D) MRC5 fibroblasts were infected with wild type RVHB5 strain of HCMV (HCMV) or mutant RVHB5 lacking vMIA (ΔvMIA-HCMV) at m.o.i. of 2 for 1 day in the presence of the caspase inhibitor ZVAD-FMK (50 µM). Cells were stained with anti-viperin (MaP.VIP) and anti-calnexin (ER marker) antibodies, antibodies to the HCMV protein pp65 to identify infected cells, and Mitotracker Red to visualize mitochondria. Scale bar, 10 µm. (E) MRC5 fibroblasts were infected with HCMV or ΔvMIA-HCMV at m.o.i. of 2 for 1 or 3 days in the absence and presence of ZVAD-FMCK5 (50 µM). Total RNA was isolated, and the mRNA levels were measured by quantitative RT-PCR and normalized to β actin mRNA. Data are presented as means ± SEM of triplicate samples and are representative of two individual experiments.Mitochondrial viperin induces (TIF)Click here for additional data file.Figure S3de novo lipogenesis. (A) The N-terminal 42 residue α-helical region of the mouse viperin-GFP chimera was replaced by the MLS of the HCMV protein vMIA. The MLS was also directly fused to enhanced green fluorescent protein (EGFP) as a negative control (MLS-GFP). ChREBP localization in viperin knockdown HFtelo cells transiently expressing the indicated chimeric viperin proteins. Cells were stained with antibody specific to ChREBP. The filled arrows indicate ChREBP localized in the nucleus, and the open arrows indicate ChREBP localized in the cytoplasm. Scale bar, 20 µm. (B) The N-terminal 42 residue α-helical region of mouse viperin was replaced by the mitochondrial localization sequence (MLS) of Tom70, a host cellular mitochondrial protein. mRNA levels of lipogenic enzymes in viperin knockdown HFtelo cells transiently expressing the indicated chimeric viperin proteins. Data are represented as means ± SEM of triplicate samples and are representative of two individual experiments. *, P<0.05. (C) mRNA levels of GLUT4, ChREBP and lipogenic enzymes in GLUT4 or ChREBP knockdown HFtelo cells transiently expressing the indicated chimeric viperin proteins. The mRNA level in the cells expressing a control vector was set at 1. Data are represented as means ± SEM of triplicate samples and are representative of two individual experiments. *, P<0.05.Targeting viperin to mitochondria induces (TIF)Click here for additional data file.Figure S4A) mRNA levels of lipogenic enzymes in viperin knockout MEFs (MEF-Viperin KO) transiently expressing the indicated chimeric viperin proteins. The mRNA level in the cells expressing a control vector was set at 1. Data are represented as means ± SEM of triplicate samples and are representative of two individual experiments. *, P<0.005. (B) LDs were monitored in viperin knockout MEFs transiently expressing the indicated chimeric viperin proteins. Cells were stained co-stained with ADRP and viperin antibodies. The filled arrows indicate the accumulated LDs in the transfected cells. The open arrows indicate the small basal LDs in the non-transfected or transfected cells. Scale bar, 20 µm. (C) Total lipid synthesis. Viperin knockout MEFs transiently expressing the indicated chimeric viperin proteins were labeled with [14C]-acetate for 3 hr. Total lipids from 5×104 cells were extracted and [14C] incorporation assessed. Data are presented as mean ± SEM of triplicate samples and are representative of two individual experiments. *, P<0.001.Targeting viperin to mitochondria leads to increased lipogenesis in viperin knockout MEFs. The N-terminal 42 residue α-helical region of mouse viperin (WT), a mouse viperin-GFP chimera, or viperin (DCA) was replaced by the mitochondrial localization sequence (MLS) of the HCMV protein vMIA. The MLS was also directly fused to enhanced green fluorescent protein (EGFP) as a negative control (MLS-GFP). ((TIF)Click here for additional data file.Figure S5P<0.05.Viperin interaction with TFP is responsible for increased lipogenic enzyme gene expression. mRNA levels of lipogenic enzymes in TFP β subunit deficient (HADHB (−)) cells transiently expressing the indicated chimeric viperin proteins were measured as described above. Data are represented as means ± SEM of triplicate samples and are representative of two individual experiments. *, (TIF)Click here for additional data file.Figure S6Specificity of the viperin effects on formation of HCMV viral envelope. Transmission electron micrographs of recombinant HCMV.mVIP-infected HFtelo cells stably expressing the indicated shRNAs. Monolayers were infected with recombinant HCMV.mVIP at an moi of 2 and processed for EM at 7 dpi. Dox (2 µg/ml) was added on day 0 and 3. Multiple frames from each sample were imaged and photographed. Particles from a representative cell are shown. White and black arrows indicate non-enveloped particles and enveloped particles, respectively. Cyto, cytoplasm. Scale bar, 1 µm. The numbers of enveloped and non-enveloped particles were counted in each frame and calculated as a ratio of enveloped particles to total particles in the cytoplasm of infected cells. The graphs indicate the mean percent of the enveloped particles per total 10–30 particles in each frame (from >10 cells of each sample) ± SEM.(TIF)Click here for additional data file.


Most children who are diagnosed with attention deficit-hyperactivity disorder (ADHD) have moderate-to-severe motor problems using the Motor Function Neurological Assessment battery (MFNU). The MFNU focuses on specific muscle adjustment problems associated with ADHD, especially motor inhibition problems and high muscle tone. Here we investigated whether adults with ADHD/hyperkinetic disorder (HKD) have similar motor problems. In our clinical experience, adults with ADHD often complain about back, shoulder, hip, and leg pain. We also investigate reported pain in adults with ADHD.Twenty-five adult outpatients diagnosed with ADHD/HKD who were responders to methylphenidate (MPH) were compared to 23 non-ADHD controls on 16 MFNU subtests and using a ‘total score’ (‘TS’) parameter. The MFNU test leader was blinded to group identity. The two groups were also compared using the Pain Drawing and Numerical Pain Rating Scale.The adult ADHD group had significantly (p < .001) more motor problems (higher TS) than controls. On the muscle regulation subtests, 36–96% of the ADHD group showed ‘moderate’ to ‘severe’ problems compared to 13–52% of the control group, and 80% of the ADHD group reported widespread pain. Highly significant differences were found between the ADHD and control groups for the variables ‘pain level’ (p < .001) and ‘pain location’ (p < .001). Significant correlations were found between TS and ‘pain location’ and between TS and ‘pain level’.These findings suggest that similar to children with ADHD, adults diagnosed with ADHD also have motor inhibition problems and heightened muscle tone. The presence of significantly higher pain levels and more widespread pain in the ADHD group compared to non-ADHD controls might indicate that pain is a long-term secondary effect of heightened muscle tone and restricted movement that can be demonstrated in children and adults by the MFNU battery. Prevalence of ADHD among children and adolescents is estimated to 5,3% worldwide -5. SixtyUsing the Motor Function Neurological Assessment (MFNU)  moderateDisinhibition in children with ADHD is present not only in higher order executive functions like motor planning, timing, and evaluation, but also seems to be involved in more basic motor functions. Stray et al.  demonstrThese results challenge the current belief that motor problems in ADHD are either side effects or symptoms of inattention [the differential section for DCD 12,  or a sepIn our clinical practice, adult patients with ADHD often report skeletal muscle pain and physical discomfort . As a peThe aims of the present study were to investigate whether adults with ADHD exhibit the motor function problems demonstrated in children with ADHD and to investigate whether such motor problems were related to the presence of reported pain.Our research questions and hypotheses were as follows:1. What are the differences in motor problems in adults with ADHD as compared to non-ADHD controls as measured by the MFNU?2. Do adults with ADHD report a) more widespread pain or b) higher levels of pain than non-ADHD controls?We hypothesized that adults with ADHD would display consistently higher scores and show significantly more motor problems on all of the MNFU subtests used in this study as compared to non-ADHD controls. We also hypothesized that adults with ADHD would report more widespread pain and higher levels of pain than adults in the non-ADHD control group.Verbal and written information about the project was given to outpatients at the Addiction Unit, Sørlandet Hospital, who were diagnosed with HKD F90.0/ADHD, had no active substance abuse, and who were positive responders to MPH. Affirmative written replies were received from 28 patients. Three of these were excluded, one for medical reasons, one who could not be taken off MPH medication due to work, and one for substance abuse. Thus the ADHD group consisted of 25 subjects with a mean age of 33 years (SD 8.9), range 20–51 years. The group included 14 men with a mean age of 34 years (SD 9.5), range 20–51 years, and 11 women with a mean age of 32 years (SD 8.3), range 20–47 years. Three subjects were university students, 12 had regular work, and 10 received social security. The patients medicated with MPH were taken off medication a least one day before the motor assessment.The non-ADHD controls group consisted of students from the University of Agder, employees from a rehabilitation department at Sørlandet Hospital HF, and employees from a care home for the intellectually challenged in Kristiansand. Non-ADHD controls were recruited by posting flyers on bulletin boards and by study personnel and student leaders through verbal communication and written information about the project. Written affirmative answers were received from 29 subjects. To exclude possible ADHD problems in the control group, the participants were rated using the ‘M.I.N.I. Plus’ module W interview . People The current version of the MFNU consists of 16 subtests that are described briefly in Table All 16 subtests were scored by the tester or by an independent observer according to three scoring categories as described in Table Subtests 1–10 are performance tests in which the person who is being tested is instructed to perform a particular task. ‘Subtest 16: Synkinesis’ evaluates the presence of overflow movements during the MFNU assessment. Four subtests (12–15) are subtests of passive movements of the hips and feet in which the tester evaluates muscular resistance. ‘Subtest 11: Palpation’ provides important physical/proprioceptive information about muscular consistency and possible high muscle tone in muscles such as the sacrospinalis and the latissimus dorsi and about restricted movements of the thorax [A reliability study of the MFNU showed high internal consistency within the total set of 17 subtests  and a high rater agreement with an average ICC of .99  . SubtestThe Pain Drawing procedure, which is commonly used in studies of pain -48, was The intensity of the pain experienced during the previous two weeks was rated by each participant on an 11-point Numerical Pain Scale (NRS) that ranged from 0 (“No pain”) to 10 (“Pain as bad as it could be”) . The NRSThis study used the Mini-International Neuropsychiatric Interview (M.I.N.I.) version 5.0.0 with the attention deficit/hyperactivity disorder module W in the M.I.N.I.-Plus. The M.I.N.I. is an short psychiatric interview for assessing psychiatric disorders according to the DSM-IV and ICD-10 classification systems . The M.IEach participant was assessed using the MFNU, Pain Drawing, and NRS. Possible ADHD symptoms in the control group were addressed by a M.I.N.I plus interview conducted by a physician or a social educator with many years of clinical and research experience in psychiatric assessment of patients with ADHD. The MNFU test leader, an experienced physiotherapist with no prior personal or professional knowledge of the subjects in either of the groups, was blinded to which group the participant was in. The assessments of the Pain Drawing, NRS, and the M.I.N.I. interview were performed prior to the MFNU assessment. The MFNU test leader did not know the results of these assessments.The statistical analyses were carried out using PASW (SPSS) Statistics, version 18 for Windows . Descriptive statistical analyses were performed on data from each subtest in order to view the distribution of the participants (%) in the scoring categories  and for the TS variable. As the samples and the measurements did not satisfy the conditions for parametric tests, the non-parametric Mann–Whitney U-test was used to compare the ranked scores of the ADHD group and control group on each of the 16 subtests. The Mann–Whitney U-test was used to compare the TS and the ‘Pain level’ of the ADHD group and control group for the same reason. Pearson’s Chi-square test was used to compare frequencies in the groups for the categorical variable ‘Pain location’. Spearman’s rho was run for all participants as a whole for finding the correlation between the TS of the MFNU and the ‘Pain level’ and between the TS and ‘Pain location’.The study was approved by The Norwegian Data Inspectorate, The National Committee for Medical Research Ethics in Norway, 2011/1947 D.The ADHD group showed a high percentage of ‘severe problems’ (a score of 2) on many of the subtests: ‘Palpation’, ‘Passive abduction of the hip’ and ‘Passive movement of the feet’, ‘Lifting arm’, ‘Lifting leg’, ‘Diadochokinesis of the left arm’, and ‘Reciprocal coordination’. The control group typically had few problems on any of the subtests Table . There wThe Mann–Whitney U-tests Table  showed tThe Pearson’s Chi-Square test showed significant differences between the ADHD group and the control group for the variable ‘Pain location’ (P < .001). Widespread pain was reported by 80% of the ADHD group compared to 17.4% of the control group. Only 8% of the ADHD group reported having no pain in the previous two weeks compared to 34.8% of the controls. There were no significant gender differences regarding pain level or pain location in either of the groups (Table The Mann–Whitney U-tests showed a significant difference in self-reported pain (‘Pain level’) between the ADHD group  and the control group  . The mean ‘Pain level’ for the ADHD group was 4.76, SD = 2.16, and the mean ‘Pain level’ for the control group was 1.96, SD = 2.27. The Cohen’s d for ‘Pain level’ was 1.26, which is a large effect size. There was a significant correlation (p < .01) between the TS on the MFNU and ‘Pain level’ and between the TS and ‘Pain location’ (p < .05) (Spearman's rho).We hypothesised that adults with ADHD would have more problems than controls in regulating movement and muscle tone as assessed by the MFNU. This hypothesis was supported by the results on all subtests except the ‘Flying’ subtest. These results strongly suggest that muscle regulation problems are very similar in adults with ADHD as in children with ADHD. Most of the ADHD subjects had a moderate to severe TS, indicating that just as in children, the adults had motor problems that affected many facets of movement and muscle control. Comparison of the results for the adult ADHD group to results for 8–12-year-old boys showed minor differences in two subtests. The results on subtest 16, ‘Synkinesis’, and subtest 07, ‘Walking’ indicated that adults with ADHD may be less affected by overflow movement (synkinesis) than children (see ). OverflMotor problems in ADHD have been seen as being co-effects or a motor consequence of inattention and impulsivity . This viIt is often argued that motor problems in ADHD that are not explained by inattention are comorbid symptoms of Developmental Coordination Disorder (DCD) ,58, whicRecent research has focused on the frontostriatal system and basal ganglia ,61. Not As we hypothesized, the ADHD group reported significantly more widespread pain (both in the upper and lower body) and a higher pain level than the control group. To our knowledge this has not been assessed before. The highly significant correlation between the TS and the pain level score might suggest that the pain reported in the ADHD group is directly linked to motor problem severity and that such pain might be an indirect long-term consequence of the muscle tone dysregulation and motor inhibition that is associated with the ADHD condition. A history of childhood ADHD, assessed with the DSM-IV criteria, has been shown to be more common in the patients diagnosed with Fibromyalgia compared to normal controls (32.3% vs. 2.52%) . Such a In this study, the control group had a higher mean age (41 years) than the ADHD group (32 years). This might have influenced the results, although it seems unlikely that there is an inherent reason why older people would be expected to perform better on the MFNU or have less pain than the younger people in the ADHD group. Significant differences were found on the same subtests using age matched participants. The subjects diagnosed with ADHD who were included in this study were all responders to MPH for their ADHD symptoms. This implies that our conclusions cannot be generalized to the broader ADHD population. The individuals in the ADHD group had an earlier history of addiction problems. Even if substance abuse was controlled for in this study, the confounding effects of residual pain or secondary health problems due to prior substance abuse cannot be ruled out. Further research involving a sample of adults without prior substance abuse is needed to clarify this issue.This study demonstrated that MPH-responsive adults with ADHD show the same functional motor problems as children with ADHD as measured with the MFNU. The motor problems were associated with heightened muscle tone in gross movement muscles and with specific motor inhibition problems that resulted in restricted movement and instability. The MFNU was able to effectively discriminate between adults with ADHD and non-ADHD controls. The ADHD group also differed significantly from the control group in terms of reported pain in that the ADHD subjects reported more widespread pain and higher levels of pain than the controls. The ADHD group showed high correspondence between reported muscular pain and TS on the MFNU. These findings suggest that the demonstrated functional motor problems are important contributors to the very high incidence of reported pain in the ADHD group and possibly to the substance abuse and addiction problems that are common in people with ADHD. Further research validation with different methods is needed to confirm these conclusions.Written informed consent was obtained from the patient for publication of this report.The MFNU manual is sold by the Reading Centre, University of Stavanger, for NOK 700 (about €85). LLS receives 9% royalties and TS receives 3% royalties from the sale of the MFNU.LLS was the primary contributor to the study conception and design and was mainly responsible for data acquisition, analysis, and interpretation. LLS was the main contributor to drafting of the manuscript. ØK contributed to the study conception and design, helped interpret the data, and helped draft the manuscript. ML contributed to the study conception and design and helped acquire the data. MS helped acquire the data. TS contributed to data analysis and helped draft the manuscript. FET contributed to data analysis and helped draft the manuscript. All authors read and approved the final manuscript.


Attention-deficit/hyperactivity disorder (ADHD) has commonly been described in psychiatric disorders. Although several studies have found positive associations between abnormal eating patterns during childhood and ADHD, there is a lack of studies on ADHD and Eating Disorders (ED). The aims of this exploratory study were 1) to assess the ADHD symptoms level in ED and to ascertain whether there are differences among ED subtypes; 2) to analyze whether the presence of ADHD symptoms is associated with more severe eating disorder symptoms and greater general psychopathology; and 3) to assess whether the ADHD symptoms level is associated with specific temperament and character traits.191 female ED patients were included. Assessment was carried out with the EDI-2, ASRS-v1.1, the SCL-90-R and the TCI-R.The ADHD symptoms level was similar in bulimia, eating disorder not otherwise specified and binge eating subtypes, and lower in anorexic patients. Obsessiveness and Hostility were significantly positively associated with ADHD symptoms. A path model showed that ADHD was associated with high Novelty Seeking and low Self-Directedness, whereas ED severity was influenced by ADHD severity and low Self-Directedness.Bingeing/purging ED subtypes have a high ADHD symptoms level, also related with more severe eating, general and personality psychopathology. Attention-deficit/hyperactivity disorder (ADHD) has its onset during childhood and is characterized by symptoms of impulsivity, hyperactivity and inattention . DespiteADHD has commonly been described in several psychiatric disorders, including affective disorders, substance abuse and impulse control disorders , as wellFew studies have analyzed the relationship between ED and ADHD and found that the main symptoms of ADHD  are often present in ED patients ,15. The In view of this criticism, the present study had the following objectives: 1) to assess the current prevalence of ADHD symptoms in ED and to ascertain whether there are differences among ED subtypes; 2) to analyze whether the prevalence of ADHD symptoms is associated with more severe eating disorder symptoms and greater general psychopathology; and 3) to assess whether the prevalence of more ADHD symptoms is associated with specific temperament and character personality traits.SD = 9.5), were included. The AN group consisted of 21 AN restrictive subtype (AN-R), 10 AN binge/purging subtype (AN-BP) and 12 subthreshold AN . Patients were consecutively admitted to an outpatient treatment in our Institution and diagnosed according to DSM-IV-TR criteria by means of SCID-I , with a mean age of 28.3 years (f SCID-I . The stun = 14), as the number of males with these diagnoses was too small for meaningful comparison and b) participants with incomplete data in any questionnaire (n = 30), . Since the Adult ADHD Self-Report Scale only comprises 6 items, the missing of one item in this questionnaire was considered criterion of exclusion.For the present analysis, from an initial sample of 235 ED patients, the following cases were excluded: a) males  of the EDI and adds 27 new items into three provisional scales: Asceticism, Impulse Regulation, and Social Insecurity. All of these scales are answered on a 6-point Likert scale, and provide standardized subscale scores. This instrument was validated in a Spanish population  with a mASRS-v1.1 was used as a severity indicator of self-reported (current) ADHD symptoms in adulthood. ASRS-v1.1. comprises the 6 out of 18 most predictive items of the Adult ADHD Self-Report Scale (ASRS) . ASRS isThis test contains 90 items and helps measure 9 primary symptom dimensions: Somatization, Obsession-Compulsion, Interpersonal Sensitivity, Depression, Anxiety, Hostility, Phobic Anxiety, Paranoid Ideation, and Psychoticism. In addition, it includes three global indices, which are a global severity index (GSI), designed to measure overall psychological distress; a positive symptom distress index (PSDI), designed to measure the intensity of symptoms as well as a positive symptom total (PST), which are reports of self-reported symptoms. The Global Severity Index can be used as a summary of the test. This scale has been validated in a Spanish population , obtainiThe TCI-R  is a 240All patients were evaluated and diagnosed at the ED Unit of the Bellvitge Hospital. Two semi-structured face-to-face interviews were conducted by psychologists and psychiatrists of the Unit. The first one provided information about current eating disorder, antecedents and other data of interest about the patient. The second interview, with the SCID I , coveredData was analyzed with SPSS19 and STATA12 for Windows. First, the comparison of the total ADHD score between ED diagnosis subtypes was carried out with Analysis of Variance including age and BMI as covariates (ANCOVA).Next, multiple linear regressions also adjusted by age and BMI explored the associations between the total ADHD score (criterion) and six different set of predictors: eating disorders measures , EDI-2 scale scores, EDI-2 total score, general psychopathology by means of SCL-90-R scale scores and SCL-90-R global indices, and TCI-R personality scale scores. These analyses were carried out for the total sample as well as for the AN and [BN + EDNOS + BED] groups separately. Moreover, the measure for ADHD has been defined as the dependent variable and as incomes/predictors a large set of independent variables  has been included. This procedure allows the obtaining of the specific contribution of each scale adjusted to the presence of the others. But the adjustment of the multiple regressions was not possible considering the EDNOS and BED groups separately due the low sample size. AN subtype samples were also too small for meaningful comparisons. Anyway, the most prevalent subtype in the AN sample was the restrictive one .p > .05). Comparative Fit Index (CFI) was higher than 0.90, and Root Mean Square Error Approximation (RMSEA) was lower than 0.06 [Finally, based on the theoretical Cloninger Biosocial model of personality , that pohan 0.06 .Most of the patients were single (68.9%) and employed (73.3%), 21.5% reported primary studies, 47.5% secondary education, and 31% university studies. Table The ADHD questionnaire scores were statistically different between ED subtypes, with AN patients showing significantly lower ADHD scores (M = 9.30). Equal means were found for bulimia, EDNOS, and binge eating .Table Among general psychopathology (SCL-90-R), only the Obsessive-compulsive and Hostility subscales were significantly and positively associated with the ADHD questionnaire score, both in the total sample as well as in the BN + BED + EDNOS group. In the AN cohort, no significant associations were found between the ADHD total score and the SCL-90-R scores.Regarding to ED symptomatology (EDI-2), some significant associations with ADHD symptoms emerged for the total sample. First, the higher EDI-2 total score and EDI-2 Interoceptive awareness score, the higher the ADHD symptoms score. A negative association appeared between the EDI-2 Perfectionism score and the ADHD symptoms scale score. Considering the AN sample, a positive association was found between the EDI-2 total score and the ADHD scale score. Finally, for the BN + BED + EDNOS group a significant association appeared between the EDI-2 total score and the ADHD symptom scale score.Multiple regressions valuing the predictive accuracy of personality traits (TCI-R scores) on ADHD symptoms score (adjusted by age and BMI) for the total sample obtained that higher Novelty Seeking and lower Self-Directedness were related to higher ADHD scores. Lastly, in the AN subsample, Reward Dependence and Self-Directedness were negatively associated with ADHD symptoms, while Cooperativeness was positively associated  and the total predictive accuracy was very good (R2 = 0.585). Results of this model indicated that high scores on Novelty Seeking increase the ADHD symptoms score, while high scores on Self-Directedness decrease ADHD and EDI-2-total scores. High scores on ADHD also are positive associated with the EDI-2-total scale score. A partial mediational path was observed for Self-Directedness, ADHD, and EDI-2-total scores. A complete mediational path emerged between Novelty Seeking, ADHD, and EDI-2-total scores: a high score on Novelty Seeking increases ADHD, and this last score is also positively associated with EDI-2-total.One structural model was generated to explain the paths between the EDI-2, SCL90-R and TCI-R scales, and ADHD total score jointly. This model included the TCI-R Novelty Seeking and Self-Directedness scales and age as predictors, and the total ADHD score and the EDI-2 total scale as outcomes Figure . Age wasThe current study assessed the ADHD symptoms level in ED patients and ascertained differences among ED subtypes. Moreover, we evaluated the association among the severity of ADHD symptoms and eating disorder symptomatology, general psychopathology and personality.Our findings support the hypothesis that the ADHD symptoms level in ED patients differs between subtypes. In accordance with previous studies ,15, our Our second hypothesis, that the severity of ADHD symptoms is associated with severity in ED symptoms and greater general psychopathology, was supported by our findings. Our results showed a positive association between the frequency of binge eating episodes and ADHD symptoms in the total sample, as well as in the BN, BED and EDNOS group. These findings are in agreement with previous research  and suggRegarding eating symptomatology, our results showed associations between ADHD symptoms and EDI-2 scores. In the total sample, a positive association was found between the EDI-2 total score, the Interoceptive awareness subscale and ADHD symptomatology, which may suggest that an altered Interoceptive awareness combined with the EDI-2 total and with a poorer self directive personality, increases comorbid psychopathology, which is also associated with ADHD symptoms .Another emergent finding was that perfectionism and ADHD symptomatology presented a negative association, i.e. the lower the perfectionism of a subject, the higher the number of ADHD symptoms. This finding goes along with the ED literature, since a perfectionism pattern and a disposition towards over-control are much more related to restrictive ED behaviors such as presented in anorexia, than the binge eating/purging conducts presented in binge eating/purging EDs .Additionally, previous studies in ADHD literature reported that obsessive-compulsive and hostility are frequently found in ADHD patients ,36. CorrAs we know from research, adult ADHD patients share the same spectrum of personality traits as ED patients. Subjects meeting the ADHD diagnosis showed, in comparison with community subjects, higher scores on Neuroticism-Anxiety, Impulsive-Sensation Seeking and Aggression-Hostility in the Zuckerman Kuhlman Personality Questionnaire (ZKPQ) . Our resIn the AN group, the ADHD symptomatology was associated with high Cooperativeness and lower Reward Dependence and Self-Directedness. This personality profile has been described in the literature as the "maladaptative" profile and generally presented with the highest values for ED symptomatology and impulsive behaviors .The present study should be evaluated within the context of several limitations. First, only female patients were included, so future studies should also considerer male ED patients. Second, compared to the binge/purge ED group, the AN group was rather small, which could have hidden some interesting findings. Further studies should include more AN patients to compare both the restrictive versus the binge/purging subtypes. Thirdly, this study is limited by the lack of information regarding psychiatric co-morbidity . Finally, we assessed the frequency of adult ADHD symptoms by means of a self-rating screening instrument, which might contribute to an overestimation of ADHD symptoms and might explain the elevated prevalence of ADHD symptomatology in the study sample. In the same way, the presence of childhood ADHD was not assessed, which would have been a prerequisite for an ADHD diagnosis in adulthood. Then future studies focusing on the prevalence of ADHD, rather than the frequency of its symptomatology, should be conducted in ED patients. Personality functioning was also based on self-report measures. Thus, future studies might also include performance-based or neuropsychological tasks (i.e. Stroop task or CPT-II test) in order to shed some light on the impulsive behaviour characterizing both binge/purge ED patients and ADHD patients.We added to the limited literature on ADHD in the traditional eating disorders and developed a path model to describe the associations between socio-demographic characteristics, personality, ADHD and ED psychopathology. Based on the findings of our path model, we can conclude that both ADHD and ED symptoms  are driven by high levels of novelty seeking and low levels of effortful control. Intervention strategies that focus on the training of effortful control – certainly under rewarding/novel conditions – can be helpful to learn both ED and ADHD patients to increase response inhibition control, decision making abilities and after all to display a higher capacity of planning their behaviour ,50.ADHD: Attention-deficit/hyperactivity disorder; ED: Eating disorders; BMI: Body mass index; EDI-2: Eating disorders inventory-2; SCL-90-R: Symptom checklist- revised; ASRS-v1.1: Adult ADHD self-report scale; TCI-R: Temperament and character inventory-revised; BED: Binge eating disorder; BN: Bulimia nervosa; AN: Anorexia nervosa; EDNOS: Eating disorders not otherwise specified; DSM-IV: Diagnostic and statistical manual of mental disorders 4th editionAll authors declare that they have no conflicts of interest.FFA, ZA, SJM, JARQ, JMM and MC designed the study. ZA, RB, IS and NR collected the patient data. RG and EP performed the statistical analyses. FFA, ZA, RC, SJM, ABF, JARQ, RB, LC and MC wrote the first draft of the manuscript. All authors commented on and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-244X/13/166/prepub


Symptoms of hyperactivity are believed to fade with age leaving ADHD adults mostly inattentive and impulsive. Our aim was to test this assertion using objective measures of hyperactivity, impulsivity and inattention.Participants were 40 subjects with ADHD  and 60 healthy adults  blindly assessed using Wender-Reimherr interview ratings, Structured Clinical Interview for DSM-IV Disorders and DSM-IV criteria. Infrared motion capture systems tracked head and leg movements during performance of a No-4’s cognitive control task. Subjects also completed the Conners’ CPT-II.ADHD and controls differed significantly in activity and attention. Effect sizes for activity measures (d’ = 0.7–1.6) were, on average, two-fold larger than differences in attention or impulsivity, correlated more strongly with executive function ratings and were more discriminatory . This finding was true for subjects with the predominantly inattentive subtype as well as subjects with combined or predominantly hyperactive/impulsive subtype. Males and females with ADHD were equally active. The superior accuracy of activity measures was confirmed using Random Forest and predictive modeling techniques.Objectively measured hyperactivity persists in adults with ADHD and is a more discriminative feature of the disorder than computerized measures of inattention or impulsivity. This finding supports the hypothesis that a deficient ability to sit still remains a defining feature of the disorder in adults when it is measured objectively. Attention-Deficit/Hyperactivity Disorder (ADHD) is defined by signs of inattention, hyperactivity and impulsivity. HoweverHyperactivity is a sign rather than a symptom and can The aim of this study was to compare head and lower extremity movements of adults with ADHD to healthy controls. If hyperactivity abates then there should be little difference between adults with ADHD and healthy adult controls. If hyperactivity fades to a disproportionately greater degree then differences between ADHD and controls on motion measures should be less robust than differences in attention.Subjects were 100 adults in good physical health, between 18–57 years of age, recruited from the community by advertisements; with a primary diagnosis of ADHD, or were healthy unmedicated controls without psychiatric disorders. Exclusion criteria included any use of drugs, herbal remedies, or non-ADHD medications (except contraceptives) for at least 2 weeks prior to enrollment. Subjects receiving treatment for ADHD were excluded if treated with anything other than short-acting stimulants. Further, they needed to be willing to stop stimulants for at least 24 hours prior to each visit. Additional exclusion criteria included a history of any major medical or neurological disorder that could affect motor activity or attention; major depression, bipolar disorder or anxiety disorders within the past 6 months; or any past or present history of alcohol or substance abuse.This was a two-visit study approved by the McLean Hospital IRB. The study was explained on the first visit and written informed consent obtained from all participants. Diagnostic assessments were made using DSM-IV criteria and the Structured Clinical Interview for DSM-IV Disorders-I (SCID). HoweverTesting for capacity to sit-still and pay attention occurred during visits 1 and 2 (two tests per visit). Infrared motion capture occurred during performance of three different computerized attention tasks. Subjects also completed the Conners’ CPT-II attention task (Ver 5) without concomitant motion measures (as motion measures are not part of this test). The four attention tasks were administered in random order spread across the two test sessions.The three different attention tasks with motion capture were the No-4’s cognitive control task, the Cued Response task and the Embedded Memory task. Stimuli for all tasks consisted on 4, 5, 8 and 16-pointed stars, which were presented at random screen positions. In the No-4’s cognitive control task subjects responded to all stars except those with 4 points. This is similar in principal to the CPT-II, in which subjects respond to all letters except X, and was a high target density task (~90% targets). In the Cued Response task subjects responded to 8-pointed stars, but only when a 5-pointed star immediately preceded them. This is similar to the AX-CPT, and wasActivity results were similar across all three motion capture/attention tasks. Results from only one of the tasks (the No-4’s cognitive control test that is available as part of the Quotient ADHD System) will be presented along with CPT-II results. The No-4’s task was selected for commercial development as its high target density facilitated analysis of attention state in sequential 30-second epochs. The Cueactivity severity composite), designed to be a treatment responsive metric of hyperactivity.Two Qualisys ProLite infrared motion analysis cameras tracked head and lower extremity (shin and foot) movements . Foot movements were similar to shin movements, but were not captured as reliably – as foot markers were occasionally obscured from the camera’s view. Hence, we limited the analysis to head and shin measures. Movement patterns were analyzed using previously described procedures. The spadistraction severity composite), designed to be a treatment responsive metric of inattention, and a discriminative index that combined both activity and attention parameters and was optimized using logistic regression to distinguish adolescent and adult subjects with ADHD from controls. The most important predictors in the logistic regression were leg measures, head measures, attention shifts and errors of omission.Stimuli consisted of 16-, 8-, 5 -and 4-pointed stars presented singly at random screen positions for 240 msec with a variable interstimulus interval (ISI) (mean 2500 msec). Subjects were instructed to press the spacebar for all stars except for those with 4 points. Ninety-percent of the stimuli were targets. Reported measures were overall accuracy, errors of omission (EOM), errors of commission (EOC), correct response latency, standard deviation of response latency, coefficient of variation (COV) in response latency, and measures of fluctuation in attentional state. For thiconfidence index composite, based on discriminant analysis, which indicates the likelihood that an individual has an actual problem with attention. The most important predictors in the discriminant function were percent omissions, gender, age, beta, and RT across ISI.Stimuli consisted of letters presented at the center of the computer screen for 250 msec with an ISI of 1000, 2000 or 4000 msec (3 levels). The test consisted of 6 blocks made up of 3 sub-blocks – one sub-block for each ISI. Subjects were instructed to press the spacebar for all letters except X. Reported measures were EOM, EOC, reaction time (RT) for correct responses, standard error in correct RT, variability in RT across sub-blocks, d-prime (capacity to distinguish targets from non-targets), beta (response bias), perseveration (RTs less than 100 msec), and measures of change in correct RT and RT standard error across blocks and ISI levels. The Connor’s CPT-II provides a activity severity composite, distraction severity composite, discriminative index and CPT-II confidence index) to discriminate between ADHD subjects and controls in the sample. Third, Random forest regression was used as a novel analytic strategy to indicate the relative importance of each measure in discriminating the present sample of subjects with ADHD from controls. This technique is used in data mining and provides new measures of variable importance that are effective in delineating the most meaningful variables even in the presence of multicollinearity. Fourth, modeling techniques with cross-validation were used to assess the capacity of activity versus attention measures to discriminate subjects with ADHD from controls in a way that would likely generalize beyond the studied sample. Statistical analyses were conducted using R[The aim of statistical analyses was to first ascertain whether there were differences between subjects with ADHD and controls in objective measures of activity, and if so, to test the hypothesis that attention measures were more significant and better able to discriminate between groups than objective measures of activity. Four different approaches, of increasing sophistication and complexity, were used to test this hypothesis. First, analysis of covariance (ANCOVA) was used to test for group differences in the individual measures of activity and attention. Effect size and area under the Receiver Operating Characteristic curve (ROC-AUC) were calculated for each covariate adjusted measure to provide a basis for comparison. Second, ROC analysis was used to compare the composite indices provided by each test .ANCOVA was used to assess main effects of group, age and sex on the dependent variables. Motion analysis data were also adjusted to account for occasional lost frames or reflections (mean loss - 18%). We used the false discovery rate method of Benjamini and Hochberg to minimDeLong’s test for two correlated ROC curves was usedCorrelation coefficients with confidence intervals were used to explore the relationship between composite indices of activity and attention and ratings of executive function on the Brown ADD scale.RandomForest 4.5-34) was used to indicate the relative importance of each measure in discriminating subjects with ADHD from controls. This technique is used in data mining where the goal is to identify the most important subset of variables in a large collection as, for example, in microarray studies. Random forest regression was developed by Breiman[Random forest regression  of a given predictor variable. The more the permutation of a variable degrades accuracy the greater the importance of the variable. The second measure of importance is the mean decrease in the Gini coefficient following permutation of a variable. The Gini coefficient is a measure of the inequality of a distribution. The better the nodes were at splitting the sample the higher the Gini coefficient. For this analysis 10,000 trees were generated with 4 variables tried per split. Results were compared with an alternative technique to minimize potential bias (age party). If hypcaret[The question whether activity measures were less effective than attention measures in discriminating subjects with ADHD from controls was also assessed using predictive modeling techniques (R package caret). A seriEach statistical model was generated using results from 75% of the subjects to build the model, which was then assessed for predictive accuracy on the remaining 25%. This process was repeated 200 times (5000 times for Random Forests) on different random splits of the data and the results average across all repetitions to yield a cross-validated estimate of the predictive discriminant ability of the measures that would likely generalize to new cases. VariablThe ADHD group consisted of 40 subjects 23M/17F) with a mean age of 35 ± 10 years. Only 2 of the subjects were receiving pharmacological treatment at the time of recruitment. Control subjects included 60 adults (28M/32F) with a mean age of 29 ± 9 years . Subjects with ADHD had, on average, 7.0 ± 1.7 DSM-IV defined symptoms of inattention and 5.9 ± 2.5 symptoms of hyperactivity-impulsivity. In contrast, controls had, on average, less than one symptom of inattention or hyperactivity-impulsivity. On the WRAAS[3M/17F wiCharacteristic movement patterns of male and female subjects with and without ADHD are illustrated in Figure The most robust differences in attention on the No-4’s test were on percent time spent fully attentive (d’ = 0.87), accuracy (d’ = 0.76) and response latency coefficient of variation . The remaining parameters, when significant, were associated with moderate to small effect sizes Table . MeasureOnly four of the Conners’ CPT-II measures showed a significant main effect of group after correcting for multiple comparisons. Differences between subjects with ADHD and controls on these few measures were associated with moderate to small effect sizes Table . The mosFigure The greater discriminative capacity of activity versus attention measures was not limited to subjects with combined or predominantly hyperactive/impulsive subtypes. ROC-AUCs for discriminating predominantly inattentive subtype ADHD from controls were only 0.62 (95% CI 0.48–0.77) and 0.53 (95% CI 0.38–0.67) for the CPT-II confidence index and Quotient distraction severity composite, respectively. In contrast, the activity severity composite discriminated predominantly inattentive subtype ADHD from controls with ROC-AUC of 0.81 (95% CI 0.71–0.92). Combining both activity and attention measures into the discriminative index provided excellent separation with an ROC-AUC of 0.90 (95% CI 0.78–1.00).cforest to generate conditional inference trees (r = 0.91).Figure r = 0.1 – 0.23) and moderate (r = 0.24 – 0.36) in effect size[r > 0.37). Including both activity and attention metrics into a discriminative index provided correlations coefficients of large size that explained between 27%-52% of the variance in the different executive function ratings.Table ect size. In cont-15) whether using accuracy, kappa or ROC-AUC as evaluative metrics.Table Contrary to conventional wisdom, adults with ADHD manifest clear signs of hyperactivity on objective assessment. Interestingly, effect sizes for ADHD versus control differences in individual activity measures were, on average, 2-fold greater than effect sizes for differences for No-4’s attention measures and 3-fold greater than CPT-II attention measures. These results are similar to findings in children with ADHD,13,28. AThis observation was not likely an artifact of recruiting an abnormally hyperactive sample of adults with ADHD. Clinical ratings of these subjects on the WRAAS showed that activity differences were significantly less robust than ratings of attention, or disorganization, as has been observed in previous adult samples. Further, activity measures remained superior to attention measures in discriminating even predominantly inattentive subtype ADHD adults from controls. Objective measures of activity may differ from clinical impressions as subjective ratings of hyperactivity are skewed by the valence of the behavior, such that aggressive individuals are rated as hyperactive regardless of their actual activity levels. The cliThere are a number of possible interpretations for the predominance of objective activity over attention measures. First, activity may be a more powerful discriminator because it can be measured more accurately. Activity is a physical property and motion analysis systems can track overt movements with precision. Attention is an internal state and is not directly assessed but rather inferred from a subject’s performance on a task.Second, we may have underestimated the importance of attention by selecting suboptimal tasks, though we are not aware of any task that would have provided superior results. For example, the most robust differences between children with ADHD and controls on the stop signal delay task and Stroop have smaller mean effect sizes  in the putamen, which wThese present findings show that tests of attention by themselves perform suboptimally as diagnostic aids for ADHD. This evidence is consonant with previous reports in adults and children. For example, Epstein, Conners’ and colleagues observed that the CPT-II discriminated adults with ADHD from controls with only 55% sensitivity and 76.4% specificity. SimilarThere are a number of design differences between the CPT-II and the No-4’s cognitive control task, such as the use of relatively large geometric shapes presented at random screen positions versus the presentation of smaller letters at the same fixed center point. The CPT-II emphasized signal detection theory and regression measures of performance change across blocks or interstimulus intervals. The No-4’s task emphasized an analysis based on fluctuations in attention state. Random In contrast, measurements of motor activity alone in the present study had the predictive capacity to differentiate a mixed group of ADHD adults from a control group with up to 83% sensitivity and 87% specificity Table , and in The main limitations of this study are moderate sample size and the selection of adults with ADHD without comorbidity. Further studies will need to assess whether these findings can be replicated, extended to subjects with comorbid disorders, and tested for their capacity to differentiate adults with ADHD from subjects with other disorders that alter activity or impair attention.Although it is widely believed that hyperactivity in individuals with ADHD abates with age we found, using objective measures, that adults with ADHD moved their head and legs to a markedly greater degree than healthy controls during performance of a computerized attention test. Indeed, activity measures were far better at discriminating adults with ADHD from controls than computerized measures of inattention and impulsivity. This was true for both males and females with ADHD and for ADHD subjects diagnosed with the predominantly inattentive subtype. These findings are very similar to results previously reported in children using the same technology ,13. In aADHD: Attention deficit hyperactivity disorder; AUC: Area under the curve; Brown-ADD: Brown attention deficit disorder scale; CI: Confidence interval; COV: Coefficient of variation; EOC: Errors of commission; EOM: Errors of omission; ISI: Interstimulus interval; ROC: Receiver operating characteristic; RT: Reaction time; WRAAS: Wender-Reimherr adult ADHD scale.de minimis guidelines established by Harvard Medical School and Partners Health Care. The Committee on Conflicts of Interest at Partners Health Care provided specific guidelines, including independent statistical analyses, to manage potential conflicts relating to this study. Dr. Teicher also received research support, as a component of an NIMH SBIR award, from Ambulatory Monitoring Inc., to investigate use of a feedback actigraph in treatment of children with ADHD. He previously received ADHD-related research funding from Copley Pharmaceuticals, Repligen and OPTAx Systems Inc., the latter also paid consulting fees. He has received funding for depression research from The LiteBook Company and CNS Response. Dr. Teicher has not received any consulting or speaking fees from pharmaceutical companies within the last five years.The Quotient™ technology used in this study is owned by McLean Hospital and licensed to BioBehavioral Diagnostic Company (BioBDx). Dr. Teicher is the inventor on a number of patents related to this technology and receives a portion of the royalties paid to McLean from the use of Quotient™, in compliance with guidelines established by Harvard Medical School to minimize conflict of interest in clinical research. Dr. Teicher has no equity interest in BioBDx and holds no management position. Dr. Teicher has been reimbursed by BioBDx for travel expenses incurred to present results of research on Quotient™, has received funding from BioBDx for research relating to Quotient™, and receives consulting fees that fall within the Dr. Fourligas was a part-time employee of BioBDx from January 2007 through February 2007, a full-time employee from March, 2007 to November, 2008 and has continued to work for the company on a limited part-time basis. He has been granted options to purchase shares under the company's Stock Incentive Plan for services to the company. A portion of these shares have become exercisable.Drs. Polcari, Vitaliano and Navalta have no conflicts to declare.MHT conceived the study, participated in its design and coordination, performed the statistical analyses and drafted the manuscript. AP participated in the design and coordination of the study, conducted the diagnostic assessments, and provided critical comments on the drafts. NF participated in the design of the study, provided technical expertise on the software and hardware for data capture and provided critical comments on the drafts. GV helped to draft the manuscript, developed the literature review and reviewed the statistical results. CN participated in the design and coordination of the study, conducted the diagnostic assessments, and provided critical comments on the drafts. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-244X/12/190/prepub