Autonomic disorders of the cardiovascular system, such as orthostatic hypotension and elevated resting heart rate, predict mortality and cardiovascular events in the population. Low-energy-fractures constitute a substantial clinical problem that may represent an additional risk related to such autonomic dysfunction.To test the association between orthostatic hypotension, resting heart rate and incidence of low-energy-fractures in the general population.Using multivariable-adjusted Cox regression models we investigated the association between orthostatic blood pressure response, resting heart rate and first incident low-energy-fracture in a population-based, middle-aged cohort of 33 000 individuals over 25 years follow-up.The median follow-up time from baseline to first incident fracture among the subjects that experienced a low energy fracture was 15.0 years. A 10 mmHg orthostatic decrease in systolic blood pressure at baseline was associated with 5% increased risk of low-energy-fractures  during follow-up, whereas the resting heart rate predicted low-energy-fractures with an effect size of 8% increased risk per 10 beats-per-minute (1.05–1.12), independently of the orthostatic response. Subjects with a resting heart rate exceeding 68 beats-per-minute had 18% (1.10–1.26) increased risk of low-energy-fractures during follow-up compared with subjects with a resting heart rate below 68 beats-per-minute. When combining the orthostatic response and resting heart rate, there was a 30% risk increase (1.08–1.57) of low-energy-fractures between the extremes, i.e. between subjects in the fourth compared with the first quartiles of both resting heart rate and systolic blood pressure-decrease.Orthostatic blood pressure decline and elevated resting heart rate independently predict low-energy fractures in a middle-aged population. These two measures of subclinical cardiovascular dysautonomia may herald increased risks many years in advance, even if symptoms may not be detectable. Although the effect sizes are moderate, the easily accessible clinical parameters of orthostatic blood pressure response and resting heart rate deserve consideration as new risk predictors to yield more accurate decisions on primary prevention of low-energy fractures. Changes in autonomic control of the cardiovascular system may adversely affect various aspects of health . OrthostHemodynamic impairment during orthostasis is a well-known risk factor for traumatic falls , 11, 12.In this study, we investigated longitudinal association of orthostatic BP fall, RHR and their combination at baseline with incident low-energy-fractures in a middle-aged cohort of 33 000 individuals.The Malmö Preventive Project (MPP) is a population-based prospective cohort study in the city of Malmö, in southern Sweden . The priThe current analysis is a retrospective review of the prospective study of MPP. The present study included subjects from MPP with complete data on age, sex, body-mass index (BMI) and follow-up data of low-energy fractures (see below), rendering a total of 33139 subjects eligible for analysis.Subjects were asked to abstain from food, alcohol and tobacco for 12 h prior to the baseline examination, which was performed by trained nurses in the morning. BP was measured using the auscultatory method with a mercury sphygmomanometer on the right arm at heart level. BP was measured twice in the supine position and twice after one minute of standing. The mean value of two readings was recorded for each position and rounded to nearest 5 mmHg. The heart rate was measured twice by palpation over one minute in supine position. After the examination, the participants were asked to fill a questionnaire focused on personal and family history of CVD, hypertension, diabetes, cancer, smoking habits, and lifestyle pattern.A detailed description of recruitment and screening procedures of MPP may be found elsewhere , 20. TheDecrease in SBP and DBP, the components of orthostatic hemodynamic response, were assessed using a continuous/semi quantitative scale. Orthostatic SBP and DBP response (ΔSBP and ΔDBP) were defined as standing SBP/DBP minus supine SBP/DBP. The RHR was the mean of two measurements in supine position. BMI was calculated as weight in kilograms divided by the square of the height in meters. Antihypertensive treatment (AHT) was defined as a positive answer to the following question: Do you take medication for high blood pressure? Those who confirmed regular or occasional current smoking were classified as smokers. Diabetes was defined as fasting plasma glucose of ≥ 7.0 mmol/l, current pharmacological treatment of diabetes or self-reported history of diabetes. Previous myocardial infarction (MI) at baseline was recorded from the national Swedish patient register (which contains data from 1964- with complete coverage from 1987-).Fracture data were obtained by linking the unique Swedish ten-digit personal identification number of included subjects with the register at the Department of Radiology at Malmö University Hospital as previously described . In the Fractures were classified as high- or low-energy trauma depending on the cause of fracture, based on information in the radiographic reports. Fractures caused by falling from standing position or less energy were classified as low-energy fractures. Of all fractures, 97.7% had descriptions with adequate information about the degree of trauma. The fractures with insufficient information were classified as low-energy fractures based on the experience that it is highly unlikely that information about high-energy accidents is not reported. Fractures caused by high-energy trauma were excluded from all analyses, as were also pathological fractures caused by cancer or other bone diseases.The hemodynamic parameters recorded at baseline  were related to first incident low-energy fracture using multivariable-adjusted Cox-regression models. We tested models entering age, sex and BMI as covariates (Model 1) as well as more comprehensive models including age, sex, BMI, AHT, diabetes, smoking, previous MI, and all measured hemodynamic parameters  from the baseline examination (Model 2).In order to evaluate the combined effect of RHR and OH on the risk of incident low energy fractures we constructed a combined RHR-OH-score for each individual subject. The score was constructed as follows: the study population was split into quartiles according to baseline RHR and ΔSBP, respectively. Thus, each individual was given a quartile number  for RHR and ΔSBP. The RHR-OH-score for each individual (range 2–8) was then constructed by summing the individual quartile number for RHR and ΔSBP, respectively. Additionally, in order to further investigate the specific contribution of RHR and ΔSBP and of their combined effect to the risk of incident low-energy-fractures, the 16 specific quartile combinations of RHR and ΔSBP that could be combined for the study subjects , and it was even higher for those who demonstrated SBP fall >5 mmHg   . In contP = 0.008) whereas there were no significant associations with radius, skull and hip fractures  an association with vertebral fragility fractures  increased risk of low-energy fractures during follow-up compared with subjects having a RHR below 68 bpm.RHR predicted incident low-energy fractures in both models, the latter model also including supine SBP and orthostatic change in SBP as covariates. The effect size was approximately 8% increased risk per 10 beats per minute (bpm) in the fully adjusted model . SubjectThere was a linear association between RHR and number of low energy fractures in individual subjects (P = 0.011) and the odds ratio (OR) of suffering more than one fracture (compared with one or zero) was 1.10  per RHR-quartile in a logistic regression model, adjusted as in model 2.P <0.001) and a borderline-significant association with vertebral fragility fractures , whereas there were no associations with hip fractures and skull fractures  an association with distal radius fractures  between the extremes of the RHR-OH-score (i.e. subjects in the fourth compared with the first quartiles of both RHR and ΔSBP). When stratifying subjects by quartiles of both RHR and ΔSBP in relation to first incident low energy fracture the effect of orthostatic SBP-decrease on risk of low energy fractures was evident predominantly in the upper quartiles of RHR .The main potential clinical implication of our results is the possibility that OH and RHR may be used to sharpen clinical risk prediction of low-energy fractures. Low energy fractures constitute a huge clinical problem , 14, oftAs for mechanistic aspects of the current results, we propose some explanations why OH and particularly RHR are independently associated with low-energy injuries. The first and most obvious is that these variables are merely markers of poor physical fitness or an impaired health status, which in turn would be correlated with traditional risk factors for fragility fractures such as diabetes or smoking , 16. HowAlternative mechanisms that might link elevated RHR with fall-related injuries include the possibility of an undiagnosed state that increases the risk of pre-syncope and syncope such as postural orthostatic tachycardia syndrome (POTS) . Also, aRegarding the long time span between baseline screening and the index event, a similar temporal delay was observed in MPP in relation to all-cause mortality, and cardiovascular events such as myocardial infarction . Thus, tAlthough asymptomatic to a large extent, OH is a clinically well recognized problem, especially in the elderly in whom the consequences of OH (such as a fracture) may frequently be the first manifestation . VariousThe main strengths of this study are the large number of subjects, long follow-up and access to reliable case data. The major limitation of this study is the lack of data on heart rate and 3-min BP on standing which were not recorded in MPP. Naturally, the current guidelines on OH  were notIn conclusion, we have shown that orthostatic blood pressure decline, elevated resting heart rate and their combination are strong independent predictors of low-energy fractures in a middle-aged population and as such, potential signs of subtle autonomic dysfunction in these subjects. Orthostatic blood pressure response and resting heart rate deserve consideration as new tools for risk prediction in order to obtain more accurate clinical decisions on non-pharmacological and pharmacological primary prevention of low-energy fractures.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 TableRelation between hemodynamic parameters and first incident low energy fractures in untreated hypertensives (A) and in normotensives (B).(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.


Zetaproteobacteria play significant ecological roles in biogeochemical iron-cycling. However, the ecophysiological characteristics and interaction between other microbial members in the habitat still remain largely unknown. In this study, we investigated microbial communities in a core sample obtained from shallow hydrothermal iron-oxyhydroxide deposits at Nagahama Bay of Satsuma Iwo-Jima, Japan. Scanning electron microscopic observation showed numerous helical stalk structures, suggesting the occurrence of iron-oxidizing bacteria. Analysis of 16S rRNA gene sequences indicated the co-occurrence of iron-oxidizing Zetaproteobacteria and iron-reducing bacteria such as the genera Deferrisoma and Desulfobulbus with strong correlations on the sequence abundance. CARD-FISH indicated that the numbers of Zetaproteobacteria were not always consistent to the frequency of stalk structures. In the stalk-abundant layers with relatively small numbers of Zetaproteobacteria cells, accumulation of polyphosphate was observed inside Zetaproteobacteria cells, whereas no polyphosphate grains were observed in the topmost layers with fewer stalks and abundant Zetaproteobacteria cells. These results suggest that Zetaproteobacteria store intracellular polyphosphates during active iron oxidation that contributes to the mineralogical growth and biogeochemical iron cycling.Previous studies of microbial communities in deep-sea hydrothermal ferric deposits have demonstrated that members of  Zetaproteobacteria were first found at the Loihi Seamount, Hawaii , image-based cell count, and catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH) techniques as well as diversity and correlation analyses of 16S rRNA gene-tagged sequences.In this study, we investigated microbial communities in the shallow hydrothermal iron-oxyhydroxide deposits (water depth: ~3 m) in the Nagahama Bay. To understand the distribution and ecophysiological characteristics of Satsuma Iwo-Jima is a small volcanic island of the southern Kyushu, Japan. The volcanism is associated with iron-rich sedimentations at hydrothermal hot springs along the island coast. The Nagahama Bay is located on the southwest coast of the island and is one of the most active regions of hot water discharge Figure . The sea3 subsamples were collected from the split core and stored at −20°C until further processing. There are hard and soft layers inside the core and the subsamples were only taken from relatively soft and undisturbed layers in this study , 10 cm3 of sediments were fixed with 4% paraformaldehyde, washed twice with 1 × PBS, re-suspended in 50% PBS-ethanol solution, and then stored at −20°C.A 50-cm-long sediment core was collected from an iron-rich hydrothermal mound in the Nagahama Bay in May 2013 by scuba divers using a clear acrylic tube , was designed in this study for the specific detection of Zetaproteobacteria. The optimal formamide (FA) concentration in the hybridization buffer was determined by clone-FISH , if needed  according to the manufacturer's instructions. The extracted DNA was further concentrated by isopropanol precipitation. For clone-FISH, nearly full-length 16S rRNA was amplified using a 27F-1492R primer set Lane, , whereas6R Lane, . After ip < 0.05) with coefficients greater than 0.9. A phylogenetic tree was constructed using MEGA5 software   of the 50 cm-long core dominated by red-brownish iron deposits Figure . SEM obs8 cells cm−3 and declined sharply in CH1_6 to 7.26 × 107 cells cm−3  concentration for specific detection of  Figures , and theM. ferroxydans PV-1 has genes related to polyphosphate metabolism  did not provide enough information for the genus-level classification, we additionally performed Sanger sequencing and obtained near-full-length sequences for the V1–V4 regions of the 16S rRNA gene, providing the finer and more precise taxonomic positions of the 1 Figure . CH1_BAC1 Figure  and the 1 Figure , respectZetaproteobacteria strongly correlated with anaerobic or microaerophilic bacteria, such as the members of iron-reducing Deferisoma and sulfate-reducing Desulfobulbus . In addition, the phylum Chloroflexi has been reported to be dominant in iron reducing enrichment culture . Because the prevalence of sulfate-reducing conditions down to at least 40 cm below the seafloor and hence, even through the iron deposits are soft and highly permeable for discharging hydrothermal fluids and/or the overlying seawater, the redox state within the iron deposits are heterologous but generally sub-oxic to anaerobic conditions, providing habitable zones for both Chloroflexi and sulfate reducers with iron oxidizers. Beta-diversity analysis of microbial communities indicated the zonation pattern of three ecological niches in the iron deposits: the top (10 cm), middle (17–33cm), and bottom layers (~40 cm), suggesting the occurrence of redox-sensitive microbial habitats associated with iron and other elemental cycles. In fact, microscopic observations revealed that twisted stalk structures are most abundant in the middle layer. In addition, based on the visual observation, upward hydrothermal fluid flows seem to form complicated webs of the fluid passage in the middle zone , although fewer stalk structures were observed , i.e., gaining energy from iron-oxidation and/or heterotrophy without forming stalk-like structures. This might be an adaption mechanism of iron-oxidizing Zetaproteobacteria; however, mechanisms for their redox-sensing and energetic response to the environmental change still remain largely elusive.Another interesting finding in this study would be a discrepancy between the frequency of twisted stalk structures and the number of d Figure . AlthougZetaproteobacteria play significant ecological roles in iron cycling with other iron reducers and construct a unique microbial ecosystem in shallow hydrothermal iron-oxyhydroxide deposits. In addition, we first demonstrated that Zetaproteobacteira in the natural ecosystem store polyphosphate grains inside cells for energy storage and a phophate source with iron-oxidation and stalk formation. We also observed that Zetaproteobacteria without pholyphosphate grains with fewer stalk formations have largest population, indicating a possibility that Zetaproteobacteria changed their metabolism depending on the environments. These results provide some new insights into ecophysiology of iron-oxidizing Zetaproteobacteria and the microbial ecosystem in marine hydrothermal environments.In conclusion, we show that TK, HO, and SK sampled and pretreated a core sample. TTH, YM and TMH performed the molecular works as DNA extraction and sequencing of 16S rRNA. TTH and YM carried out microscopic works including cell count and FISH. TTH analyzed sequencing data. TTH, SK, and FI designed of the work and drafted the manuscript, which was critically riveted by all authors. The final manuscript was approved by all authors.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Personality disorders are highly comorbid with alcohol misuse and depressive symptomatology; however, few studies have investigated treatment outcomes in this population. The aim of this study was to examine relationships between baseline personality disorder cluster profiles and overall and treatment-related changes for those with co-occurring alcohol misuse and depression.N = 290) from two randomised controlled trials of psychological interventions for co-occurring alcohol misuse and depression, which did not specifically target personality disorders. Baseline dimensional personality disorder cluster scores were derived from the International Personality Disorder Examination Questionnaire (IPDEQ). Four treatment conditions were compared: a brief integrated intervention, followed by no further treatment, or nine further sessions of integrated-, alcohol-, or depression-focused treatment. Associations between IPDEQ scores and changes in alcohol use, depressive symptoms and functioning from baseline to the 6- and the 12-month follow-ups were of primary interest.Secondary analysis was conducted using a subset of data , as well as treatment-related outcomes (primarily Cluster A), particularly changes in depressive symptoms and psychosocial functioning. Longer interventions appeared to be more effective in the longer-term , with integrated interventions relatively more effective than single-focused ones for individuals with higher personality disorder cluster scores.Greater attention needs to be paid to particular personality disorder clusters during the assessment and treatment of individuals with co-occurring alcohol misuse and depression. Integrated interventions, incorporating motivational interviewing and cognitive behaviour therapy, may provide a useful therapeutic framework. Integrated interventions also provide opportunities for adjunctive components focussing on other issues and coping strategies , potentially tailored to the characteristics and needs of individual participants. Co-morbid alcohol misuse and mental health problems are a major health concern as they place significant burden on the health care system  and are Existing research on the co-occurrence of personality disorders and either alcohol misuse or depressive symptomatology consists mainly of prevalence based studies that do not address treatment outcomes , 13. TheThere is also ongoing debate about categorical versus dimensional approaches to the assessment of personality disorders , and aboAlthough it has been suggested by the World Health Organization that alcohol use disorders and major depression may require concurrent treatment , few ranIn the first study of its kind, Baker et al.  comparedPeople with co-occurring alcohol misuse and mental health problems are often excluded from existing treatment studies , 29. To Poorer response to treatment for depression has been found among people with co-occurring personality disorders and depression . Higher Given the relatively high prevalence of co-existing personality disorders, alcohol misuse and depressive symptoms, and the lack of previous research investigating treatment outcomes when these disorders co-occur, it is also important to generate greater understanding of the ways they interact in order to develop effective interventions.We undertook secondary data analysis of composite data from two large randomised controlled trials in an effort to achieve key preliminary insights into the complex interaction between personality disorder cluster scores, alcohol misuse and depressive symptoms. Specifically, we investigated whether baseline personality disorder cluster scores are associated with overall changes in alcohol use, depressive symptoms and functioning, and treatment-related changes at the 6- and the 12-month follow-up, for individuals in treatment for comorbid alcohol misuse and depressive symptomatology.As hypothesised in the parent studies , 38, 39,This study combined data from two randomized controlled clinical trials co-ordinated by the Centre for Brain and Mental Health Research, University of Newcastle, New South Wales, Australia. Study 1, the Self-Help for Alcohol/other drug use and DEpression (SHADE) project, included 273 participants with comorbid depressive and drug and alcohol problems . Study 2Across the two parent studies, a range of manualised MI/CBT based interventions designed to reduce alcohol consumption and/or improve depressive symptoms were delivered. These included a single 90-min brief integrated intervention (BI), followed by either: 1) no further treatment; or 9 further sessions of 2) integrated  therapy (delivered either by a therapist or computer program), 3) alcohol-focused therapy, or 4) depression-focused therapy; in addition, a person-centred (supportive counselling) therapy was offered as a control condition in the SHADE study. Participants provided informed consent and received $20 reimbursement on each assessment occasion .N = 398), the majority had a DSM-IV alcohol dependence disorder  and/or major depression  during the last 12 months.This study involved a secondary analysis of a subset of data from the two randomised controlled clinical trials described above. Subset inclusion criteria for the current study were: (i) hazardous alcohol consumption in the 12 months before baseline ; (ii) a BDI-II score ≥ 17; and (iii) assignment to a therapy that specifically targeted alcohol misuse and/or depressive symptoms; consequently SHADE study participants receiving the person-centred therapy intervention were not included in the current study, nor were participants with only cannabis related substance use problems. Among those who were eligible for the current analysis  missing personality disorder data at baseline; or (ii) an absence of outcome data at 6- or 12-month follow-up .A description of the full set of assessments employed across the parent studies and the rationale for their use have been reported elsewhere , 38, 39.The Structured Clinical Interview for DSM-IV-TR (SCID) [Opiate Treatment Index (OTI) [Beck Depression Inventory II (BDI-II) [. The Global Assessment of Functioning scale (GAF) [A range of measures were used to quantify the duration and severity of existing conditions and measure primary outcomes. R (SCID)  was usedex (OTI)  estimate(BDI-II) . The Glole (GAF)  providedThe parent studies screened participants at baseline for possible Axis II personality disorders using the International Personality Disorder Examination Questionnaire (IPDEQ). The World Health Organization developed the IPDEQ self-report screener based on the International Personality Disorder Examination (IPDE) , which iData were analysed using SPSS for Windows . Pearson correlations were used to examine simple associations among continuous measures. Single sample t-tests were used to compare IPDEQ profiles with national survey data, while generalized estimating equations were used to examine overall changes between baseline and follow-up phases at 6- and 12-months.For the major analyses, the primary outcome measures were expressed as change from baseline . Notwithstanding, our focus here was essentially on clinical change at discrete time points, with longer-term benefits  seen as the primary clinical outcome point, and intermediate benefits  viewed as being of lesser importance. Two-step hierarchical linear regressions were used to assess the contributions of socio-demographic, personality disorder and treatment related predictors to change (from baseline) at 6- and 12-months, whilst controlling for baseline scores for the set of outcome measures. Three planned (Helmert) orthogonal treatment condition (TC) contrasts were included at Step 1 in each of these analyses: Contrast TC1, BI vs. 10 sessions ; Contrast TC2, integrated- vs. single-focused ; and Contrast TC3, alcohol- vs. depression-focused ; this approach parallels that reported previously for the DAISI trial . All intp < 0.01; however, trends at p < 0.05 are also reported.Importantly, separate regression models were examined using overall IPDEQ scores and associated interactions; parallel analyses substituted the set of individual cluster scores and associated interactions, providing an opportunity to identify their unique contributions to prediction . As a partial control for the number of statistical tests, the threshold for significance was set at N = 108), 54 did not have baseline personality disorder data, 36 did not have any outcome data for at least one of the follow-up assessments, and 18 failed to meet both of these criteria. There were no significant differences in baseline characteristics between those who were retained in the current analysis and those with insufficient data; however, the latter subgroup attended fewer treatment sessions  = 5.30, p < .001).The pattern of participation in the parent studies and current analysis is summarized in Fig. Baseline demographic, symptomatology and substance use data for the selected sample is presented in Table r = 0.40; Clusters A and C: r = 0.44; and Clusters B and C: r = 0.34). These correlations were also comparable to the subset IPDEQ values reported in Lewin et al. [As shown in Table n et al. , based on et al.  and 155 n et al. .Table 2Cp = 0.008). As detailed in Table Estimated mean changes in OTI alcohol scores, BDI-II scores and GAF scores between phases are shown in Table The predictors of OTI change scores at the 6- and the 12-month follow-up are shown in Table The predictors of BDI-II change at the 6- and the 12-month follow-up are shown in Table Discussion . As shown in Table The prediction of GAF change scores at the 6- and the 12-month follow-up is presented in Table This study strengthens our understanding of the complexities associated with co-occurring alcohol misuse, depressive symptoms and personality disorder. In broad terms, we sought to investigate whether dimensional personality disorder cluster scores were associated with changes in selected outcomes at the 6- and the 12-month follow-up among outpatients in treatment for comorbid alcohol misuse and depression.Our first set of hypotheses referred to patterns of change in the outcome measures. As expected, participants’ scores on the OTI, BDI-II and GAF improved across phases, particularly between baseline and the 6-month follow-up. With respect to key treatment related hypotheses derived from the parent studies: longer interventions were more effective than the BI condition at 12-months, contributing to larger reductions in depression and improvements in functioning; collectively, single-focused treatments produced reasonably comparable changes to integrated treatment; and, within the single-focused treatments, alcohol-focused treatment tended to be relatively more effective for alcohol use outcomes, at least in the short-term. These findings are consistent with those reported recently for comparable phases of the DAISI project .The strong associations between baseline scores for the three outcome measures and the corresponding change scores at 6- and 12-months were not surprising, for a range of reasons: because there is a mathematical relationship between difference scores and their constituent parts ; and because samples selected on the basis of more extreme values  would be expected to show some ‘regression to the mean’ effects.More importantly, as discussed below, this secondary analysis of the combined clinical trials dataset sheds additional light on these treatment effects and associations by examining the contributions of personality disorder cluster scores to the overall prediction of change and to the efficacy of the treatments provided. The presence of some personality disorder profiles negatively impacted on overall change during the follow-up period (primarily Cluster C) as well as treatment-related outcomes (primarily Cluster A), especially in regards to depressive symptoms and psychosocial functioning. These insights have several clinical implications, particularly the potential to inform the development of effective interventions for this comorbid population.IPDEQ scores showed limited associations with changes in alcohol use, with no statistically significant effects ; and comorbid personality disorder profiles made little contribution. Consequently, from a stepped-care perspective in particular e.g., , brief MParticipants with higher baseline IPDEQ overall scores and Cluster C scores experienced smaller improvements in depression at 6- and 12-months  treatments for depressive symptoms and alcohol misuse. Although individuals with personality disorders are more likely to seek treatment for their depression or alcohol use disorder than for their personality pathology , it is fIn the current analyses, there was also a tendency for participants with higher baseline alcohol scores to experience less improvement in depression at 6-months, whereas those with poorer baseline functioning experienced greater reductions in alcohol consumption at 6-months. That is, in the short-term, higher alcohol consumption tended to impact in a similar way to the baseline personality disorder scores, in restricting improvements in 6-month depression, while lower baseline functioning scores were associated with greater room for improvement in both alcohol consumption and functioning.The major limitation of the current study is that it was a secondary data analysis of selected composite data from two randomised controlled trials that did not specifically focus on personality disorder. Therefore, to confirm our findings, a more comprehensive trial is required that includes a clinician administered structured diagnostic assessment for personality disorder. Inclusion of a measure of personality disorder at the conclusion of the follow-up phases would have also been useful. With respect to our current functioning measure, it should also be acknowledged that the GAF has been removed from DSM-5 in favour of more comprehensive assessments of disability and functioning ; howeverUtilisation of separate analyses of change scores at the 6-month and the 12-month follow-ups also raises some potential statistical/methodological concerns, since such outcome scores would be expected to be correlated ; however, as noted earlier, for the current analyses change at 12-months was regarded as the primary clinical outcome point. For researchers interested in longitudinal profiles across multiple time points  and their predictors and mediators, different analytical approaches would be preferable, which simultaneously consider data from all phases e.g., .Although we observed significant improvement from baseline in OTI, BDI-II and GAF scores, it should also be acknowledged that 12-month impairment remained substantial; suggesting that the scope and intensity of our interventions needs to be revisited. For example, mean alcohol consumption per day at 12 months (5.32 drinks) was still well above recommended levels, and mean BDI-II scores at 12 months (17.2) were indicative of mild-moderate depression, albeit at the threshold for entry to the parent studies.p < 0.05); consequently, they await replication elsewhere. On the other hand, studies often lack statistical power to detect complex interactions . However, in the 12-month depression analyses, the (Step 2) interaction effects added 5.9 % to the explained variance , longer (10-session) psychological interventions appeared to be more effective in the longer-term (at 12-months), especially for changing depressive symptoms and improving functioning. Moreover, integrated interventions were also relatively more effective than single-focused ones for individuals with higher personality disorder cluster scores. Consequently, longer integrated interventions may be a sensible general strategy for this subgroup, perhaps delivered within a stepped-care framework . HoweverAt the very least, adjunctive components need to be incorporated into our integrated therapy programs to more fully engage individuals with particular personality disorder cluster profiles, in an attempt to counteract the potentially negative consequences associated with these conditions, perhaps with a specific focus on emotion or affect regulation and coping strategies. An integrated framework may also provide opportunities to tailor some intervention components to the characteristics and needs of individual participants. We also encourage researchers and clinicians to more actively consider the influence of particular personality disorder clusters in assessing, treating and monitoring individuals engaged in treatment for co-occurring alcohol misuse and depression.th Edition; GAF, The global assessment of functioning scale; ICD-10, International Classification of Diseases – 10th Edition; IPDE, International Personality Disorder Examination; IPDEQ, International Personality Disorder Examination Questionnaire; MI, motivational interviewing; OTI, opiate treatment index; SCID, The Structured Clinical Interview for DSM-IV-TR; SHADE, Self-Help for Alcohol/other drug use and Depression (Project)BDI-II, Beck Depression Inventory – Version 2; CBT, cognitive behaviour therapy; Cluster A, paranoid & schizoid personality disorders (ICD-10); Cluster B, dissocial, impulsive, borderline & histrionic personality disorders (ICD-10); Cluster C, anankastic, anxious & dependent personality disorders (ICD-10); DAISI, Depression and Alcohol Integrated and Single-focused Interventions (Project); DSM-IV, Diagnostic and Statistical Manual for Mental Disorders – 4


Prolonged dual anti-platelet therapy (DAPT) may cause excess bleeding in certain patients. The biolimus-A9 drug-coated stent (BA9-DCS) has a rapid drug-elution profile allowing shortened DAPT. Data were gathered on the early experience implanting this stent in drug-eluting stent eligible patients deemed to be at high risk of bleeding.The demographics, procedural data and clinical outcomes were gathered prospectively for 249 patients treated with a BA9-DCS stent at 2 UK centres, and compared to a cohort of patients treated in the same period with drug-eluting stents (PCI-DES).Operator-defined BA9-DCS indications included warfarin therapy, age, and anaemia. Patients receiving a BA9-DCS were older , more often female , and more likely to have comorbidity including chronic kidney disease or poor LV function than PCI-DES patients. The baseline Mehran bleed risk score was also significantly higher in the BA9-DCS group . Of the BA9-DCS cohort, 95.5% of patients demonstrated disease fitting NICE criteria for DES placement. The number of lesions treated , total lesion length , number of stents used  and total stent length  were greater for BA9-DCS patients. DAPT was prescribed for 3.3±3.9 months for BA9-DCS patients and 11.3±2.4 months for PCI-DES patients (p<0.001). At follow up of 392±124 days despite the abbreviated DAPT course stent related event were infrequent with ischemia-driven restenosis PCI , and stent thrombosis  rates similar between the BA9-DCS ad PCI-DES groups. After propensity scoring all clinical end-points were similar between both cohorts.This early experience using polymer-free BA9 drug-coated stents in drug-eluting stent type patients at risk of bleeding are encouraging. Further studies are warranted. Prolonged dual anti-platelet therapy (DAPT) exposes patients to the risk of bleeding complications after PCI. In the CURE trial the absolute excess risk of major bleeding with clopidogrel over placebo at 12-months was 0.5–1.0% –5. HowevPrevious registry data combined with observational data from clinical trials have demonstrated beyond doubt that bleeding after PCI is a significant event, with a close association with morbidity and mortality for at least 12-months after the procedure –9. SeverHowever, as well as shortened courses of DAPT after implantation of contemporary second-generation stents, newer stent technologies also provide further potential to maintain optimal ischaemia outcomes whilst avoiding bleeding associated with prolonged DAPT. The BioFreedom stent  is a stainless steel platform engineered to allow the biolimus A9 drug to be applied directly to its surface obviating the need for any polymer. As a result, the stent is polymer-free and demonstrates rapid drug elution profile allowing accelerated vessel healing. This has the potential to facilitate a shortened duration of DAPT without compromising TLR and MACE . The recThe University Hospital of Wales, Cardiff and Morriston Cardiac Centre, Swansea provide tertiary cardiac care to a population of 2.3 million people and combined perform in excess of 2,500 PCIs/year. The BA9-DCS became available for clinical use in the United Kingdom in January 2014 and was implanted at both centres at the interventional cardiologists discretion. Treatment was targeted to patients in whom it was deemed undesirable to prescribe prolonged DAPT therapy. Baseline demographics, procedural data and outcomes were gathered and entered prospectively from 249 consecutive patients treated with a polymer-free biolimus A9 drug-coated stent over a 12-month period. The Cardiff and Vale University Health Board audit department approved the study. As all data acquisition and analysis was retrospective, and because patient records/information were anonymized and de-identified prior to analysis, patient consent was not sought. To contextualise the characteristics of the BA9-DCS cohort we gathered baseline, procedural and outcome data on all other patients undergoing PCI with drug-eluting stents (PCI-DES) at our institutions during the same time period. Consecutive patients with de-novo disease treated with the BioFreedom stent were included apart from those presenting in cardiogenic shock. Patient demographics and procedural data were retrieved from the national British Cardiovascular Intervention Society Database (BCIS) Central Cardiac Audit (CCAD) database. The standard CCAD definitions of comorbidity were used for the study purposes . The indFor follow-up, the in-hospital causes of death were derived from internal database analysis. For deaths in the community the patient’s general practitioner was contacted for cause of death. Repeat procedure data were derived from an internal angiographic database and the CCAD database. Stent thrombosis (ST) was defined as definite, probable or possible as per the ARC criteria. For completeness, patients’ details were crosschecked with all interventional centres in Wales to ensure TLR or stent thrombosis had not been treated in another centre. Ischemia-driven TLR (ID-TLR) was defined as repeat PCI to the study stent or the segment 5mm either proximally or distally that was performed because of a recurrence of chest pain or presentation with an acute coronary syndrome. Myocardial infarction (MI) was defined using the universal definition of myocardial infarction criteria. Bleeding was defined using BARC criteria. Major adverse cardiac outcomes were the combination of death, ST, and ID-TLR with episodes treated in a hierarchical fashion ie death was the most important followed by stent thrombosis and then target lesion revascularisation. Data were gathered for audit purposes to ensure patient safety and permission for this granted by the hospital audit department. Clinical outcome data were collected by research registrars  who were not directly involved in the patients’ care.Propensity score analysis was performed in order to balance for covariates that might bias estimates for causal inferences. The variables that were controlled were age, sex, weight, presentation, previous MI, angina status, diabetes status, history of renal insufficiency, congestive heart failure, Q-wave on ECG, hypertension, CVA and PVD. A propensity score for each patient was obtained from a logistic regression using Inverse Probability of Treatment Weights. The adjusted event rates (adjusted Kaplan-Meier estimates) and the adjusted hazard ratios and p-value were reported. Continuous data were expressed as mean (SD) and comparison between groups was performed using Student T-Test. Categorical data are presented as frequencies and percentages, and were compared using a chi-square statistics or the Fisher’s exact test where indicated.A total of 249 patients underwent stenting with a BA9-DCS between January 2014 and February 2015 and as such represents 8.8% of the total volume of PCI procedures undertaken at the 2 centres in that time period. The baseline demographics of the BA9-DCS and PCI-DES patients are listed in Reflective of increased age and a greater frequency of female patients, the BA9-DCS patients were more likely to undergo PCI from the femoral artery . The mean number of vessels treated  was simiPhysician-recommended DAPT duration was 3.3±3.9 months for BA9-DCS patients , panel ACrude unadjusted ischaemia-driven restenosis PCI occurred in 2.8% of the BA9-DCS cohort at a mean of 141.3±57.6 days compared to 3.4% in the PCI-DES cohort (p = 0.84). There was a numerical excess of unadjusted death in the BA9-DCS cohort compared to the PCI-DES population although this did not reach statistical significance (p = 0.26). However, adjusted clinical outcomes after propensity scoring were similar in both cohorts  with no Our early clinical experience with a polymer-free BA9 drug-coated stent can be summarised as follows: 1) Patients in the BA9-DCS cohort were at high bleeding risk with advanced age, over-representation of female patients, low body weight, and a high prevalence of hypertension; 2) The BA9-DCS PCI procedures were complex with long lesions, frequent requirement for atherectomy and long lengths of stents implanted; 3) Clinician-determined DAPT duration in BA9-DCS patients was significantly shorted than the PCI-DES recommendation; 4) Despite longer lesions and abbreviated DAPT duration, the clinical outcomes were encouraging with low TLR and ST rates.The identification of patients deemed to be at risk of bleeding with prolonged DAPT duration is an important component of PCI planning. Important risk factors for bleeding have been identified in several studies and include age, female sex, low body weight, chronic kidney disease and acute presentation . AdditioOne possible strategy to shorten DAPT therapy in patients at risk of bleeding is to implant a bare metal stent. Indeed, data from the run-in study to LEADERS-FREE suggests warfarin therapy was a frequent reason that interventional cardiologists reported for choosing a bare metal stent . HoweverIf a DES is implanted, operators are faced with a choice to either prescribe the recommended 12-month DAPT course (in patients with an ACS indication), or alternatively to abbreviate the course perhaps to 3 or 6-months . However, persisting with a 12-month course places patients at significant risk of bleeding as illustrated by WOEST . In the Emerging data from recent observational studies of the randomised trial programs of the Endeavor and Xience stent provide support for the concept of a reduction in DAPT duration. However, as this is observational data derived from randomised trials, there are limitations in its applicability to everyday clinical practice. Such limitations include confounders such as treatment bias and also small numbers of patients receiving short DAPT duration ,24. DataGiven the lack of data in dealing with complex coronary disease in patients at high risk of bleeding, there is potential for new stent technologies to improve patient outcomes. The BioFreedom drug coated stent is a polymer-free platform resulting in rapid drug-elution profile that may allow shortened duration of DAPT without compromising TLR and MACE. In pre-clinical studies, superior strut coverage was demonstrated compared to bare-metal and sirolimus-eluting stents , 30. In The heterogeneity of DAPT duration observed reflects implanting physicians’ discretion, and interpretation of balancing the individual patient stent thrombosis and bleeding risks. This heterogeneity is likely a result of only early data being available regarding DCS use, and the absence of guidelines regarding optimal DCS DAPT duration. The 12-month recommendation in certain cases was due to the ACS presentation and due regard to the ACS guidelines. However, in these cases often the narrative was that 12-month was recommended due to an ACS presentation, but that the use of a BioFreedom facilitated DAPT discontinuation should a bleeding event or intolerance/allergy occur for example.In considering the other limitations of this study, firstly—as this is observational non-randomised data—it is possible that selection and treatment biases confound the results. Secondly, as the patients were treated in only two centers the results of the polymer-free BA9 drug-coated stent in other centers might be different. Thirdly, as the numbers are modest we did not have the statistical power to analyse outcomes by variable DAPT duration. However, as this is observational data any conclusions from such an analysis are likely to be severely limited by treatment bias.In conclusion, the outcomes from this preliminary study using polymer-free BA9 drug-coated stents in DES-type patients who are poor candidates for prolonged dual anti-platelet therapy are encouraging, with low repeat revascularisation and stent thrombosis rates. Further studies are warranted.S1 File(XLSX)Click here for additional data file.S2 File(XLSX)Click here for additional data file.S3 File(DOCX)Click here for additional data file.


P-values 10−13, 10−9 and 10−7 respectively). Finally, using an ensemble machine-learning tool, we discovered a unique 8-gene signature for this group of cancers that differentiates the group from the other tumor subsites of head and neck region. We investigated the role of promoter methylation in one of these genes, WIF1, and found no correlation between DNA methylation and down-regulation of WIF1. We validated our findings of gene expression, 8-gene signature and promoter methylation using q-PCR, data from TCGA and q-MSP respectively.Laryngo-pharyngeal squamous cell carcinomas are one of the most common head and neck cancers. Despite the presence of a large body of information, molecular biomarkers are not currently used in the diagnosis, treatment and management of patients for this group of cancer. Here, we have profiled expression of genes and microRNAs of larynx and hypopharynx tumors using high-throughput sequencing experiments. We found that matrix metalloproteinases along with SCEL, CRNN, KRT4, SPINK5, and TGM3 among others have significantly altered expression in these tumors. Alongside gene expression, the microRNAs hsa-miR-139, hsa-miR-203 and the hsa-miR-424/503 cluster have aberrant expression in these cancers. Using target genes for these microRNAs, we found the involvement of pathways linked to cell cycle, p53 signaling, and viral carcinogenesis significant (Data presented in this manuscript has been submitted to the NCBI Geo database with the accession number GSE67994. Head and neck squamous cell carcinomas are a diverse group of tumors that originate from anatomically different locations, including nasal cavity, sinuses, lips, mouth, salivary glands, and throat. Cancers of the upper aero-digestive tracts  are the sixth most common cancer worldwide . In addiLarge-scale molecular characterizations of HNSCC have been performed in recent years for different subsites -10. DespN=13) and the other where we validated our results . We used computational pipeline with sequencing data from the discovery set to identify the significantly over- and under-expressed genes in SCCL and SCCHP. Our study identified matrix metalloproteinases, KRT4, SPINK5 and TGM3 among others as the main genes with altered expression in the tumors studied. Among the micro RNAs, miRNA-139, miRNA-203 and miRNA-424/503 cluster had altered expression in larynx and hypopharynx carcinomas. We found the target genes for these miRNAs to be linked to processes like cell cycle, p53 signaling and viral carcinogenesis in larynx and hypopharynx carcinoma. Additionally, we inferred a unique 8-gene signature using a machine-learning algorithm as a potential diagnostic marker set for this group of cancer, which was further validated using data from the TCGA samples.In this study, we present data from whole transcriptome analyses using next-generation sequencing experiments to profile gene and miRNA expression landscape in larynx and hypopharynx carcinoma samples. We divided our study into two sets of patients, one where we discovered the gene expression signature  are provided in CLTC-VMP1 genes, supported by 16 reads perfectly mapping across the breakpoint  in ~30% of breast cancers and the potential association of this expression with poor prognostic outcome in breast cancer patients [CLTC-VMP1 fusion, we also found an intra-chromosomal fusion event (JCV=68.52) involving CTBS-GNG5 genes in a tumor sample  that aids in the detection of false positives. A number of gene fusions detected involved small nuclear/RNA genes . In one t Figure . The obsg cancer . VMP1 geg cancer . Additiog cancer . It is npatients . In addir sample . The samp-value (padj) of at most 0.05 in either pooled or paired interpretations. A list of 47 significantly differentially expressed genes that passed the statistical test (padj≤0.05) and genes that are reported to play biological importance is given in Table We performed significance testing of the tumor vs normal FC values to find differentially expressed genes in the larynx and hypopharynx samples, selecting for those with a corrected P-value 10−13, 10−9, 10−7 and 10−6 respectively). A list of all miRNA target genes, pathways, and gene annotations are provided in VMP1 in our study. It is early to state whether there is a relationship between the CLTC-VMP1 fusion detected in our study and tumor invasion and proliferation. Taken together, it was interesting to see that some of the miRNAs identified in our study are related to regulation of cancer stem cell-like cells related markers affecting invasion and metastasis.We performed pooled analysis on miRNA differential expression analysis to find significant changes in their expression in tumor samples compared with the corresponding normal samples . We founIn order to find a specific gene signature that distinguishes between the carcinoma of larynx and pharynx from those of the other subsites in the head and neck region, we performed random forest (RF) analysis  using gewnt antagonist that functions as a tumor suppressor that is epigenetically silenced in various cancers and is involved in regulation of cancer stemness and senescence [2FC = 0.7) but WIF1 promoters were methylated to a larger extent (10%) than the LP6 tumor. In another tumor sample LP10, the log2FC for WIF1 was 2.7 while 16% of the WIF1 Promoters were hypo-methylated. As a control, we used a tumor from oral cavity (OT6) to study WIF1 promoter methylation and found very strong WIF1 hyper-methylation . Post-treatment surveillance was carried out by clinical and radiographic examinations as per the NCCN guidelines. In the discovery set, ten treatment-naïve (primary) and three recurrent patient samples were used for sequencing study. Out of the thirteen samples used in the discovery set, nine were tumors of the larynx and four of the hypopharynx. In the validation set of 18 tumors that included the discovery set, twelve samples were from larynx; three were from hypopharynx; and three other pharyngeal sites. Clinical details along with the treatment details for all samples are provided in Details of the tumor specimens and matched normal samples collected and used in the study are presented in Table Sequencing libraries were prepared for both whole transcriptome and small RNA libraries.Total RNA was isolated using Purelink RNA mini kit  following manufacturer's instructions. Isolated total RNA was quantified using Qubit RNA Assay Kit (Invitrogen) before being used in the sequencing library preparation. For SOLiD whole transcriptome library preparation, two micrograms of total RNA was subjected to rRNA depletion using Ribominus Eukaryote Depletion System v2 . Post rRNA depletion, the samples were quantified by Qubit RNA kit again and used for library preparation using SOLiD Total RNA seq kit, following the manufacturer's instructions. In brief, rRNA-depleted RNA was fragmented, cleaned, hybridized to adaptors, reverse transcribed, purified, size selected by AMPure beads over two rounds (to remove any fragments below 150bp), amplified, and finally, purified again. It was then quantified using Qubit HS kit (Invitrogen) and DNA 1000 Bioanalyzer . Post library preparation, all the libraries were pooled in equimolar concentration and 0.6pM of the pooled library was used for the ePCR and enrichment step, as per the protocol. Following enrichment, the libraries were 3′ modified, loaded onto 4 lanes of the SOLiD flow cell and sequenced on SOLiD 5500xl sequencer. Data was obtained for 75×35 bp paired end reads (forward and reverse) in XSQ format.Small RNA was isolated from tumor and matched normal samples using Ambion's MirVana kit following manufacturer's instructions. Small RNA library was prepared for 5 tumor-normal pairs and its quality was analyzed on RNA6000 nanochip using Agilent Bioanalyzer. Small RNA SOLiD sequencing libraries were prepared following manufacturer's instructions (Life Technologies). In brief, the library preparation involved hybridization and ligation, reverse transcription, purification of cDNA using Minelute PCR purification kit (Qiagen), gel size selection, amplification of the size selected cDNA and purification using Purelink PCR micro kit (Life technologies). For the gel size selection, the Novex TBE urea 10% - PAGE gel system (Life technologies) was used to excise bands between 60-80 nucleotides. Each gel piece was cut into 4 portions and two such portions were used for the process of amplification. Thus for each sample the cDNA library amplification was done in duplicates, with one gel piece in each tube. The final amplification of the size selected cDNA library was done for 15-18 cycles. Each of the libraries was labeled with a different barcode using SOLiD RNA Barcoding Kit, Module 1-16, which enabled multiplexing. The libraries were quantified using Qubit HS kit (Invitrogen) and the size distribution was analyzed using DNA HS chip on Agilent Bioanalyzer. Post library preparation, the five tumor/normal pairs (10 samples) were pooled together in equimolar concentration into a single library and 0.4pM of the pooled library was used for the ePCR, using e80 scale, and enrichment, as per manufacturer's instructions. Following enrichment, the library was 3′ modified, loaded onto a single lane of the SOLiD-6lane flowchip cell and sequenced on SOLiD 5500xl genetic analyzer system. Sequenced data was obtained (35 bp single-end reads) in the XSQ format and further used for downstream analysis using Lifescope.For all the tumor and normal samples, reads were filtered against tRNA, rRNA, adaptor and repeat sequences using Lifescope (v2.5-r2.5.1). The remaining reads were aligned to the hg19 reference sequence with default options. Of these, only primary alignments with minimum mapping quality of 10 were counted and the output was stored in a tab-delimited file, containing all gene annotations and their raw read counts. Additionally, putative gene-fusions in all samples were detected using the ‘splice finder’ module in Lifescope. A junction confidence value (JCV) ranging from 1 to 100 is specified with each predicted fusion event. The closer the JCV value to 100, the likelier the fusion event is to be real.The gene-wise read counts of all the tumor and normal samples were pooled, and only those genes with a non-zero read count in at least one sample were selected. Normalization of the raw counts was done using the DESeq R package   and testwww.bioconductor.org). miRNA functional analyses using individual cancer-related pathway information were analyzed using the DIANA mirPath web server [Pooled miRNA expression analysis was carried out using DESeq2  R packagb server  that usehttps://tcga-data.nci.nih.gov/tcga/), raw RNA-seq read counts for matched tumor-normal TCGA HNSCC samples were downloaded and normalized using DESeq. For each sample pair, gene-wise tumor vs normal ratios of normalized counts, called fold changes (FC), were calculated. Genes with fold change calculations of less than 1 (FC<1) were taken as down-regulation (D), genes with FC>1 were taken as up-regulation (U) and FC=1 meant no change (X) in the expression of the gene in tumor with respect to matched normal. Furthermore, based on the subsite, all the above TCGA samples were classified into 2 types - TCGA_L , TCGA_O . Finally, for the set of significantly differentially expressed genes from SCCL and SCCHP samples, FC values  and TCGA_L or TCGA_O samples were used as input to the machine learning algorithm random forests [R) by using a formula:From the TCGA portal .Genes found to be significantly up- and down-regulated in larynx and hypopharynx tumors were validated using quantitative PCR (qPCR) experiments in eighteen tumor samples . Sequences of the methylation-specific primers, annealing temperature and amplicon size is provided in Genomic DNA was isolated from normal and tumor samples using DNeasy Blood & Tissue Kit (Qiagen), following the manufacturer's instructions. Genomic DNA (500 ng) was bisulfite converted using EZ DNA Methylation Kit (Zymo Research), and amplified using two primer sets, one set specific for methylated and the other for non-methylated DNA. Methylation-specific primers were designed using MethPrimer tool (The scripts used to analyze and interpret data are provided in Supplementary Text1.


The possible role of viruses in breast cancer etiology remains an unresolved question. We hypothesized that if some viruses are involved, it may be in a subgroup of breast cancers only. Epidemiological arguments drove our interest in breast cancer subgroups that are more frequent in Africa, namely inflammatory breast cancer (IBC) and triple-negative breast cancer. We tested whether viral prevalence was significantly higher in these subgroups.One hundred fifty-five paraffin-embedded malignant breast tumors were randomly selected at the pathology laboratory of the University Hospital of Annaba (Algeria) to include one third of IBC and two thirds of non-IBC. They were tested for the presence of DNA from 61 viral agents  using type-specific multiplex genotyping assays, which combine multiplex PCR and bead-based Luminex technology.Viral DNA was found in 22 (17.9%) of 123 tumors. The most prevalent viruses were EBV1 and HPV16. IBC tumors carried significantly more viruses (any type) than non-IBC tumors . Similarly, triple-negative tumors displayed higher virus-positivity than non-triple-negative tumors .Our results suggest an association between the presence of viral DNA and aggressive breast cancer phenotypes . While preliminary, they underline the importance of focusing on subgroups when studying viral etiology in breast cancer. Further studies on viruses in breast cancer should be conducted in much larger samples to confirm these initial findings. Breast cancer (BC) is the second most common cancer worldwide, with 1.67 million new cases per year in 2012 The International Agency for Research on Cancer (IARC) estimated that 15-20% of cancers are associated with infectious agents We based the present work on the hypothesis that if a virus that increases risk of BC exists, it may account for a higher attributable fraction of BC in developing countries than in industrialized countries, where almost all studies of viruses and BC have been conducted to date. Indeed, it has been shown that the proportion of cancers caused by viruses is substantially higher in developing countries, notably in Africa: in Western Europe, it is estimated to be 7% of all cancer cases, whereas this proportion reaches 40% on the African continent Inflammatory BC (IBC), a particularly aggressive form of BC Triple-negative BC, defined as BC that is negative for estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2), is another subgroup of interest since recent observations have suggested that this subtype of BC is more frequent in less developed countries (17% to 28%), notably in Africa The relation between IBC and triple-negative status has not been much studied, but the few studies published to date suggest that IBC may be triple-negative more frequently (20% to 40% of cases) than non-IBC The present study was conducted on samples from Algeria, a North African country. The two main objectives of the study were (1) to compare viral prevalence in IBC and non-IBC, and (2) to compare viral prevalence in triple-negative and non-triple-negative BC tumors. Viral prevalence was tested for using highly sensitive bead-based multiplex genotyping assays for the detection of 46 mucosal and cutaneous human papillomavirus types, 10 polyomaviruses including the simian polyomavirus SV40, and 5 herpesviruses.Tumors from patients of Algerian ethnic origin diagnosed between 2008 and 2011 at the University Hospital of Annaba were considered. The use of this tumor collection for research purposes was approved by the ethics committee of the University Hospital of Annaba. The ethics committee waived the need for written informed consent for the present study. Fifty IBC tumors and 105 non-IBC tumors, fixed in paraffin, were selected randomly from the cytology and pathology laboratory database. Epidemiological and demographic data were extracted retrospectively from medical records and cancer registry records and entered in EpiData software. The following were documented: morphology, tumor–node–metastasis (TNM) stage, “Poussée Evolutive” (PEV) stage , hormone receptor subtypes (ER/PR/HER2), age, housing type, socio-economic level, and menopausal status. Socio-economic level  was inferred from the district where the patient lived and her health insurance scheme. Since subtype testing is a recent technological advance, information about ER/PR/HER2 subtypes was not available for all patients, and notably was missing for those diagnosed before 2009. Inflammatory status was mentioned in the medical records, but patients were included as IBC in the final sample of the present study only if the IBC clinical diagnosis was corroborated by TNM (T4d) or PEV status (PEV3). Patients who were included as non-IBC in the final sample were all PEV0 (one tumor was excluded because of lack of information on the PEV status), and none were T4d. The final study sample included 45 confirmed IBCs and 104 confirmed non-IBCs. ER/PR/HER2 subtypes were available for 96 of these 149 patients.All paraffin blocks were processed at IARC, Lyon. Three sections of 10 µm were cut from each paraffin block. For each specimen, the blades were changed and the microtome extensively washed with DNA Away  to prevent the risk of cross-contamination between different specimens during the cutting. In addition, empty paraffin blocks were cut every 10 tumor specimens and blindly analyzed to monitor possible cross-contamination. No contamination was detected throughout the study.g in a bench centrifuge, and chilled on ice, to inactivate the proteinase K and to remove the paraffin. Finally, the aqueous phase was transferred to a new tube.DNA was prepared by incubating the paraffin tissue sections in digestion buffer  overnight at 37°C. Samples were incubated for 10 min at 95°C, centrifuged rapidly for 2 min at 15700The identification of 61 infectious agents was performed by using type-specific multiplex genotyping (TS-MPG) assays, which combine multiplex polymerase chain reaction (PCR) and bead-based Luminex technology , as described previously In situ hybridization for EBER was performed on EBV DNA positive cases using a FITC-labeled oligonucleotide probe  on an automated stainer (Ventana BenchMark). Visualization was achieved using the ISH iView system with Alk-Phosphatase and NT/BCIP substrate, counterstained with red .Statistical analyses were performed using SAS software . For most analyses, the Fisher exact Chi-square test, which enables the testing of very small numbers, was used. When tables were larger than 2×2, the classical Pearson Chi-square test was used.The demographic and clinical data for IBC and non-IBC patients are summarized in The amplification of the beta-globin gene showed that good-quality DNA could be obtained for 123 (82.6%) of the 149 paraffin-embedded tissue samples. The 26 samples negative for the beta-globin test were thus excluded from the analyses.The presence of viral DNA was detected in 22 (17.9%) of the 123 tumors. As detailed in The presence of viral DNA according to IBC status is detailed in The presence of viral DNA according to ER/PR/HER2 status is detailed in In order to rule out potential confounding factors, we investigated the viral prevalence  by age, housing type, education level, and socio-economic level. We found no significant difference in viral prevalence according to these variables, confirming that these variables could not be confounding factors (data not shown).p<0.03) but not in triple-negative tumors , with that in the subgroup of non-aggressive tumors, defined as non-IBC or non-triple-negative (ER+ or PR+ or HER+) (N = 46). The results were quite similar to those obtained in In situ hybridization for EBER was performed on 10 EBV DNA positive cases in order to determine whether EBV expression occurs in tumor epithelial cells or in infiltrating lymphocytes. Only 1 out of 10 cases, an IBC co-infected with EBV and HPV31, showed rare mammary tumor epithelial cells with nuclear staining for EBER . EBV-infThe major finding of the present study is that IBC and triple-negative BC have a higher tendency to be positive for oncogenic viruses, mainly EBV and HPV. Our results suggest that these viruses could act as co-factors in the oncogenic process that lead to a particularly aggressive form of BC. The study has several limitations, including the relatively small sample size and the absence of control , but it also has some unique strengths compared with previous studies; these are (1) the very large number of viruses tested for (to date no study has explored the presence of so many oncogenic viruses in BC), (2) the high quality of the laboratory assays, and (3) the rigorous collection and use of critical epidemiological information.Overall, we found a relatively low prevalence of viral DNA in our tumors from Algerian patients (18% of tumors were virus-positive). This is not attributable to the quality of the samples, because the amplification of the beta-globin gene ensured that all samples included in the analyses contained DNA of good quality. The low number of positive samples did not confer high statistical power to observe differences between subgroups; however, we found significant differences among the two subgroups we had chosen to study based on epidemiological arguments. There were significantly more viruses in IBC tumors (30% virus-positive) than in non-IBC tumors (13% virus-positive) (p<0.04), and there were significantly more viruses in triple-negative tumors (44% virus-positive) than in non-triple-negative tumors (14% virus-positive) (p<0.009). IBC and triple-negative status correlated in our sample ; the subgroup of aggressive tumors, defined as IBC or triple-negative, contained viral DNA in 32% of cases, compared with 9% for the subgroup of non-aggressive tumors (p<0.006).EBV was the most common virus. It was significantly more prevalent in triple-negative tumors (24%) than in non-triple-negative tumors (2%) (p<0.003) and also appeared more prevalent in IBC tumors (14%) than in non-IBC tumors (6%), although this difference was not significant. Studies in larger samples are required to confirm these associations.Both IBC and triple-negative cancers are known to be more aggressive than the other types of BC To date, only three studies have explored the prevalence of viral infection in IBC. In 2001, Fina et al. compared EBV load in 22 IBC and 76 non-IBC frozen tumors from Algeria and Tunisia and found no significant difference. The prevalence of EBV in their samples (40% of IBCs EBV-positive) was much higher than in ours To our knowledge there is no large study exploring viral infection in triple-negative BC, but some studies exploring association with hormone receptor subtypes have been conducted. In a well-conducted study of 196 frozen tumors from the south of France, Mazouni et al. found that EBV positivity was significantly more frequent among ER- than ER+ samples (45.4% vs. 27.9%) Our results on EBV corroborate those of the about 30 studies that report the presence of EBV in BC tumors , which is similar to that observed in BC from Chile (2.1%) Our results suggest an association between the presence of viral DNA and aggressive BC phenotypes . This corroborates the results of Mazouni et al. Our results are very preliminary; however, they underline the importance of focusing on subgroups, notably aggressive ones, when studying viral etiology in BC. They also highlight the fact that viruses should not be studied in isolation because some kind of viral collaboration may take place. These two recommendations imply that studies about viral etiology in BC should be conducted in much larger samples than has generally been done to date.Our results support the possible involvement of EBV and, to a lesser extent, HPV in some subgroups of BC. The fact that an EBV vaccine is under development


Salmo salar L.) juveniles (fry) fed diets containing genetically modified maize  expressing Cry1Ab protein from first-feeding were investigated during a 99-day feeding trial. Four experimental diets were made; each diet contained ∼20% maize, either Bt-maize or its near-isogenic maternal line (non-GM maize). One pair was fishmeal-based while the other pair included standard (extracted) soybean meal , with the intention of investigating responses to the maize varieties in healthy fish as well as in immunologically challenged fish with SBM-induced distal intestinal inflammation, respectively. Three replicate tanks of fry  were fed one of the four diets and samples were taken on days 15, 36, 48 and 99. Survival, growth performance, whole body composition, digestive function, morphology of intestine, liver and skeleton, and mRNA expression of some immune and stress response parameters in the distal intestine were evaluated. After 99 days of feeding, survival was enhanced and the intended SBM-induced inflammatory response in the distal intestine of the two groups of SBM-fed fish was absent, indicating that the juvenile salmon were tolerant to SBM. Mortality, growth performance and body composition were similar in fish fed the two maize varieties. The Bt-maize fed fish, however, displayed minor but significantly decreased digestive enzyme activities of leucine aminopeptidase and maltase, as well as decreased concentration of gut bile salts, but significantly increased amylase activity at some sampling points. Histomorphological, radiographic and mRNA expression evaluations did not reveal any biologically relevant effects of Bt-maize in the gastrointestinal tract, liver or skeleton. The results suggest that the Cry1Ab protein or other compositional differences in GM Bt-maize may cause minor alterations in intestinal responses in juvenile salmon, but without affecting overall survival, growth performance, development or health.Responses in Atlantic salmon ( Bt-maize (MON810) is the only GM maize event authorized and cultivated in the EU and is mainly used in animal feeds Ostrinia nubilalis. The Cry proteins are a group of endotoxins produced by different strains of soil bacterium Bacillus thuringiensis (Bt) and have putative species-specific toxicity for insects Bt-crops have been suggested to be as safe and nutritious as their conventional/commercial counterparts for various animals in some Bt-maize, concerns regarding possible allergenicity of GM plant crops, in particular the transgenic proteins, have been raised Since the first commercialization in 1996, safety issues regarding the introduction of GM crops into food and feed have been debated. Among the globally available GM crops, th Framework Programme EU-project entitled “Biomarkers for post market monitoring of short and long-term effects of genetically modified organisms on animal and human health” (GMSAFOOD) was funded to investigate whether such biomarkers could be identified. The project used Bt-maize as the authorized GM model. Model animals included mice, rats, pigs and fish at various stages of development.In concert, all these various concerns have elicited uncertainty regarding long term ramifications of GM crop consumption to the health and longevity of animals and humans. In addition to the extensive risk assessment prior to market authorization, post-market monitoring (PMM) may now be required by the EU commission, based on the specific results of the pre-market risk assessment, to address these concerns. When required, PMM is considered necessary to confirm the safety of the products in the long term, and to increase the probability of detecting unintended effects Salmo salar L.). A large share of the global market of some plant crops, such as maize and soybeans, are now genetically modified (GM). To assess its safety, feeding trials with GM Bt-maize in post-smolt Danio rerio) Bt-maize-containing diets displayed some differences in performance parameters and functional responses compared to control non-GM maize diets, including reduced feed intake and growth despite elevated activity of maltase in mid and distal intestine Bt-maize for 90 days displayed reductions in digestibility of protein and mineral, retention efficiency of lipid and energy, as well as activity of leucine aminopeptidase (LAP) in proximal intestine. Gene expression of the T cell marker CD4 and the cytokine interferon-γ in the distal intestine was increased Bt-maize, experimental groups were included that were simultaneously exposed to extracted soybean meal (SBM) in their diets, which causes an apparently T-cell-mediated type IV hypersensitivity response in the distal intestine of salmon Bt-maize potentiated oxidative cellular stress in the inflamed intestine, as indicated by increased transcription level of superoxide dismutase (SOD) and heat shock protein 70 (HSP70) Inclusion of plant crops in commercial diets for farmed fish is increasing in an effort to reduce dependence on limited marine resources such as fishmeal and fish oil, and to improve cost efficiency and sustainability of the aquaculture industry. This includes piscivorous species such as Atlantic salmon  unintended Bt-maize responses in general, 2) whether challenged fish with an intestinal inflammation would alter the response to dietary Bt-maize, as well as 3) whether any response parameters could be suitable biomarkers for GM responses to exposure. However, the data indicated that the juvenile salmon did not respond with intestinal inflammation to SBM as more developed salmon do, suggesting tolerance to SBM. Thus the second aim was not achieved.The present paper documents the results from a 99-day feeding trial on developing Atlantic salmon juveniles fed Rearing of the fish was conducted at Nofima's Research Station , which is an approved research facility by Norwegian Animal Research Authority (NARA) and operates in accordance with the Norwegian Regulations of 17 June 2008 No. 822: Regulations relating to Operation of Aquaculture Establishments (Aquaculture Operation Regulations). Up to sacrifice and sampling, the fish were treated as production fish in accordance with aforementioned Aquaculture Operation Regulations. Prior to sampling, the fish were humanely anaesthetized before euthanasia in accordance with the Norwegian Animal Welfare act. Hence, no NARA approval was required according to Dr. G Baeverfjord (Nofima), appointed by NARA.Bt-maize (MON810) and its near-isogenic maternal line (non-GM maize) derived from planted seed varieties PR34N44 and PR34N43, respectively, were provided by Pioneer . They were grown simultaneously in neighboring fields in Spain. Details regarding nutrient composition, pesticide and mycotoxin levels, and level of Cry1Ab gene insert in the two maize meals have been previously reported www.denofa.com). The diets were balanced regarding vitamins and minerals according to estimated requirements Two maize types, The experiment was carried out using a 2×2 factorial design with four diet groups. The factors GM and SBM inclusion were tested separately and in combination. The fry of SalmoBreed origin with an approximate initial weight of 0.17±0.01 g (mean ± standard error of the mean) were randomly allocated, ca. 1200 fry per tank, to 12 tanks, 60 cm in diameter. Each of the four experimental diets was fed to triplicate tanks of fish. The fresh water level was kept at 19 cm at initiation of the feeding period and later elevated to 32 cm when the fish started to swim upwards in the water column. The feed pellet size at start was 0.6 mm, and was subsequently adjusted to 0.9 mm and 1.3 mm as the fish grew to 1 and 3 g, respectively. The tanks were supplied with filtered fresh water at approximately 12°C. The fish were fed by automatic belt feeders set to supply feed every 10 min, and the feeding level was 20% in excess of estimated feed requirement. Due to the small size of the feed pellets, feed intake could not be accurately measured. Light was provided continuously. All mortalities were recorded.Samplings were conducted following 15, 36, 48, and 99 days of exposure to the experimental diets. Sampled fish were in the fed state as fasting may affect physiological parameters Diets were analyzed for dry matter, crude protein, and crude lipid. At the end of the 99 day feeding trial, one pooled sample of 20 whole fish per tank was analyzed for whole body composition of dry matter, crude protein, crude lipid, and ash. Analyses were performed at Nofima AS  following standard methods. In short, dry matter was measured by drying at 105°C for 16–18 h. Nitrogen (N) was analyzed according to semi-micro-Kjeldahl method with Kjeltec-Auto System  and crude protein was calculated as N*6.25. Crude lipid was determined in a Fosstec analyser  after diethyl ether extraction. For ash, samples were weighed before and after burning at 550°C.One pooled sample of 15 whole fish per tank on days 15 and 36, and 10 whole fish per tank on days 48 and 99, respectively, were analysed for activities of pancreatic enzymes trypsin and amylase, brush border membrane enzymes leucine aminopeptidase (LAP) and maltase, as well as bile acid concentration. At the last two samplings, one pooled sample of 10 dissected intestinal sections  per tank was also analysed for the above-mentioned parameters. On the day 48 sampling, the small size of the intestines made distinguishing the MI from the DI intestinal regions difficult. Thus the MI and DI (MI+DI) were analysed together, while the PI, with its visible pyloric caeca, was analysed separately. On day 99, the DI was analysed separately, while MI and PI samples (PI+MI) were analysed together.LAP and maltase activities, investigated to assess intestinal function, were analyzed following the method described previously 2O. Trypsin activity was measured colorimetrically according to Kakade et al. −1 tissue homogenate.For trypsin activity analyses, the frozen tissue with intestinal content was homogenized and suspended in dH−1 tissue homogenate.The bile salt concentration in samples was determined using the Enzabile kit from Nycomed Pharma AS Diagnostics  and expressed as mg gFormalin-fixed liver, PI and DI samples from four fish per tank, i.e. 12 fish per diet, sampled on day 99 were routinely dehydrated in ethanol, equilibrated in xylene and embedded in paraffin according to standard histological procedures. Sections of 3–5 µm were stained with haematoxylin and eosin (H&E) and blindly evaluated under a light microscope  by a trained veterinarian. Morphology of PI and DI was evaluated according to the criteria previously described in Atlantic salmon Radiography of frozen fish from the day 99 sampling, 50 to 58 individuals per tank, was conducted in a semi-digital system, with a Giotto Image  mammography X-ray source combined with an FCR Profect image plate reader and FCR Console . The system comprises automated image enhancement-procedures for contrast enhancement and edge visualization. The skeletal morphology was evaluated by trained personnel according to the following criteria: (1) fusions and fusion-associated changes; (2) compressed vertebrae in neck; (3) complex deformities, consisting of a mixture of fusions, compressions and distorted shapes; (4) head deformities.http://frodo.wi.mit.edu/primer3). See Real-time quantitative PCR analyses were limited to the DI, where possible interactions in SBM-fed (immune-sensitised) salmon would be expected. Transcriptional expression of T helper cell marker CD4, the cytokines interleukin 17a (IL17a) and interferon gamma (IFNγ), proliferating cell nuclear antigen (PCNA), and heat shock protein (HSP) 70 in the DI tissue were assessed. Total RNA was extracted from dissected DI on the day 99 sampling from three fish per tank, i.e. nine animals per diet. RNA purification and quality control, DNase treatment, complementary DNA synthesis, primer optimisation and quantitative PCR assays were performed as described in detail elsewhere K = bw/fl3Condition factor K : Specific growth rate SGR : p-values are two-sided and significance was set at p<0.05. Trends/tendencies towards significance are discussed when p-values were in the range of 0.05–0.10.The data were statistically evaluated using JMP version 9.0 (2010) Statistical Discovery™ (SAS Institute Inc.). Most of the results were subjected to two-way ANOVA with GM and SBM inclusion as the class variables and performed on calculated tank means or results from pooled samples (n = 3 tanks for each dietary treatment) based on a variable number of sampled individuals per tank depending on sampling time and parameter (see respective sections above for details). However, the histological scores were compared using non-parametric contingency analysis and performed on data from 12 fish per dietary treatment (n = 12). All reported Analyzed proximate compositions of the experimental diets were similar to expected compositions . The proBt-maize, SBM effects are briefly described first before the focus is shifted to GM effects and interactions between GM and SBM.Very low mortality was observed among the fish throughout the experiment with a cumulative mortality of ≤2.5% (pooled standard error 0.33), indicating high quality feed and husbandry conditions. Interestingly and contrary to studies in more mature salmon, survival and performance of the juveniles was enhanced by addition of SBM to the experimental diets and no SBM-induced inflammatory changes in the distal intestine were observed. As this was an unexpected finding with implications for the interpretation of the results regarding effects of the p<0.10) to be higher at day 48 and 99 compared with non-SBM fed fish. Moreover, significantly higher body weight, fork length and SGR were observed in SBM-fed fish on day 99. When differences were observed in LAP (p<0.10) towards lower activities, with an exception of significantly higher LAP activity in PI on day 48  but was significantly lower in PI+MI on day 99 (p = 0.0169) in SBM-fed fish  and the same interaction was observed between GM and SBM effects as for LAP activity , and in DI+MI an interaction between GM and SBM effects was detected, in which fish fed the Bt-maize diet with SBM inclusion displayed significantly lower maltase activity compared to fish fed Bt-maize without SBM  effects on growth performance in fish has shown varying results. Hemre et al. Bt-maize study in post-smolt salmon, in which the same batch of Bt-maize was used Bt-maize diet further supported absence of any Bt-maize or SBM effects on general health in the juveniles.Earlier reports of Bt-maize, some mild changes in intestinal function were apparent. Similar findings were also observed in our previous study in post-smolts Bt (Cry) protein Bt-maize containing diets Bt-maize effects on survival, growth performance and whole body composition suggests that any minor loss of function measured was not severe enough to impair overall fish performance and health and may simply reflect compensatory responses to compositional differences in the two maize meals Although survival and growth did not seem to be affected by the Bt-maize fed post-smolt salmon Bt-maize fed salmon juveniles. To our knowledge, this was the first study on effects of any GM plant ingredient on animal bone health. The very low frequency of skeletal deformities in general and no differences attributed to Bt-maize consumption specifically suggest that Bt-maize did not affect bone development of juvenile salmon in this 99 day trial. In addition, the histological examinations indicated that the presence of Bt-maize in diets did not induce major impairment to any organs or tissues examined. This conclusion is in line with other studies of effects of GM ingredients in Atlantic salmon Skeletal deformities can be a problem in intensive salmon farming, especially for first-feeding fish due to their rapid growth, high nutrient requirements and relative lower efficiency of nutrient and mineral utilization in vitro digestion trial, in which Cry1Ab was only slightly degraded at pH 2, even at high pepsin-to-substrate ratio Bt-maize fed pigs Bt-maize may activate a mild, local IL17a-mediated immune response in juvenile fish. The magnitude of the up-regulation, however, was less than 2-fold and therefore not considered a sign of inflammation in the DI. This was confirmed by the absence of inflammatory changes as assessed by histomorphological evaluation. During SBM-induced inflammation in older salmon, IL17a expression has been reported to increase more than 200-fold Bt-maize. The different Bt-maize effects found in juvenile and post-smolt salmon may be explained by differences in immune responses between the developmental stages, as indicated also by the lack of DI inflammatory response to SBM in the juvenile fish. The differing responses also preclude any conclusions regarding potential biomarkers for Bt-maize exposure or responses in Atlantic salmon.Previous mammalian studies have indicated immunogenicity of Cry1A protein Bt-maize for 99 days from first-feeding showed similar survival, growth performance and feed utilization  as those fed diets with the non-GM near-isogenic maternal line. Furthermore, microscopic and radiographic examinations did not reveal negative Bt-maize effects on the liver, intestinal tract or skeletal morphology or development. However, the Bt-maize diets apparently did somewhat alter digestive function as indicated by significant reductions in LAP and maltase activities and gut bile salt concentrations, and increased amylase activity at some sampling points. IL17a expression in the DI following 99 days of exposure was also significantly increased. But these responses may not be of biological significance since they did not appear to impact general fish health, and may be as much due to compositional differences in the two maize meals Bt-maize would change a hypersensitivity response in salmon was not resolved, since the juvenile fish were apparently tolerant to SBM inclusion in the diet.In conclusion, the Atlantic salmon juveniles fed