The HIV-Brazil Cohort Study was established to analyze the effectiveness of combination antiretroviral therapy (cART) and the impact of this treatment on morbidity, quality of life (QOL) and mortality. The study design, patients’ profiles and characteristics of cART initiation between 2003 and 2010 were described.+ T-cell count ≤200 cells/mm3. The medications most often used in the various treatment regimens were efavirenz (59.7%) and lopinavir/ritonavir (18.2%). The proportion of individuals achieving viral suppression within the first 12 months of cART use was 77.4% (95% CI 76.1–78.6). Nearly half (45.4%) of the patients presented HIV-related clinical manifestations after starting cART, and the AIDS mortality rate was 13.9 per 1,000 person-years.Since 2003, the HIV-Brazil Cohort has been following HIV-infected adults receiving cART at 26 public health care facilities, using routine clinical care data and self-reported QOL questionnaires. When not otherwise available, data are obtained from national information systems. The main outcomes of interest are diseases related or unrelated to HIV; suppression of viral replication; adverse events; virological, clinical and immunological failures; changes in the cART; and mortality. For the 5,061 patients who started cART between 2003 and 2010, the median follow-up time was 4.1 years (IQR 2.2–5.9 years) with an 83.4% retention rate. Patient profiles were characterized by a predominance of men , with a mean age of 36.9 years (SD 9.9 years); 55.2% had been infected with HIV via heterosexual contact. The majority of patients (53.4%) initiated cART with a CD4Results from cART use in the daily practice of health services remain relatively unknown in low- and middle-income countries, and studies with the characteristics of the HIV-Brazil Cohort contribute to minimizing these shortcomings, given its scope and patient profile, which is similar to that of the AIDS epidemic in the country. In 1996, Brazil adopted a policy of universal access to combination antiretroviral therapy (cART), which was free of charge to human immunodeficiency virus (HIV)-infected individuals. Since then, immunological and virological treatment monitoring, as well as genotype-resistance testing for treatment failure management, have been incorporated into that policy. As of 2012, approximately 217,000 patients in Brazil were being treated with first-, second- or third-line antiretroviral therapy, including newer antiretroviral options for salvage therapy management, leading to an overall reduction in the morbidity and mortality associated with HIV infection In recent years, there has been a reduction in the magnitude of the impact that opportunistic diseases have had on morbidity and mortality in cART patients in Brazil, although there has been an increase in the incidence of complications unrelated to infection by HIV, such as cardiovascular events, impaired renal function, liver disease and neoplasia The impact of the Brazilian policy has been the object of nationwide studies, mostly focused on mortality Furthermore, recently, important changes have occurred in the use of cART therapy due to the proven efficacy of these drugs to reduce the risk of acquiring and transmitting HIV infection, as well as increased evidence that early initiation of cART has significant health benefits These circumstances increase the relevance of studies assessing the various dimensions of access to diagnosis and treatment of HIV infections; the efficacy and effectiveness of such treatment; and the impact of this treatment on morbidity, mortality, and QOL. Therefore, the HIV-Brazil Cohort Study was established to gather further knowledge on the context of the Brazilian epidemic and to generate scientific evidence to make informed decisions regarding the planning and implementation of public policies, taking into consideration the regional disparities, as well as the specificities of the Brazilian population and of the national health care system There are 13 sites participating in the study, involving 26 public health facilities in 11 cities across four of the five regions of the country . The facThe number of patients under treatment at the participating sites ranged from 519  to 34,932 , with 7 of the 13 sites being responsible for treating more than half of the AIDS cases identified in their municipalities. Across the 13 sites, the mean ratio of outpatients to infectious disease specialists or general clinicians is 299∶1 . Among the patients on cART, the interval between clinical consultations has ranged from 3 to 6 months, and 6 of the 13 sites have adopted outreach strategies for patients who miss appointments .At most of the sites , the heaAs of November 2012, 6,109 HIV-infected adults (≥18 years of age) had been enrolled in the cohort. All the included individuals had initiated antiretroviral treatment on or after January 1, 2003 , and had had at least one clinical follow-up appointment after treatment initiation. The inclusion occurred in two phases. Between 2009 and 2011, patients who had been started on cART between January 2003 and December 2010 were enrolled, whereas in the second phase, patients who had been started on cART on or after 1 January 2011 were enrolled. The first phase was aimed at observing events of interest that had occurred before the patients had been included in the cohort (retrospective phase), and the second phase was aimed at observing such events as they occurred, over the course of follow-up performed as routine clinical outpatient services (prospective phase).+ T-cell count were observed. The principal differences observed were that the proportion of females was greater in the non-probabilistic sample of the São Paulo Municipal Network than in the patients of the southeastern cohort (36.4% vs. 31.8%) and that the mean CD4+ T-cell count was higher in the non-probability sample of the Federal University of Minas Gerais than in the patients of the southeastern cohort (204.2 cells/mm3 vs. 174.2 cells/mm3).The cohort data were obtained as part of the routine clinical care provided at the health service centers included (routine-care based cohort) and were abstracted from the patients’ clinical records by trained abstractors onto standardized forms. These clinical records were reviewed at intervals not exceeding six months to investigate events recorded during the routine clinical follow-up visits performed within each period. To determine the consistency and completeness of the information, the forms were checked at the cohort coordination level. Approximately 5% of the forms were crosschecked against the information in the clinical records. From 2012 on, information has been collected using an online system developed on the Research Electronic Data Capture platform Information on QOL started being collected in 2011, using the World Health Organization self-report questionnaire  validated for use in Brazil The main outcomes of interest are the occurrence of diseases related or unrelated to HIV; suppression of viral replication ; antiretroviral treatment modifications ; treatment failure ; adverse events; and death. Clinical and epidemiological data were collected for the entire clinical follow-up period, including socio-demographic data; HIV transmission category; use of illicit drugs, alcohol and tobacco; individual or family history of metabolic disorders, hypertension or cardiovascular disease; AIDS-related and non-AIDS-related manifestations; initial and subsequent cART regimens used for prophylaxis and treatment; occurrence of adverse events related to the use of cART, prophylaxis for opportunistic diseases and vaccines; CD4, viral load and genotyping results, as well as safety laboratory tests for cART monitoring; and mortality.+ T-cell counts, viral load and mortality data–were systematically checked across the national information systems.To enhance the level of completeness, missing data–notably, demographic data, CD4+/CD8+ T Lymphocyte Count and Viral Load Network Laboratory Test Control System ; the National System for the Logistic Control of Medications (for information regarding the dispensing of cART); and the National Mortality Information System . The screening was conducted annually, through electronic matching, checking for perfect combinations of key fields . Patients not located through this procedure were then investigated using an alphabetically sorted list.Individuals were censored in case of loss of clinical follow-up, relocation for follow-up in another clinical setting or death. To check the status of patients in the cohort, those categorized as lost to clinical follow-up were traced in the national information systems managed by the Brazilian National Ministry of Health: the National CD4+ T-cell count, viral load determination or ART dispensation elsewhere after the date of the last entry in the clinical record. AIDS-related deaths were defined as those in which the official record listed AIDS as being the underlying cause of death.Loss of follow-up was defined as an absence of patient contact with the health care center for more than 12 months  and the non-identification of the patient in the national information systems, as described above, up to July 2011. Patients were classified as having been relocated for follow-up in another clinical setting when relocations were noted in the clinical record or when the patients were identified in the information systems as performing their CD4Proposals from external researchers to use our data are encouraged and welcomed, and requests will be analyzed by the Cohort Steering Committee that is composed of the Cohort Principal Investigators. The use of data pertaining to the individual sites is restricted to the sites themselves. In all circumstances, the confidentiality of each participant’s related data must be preserved.This protocol was approved by the Institutional Review Boards (IRB) of the participating sites , according to the Brazilian regulation for research with human subjects. In the first phase, the IRB waived the requirement for written informed consent and requested the confidentiality of the individual’s data, which was ensured at all stages of the project. In the second phase, all participants provided written consent for participation in the study.The socio-demographic, clinical and immunological profiles of the 5,061 patients enrolled during the first phase of the study (those started on cART between 2003 and 2010) are shown in As shown in The largest proportion of patient loss  was observed in the first year of cohort follow-up, with reductions over time. The loss to follow-up rate ranged from 19.8 per 1,000 PY in the first 12 months to 1.1 per 1,000 PY after the sixth year of observation, with an overall loss rate of 10.1 per 1,000 PY. The greatest risk of loss  was obse+ T-cell count of 177.5 cells/mm3 (SD 121.7 cells/mm3) and 169.0 cells/mm3 (IQR 75.0–250.0 cells/mm3), respectively. These values remained stable over the years evaluated . The majority of patients (53.4%) initiated cART with a nadir CD4+ T-cell count ≤200 cells/mm3, and the CD4+ T-cell count at cART initiation was ≤350 cells/mm3 in 84.8% of the sample.Information on the pre-cART immunological status is available for 89.6% of the patients, who presented with a mean and median nadir CD4The initial treatment regimens prescribed were in line with the current recommendations of the Brazilian National Ministry of Health The median number of regimens used per patient was 1 , and the mean duration of the first-line regimens was 30.4 months (95% CI 29.7–31.2). The number of individuals achieving viral suppression (VL <400 copies/ml [2003 to 2006] and <50 copies/ml [after 2007]) within the first 12 months after cART initiation was 77.4% (95% CI 76.1–78.6). During the 6.5 years of observation, 88.2% (95% CI 87.2–89.1) of the patients achieved viral suppression at some point after cART initiation. The lowest rates of viral suppression in the first year of cART were observed in individuals aged between 18 and 29 years (66.2%), in transmission category injection drug use (73.7%), with cocaine use before treatment (74.1%), in service areas with lower economic development  and showing no adverse ARV events (85.2%).Nearly half (45.4%) of the patients presented with HIV-related clinical manifestations after starting cART, and the most frequent were oral candidiasis and tuberculosis. The AIDS mortality rate for the period evaluated was 13.9 per 1,000 PY.The HIV-Brazil Cohort Study, with a long follow-up period and a significant number of observations, is an important asset to increase the availability of data on the countrywide outcomes of the National AIDS Program related to cART in public health care services. A special feature of our cohort is that HIV-infected individuals in Brazil have been exposed to a broad array of antiretroviral drugs–21 antiretroviral drugs of all classes (including 10 generic drugs) used in first-, second- and third-line treatment regimens The characteristics of the HIV-Brazil Cohort Study facilitates analyses of the short-, medium- and long-term impact of using antiretroviral drugs in the context of the routine care of patients at publically funded health care centers, providing complementary evidence to that obtained through clinical efficacy trials. The evidence obtained from the analyses performed in this cohort will enable field evaluations and further improvement of the national public health policies The socio-demographic profile of the HIV-Brazil Cohort Study is similar to that represented by the cases of AIDS in adults reported to the Brazilian National Ministry of Health between 2003 and 2010 . The two+ T-cell count, such as gender, age and transmission category, did not differ (p<0.05) from the other patients included in the cohort. Although the analysis of outcomes directly related to the CD4+ T-cell count showed few significant variations when excluding individuals without baseline information on CD4+ T-cell count, the mortality rate rose from 13.9 to 14.2 per 1,000 PY.The quality assessment of the data collected showed a satisfactory degree of completeness of the information for essential variables . A lack An important aspect to be highlighted is the relatively low rate of loss of follow-up, similar to what is observed in high-income countries and in the middle- and low-income countries reporting the lowest rates of loss of follow up The analysis presented in this article aims to outline the scope and potential use of the information derived from the HIV-Brazil Cohort Study. First-line non-nucleoside reverse-transcriptase-based regimens were the most prescribed first-line cART in Brazil and resulted in high levels of viral suppression in the first year of treatment, comparable to single-site observational studies + T-cell counts ≤200 cells/mm3) or AIDS-related diseases at cART initiation. This finding was higher than the results of previously reported studies that reviewed the clinical and immunological status at the time of arrival to the health services for initial clinical follow-up in Brazil The majority of patients presented with severe immunodeficiency (CD4It is also noteworthy that the mean CD4+ T-cell count at the beginning of cART did not show a trend of increase over time, indicating the urgent need for increased access to earlier HIV diagnosis and linkage to care countrywide, considering the fact that Brazil has a concentrated epidemic in social sectors with a high degree of stigma and a pattern of service use that is more restricted than in the general population Another important aspect is the fact that the worst results were observed in regions with the lowest economic development levels in the country, indicating that the reduced ability of these regions to obtain optimal results from the therapies available for AIDS may lead to an increase in the health inequalities existing in the country through increase HIV incidence and HIV-associated mortality.The study design, characteristics of patients included and the initiation of cART presented in this paper show the scope of investigation conducted by the HIV-Brazil Cohort Study, which is the main initiative to analyze the effects of cART use in public health services in the country. Thus, the results shown herein and those yet to be produced can contribute to advancing the knowledge generated from clinical trials, particularly considering the context of medium- and low-income countries, which in recent years have greatly expanded their programs to access cART. This is important in the formulation of better health policies for the needs of people living with HIV in resource-constrained situations.


Allium ampeloprasum), is a monocotyledon plant of the lily family (Liliaceae). It belongs to the genus Allium, has a characteristic taste and morphological features, making it to be considered as one of the popular herbal medicine. This research was conducted with the purpose of obtaining optimal conditions for tissue culture of Persian shallot and comparing its active ingredient production in vitro versus in vivo. In this study, the auxin 2, 4–D and benzyl aminopurine- 6 (BAP) hormones, each at two concentrations (0.5 and 0.1 mg/ L) and Kin at 0.5 mg/ L were used in the format of a randomized complete block design in three replications. Results showed that the best culture media for callus formation for leaf and seed explants were the MS cultures with the hormonal compositions  and . Identification of the chemical composition of the essential oils, extracted either from leek callus or leaf was carried out using GC mass analysis. Twenty one compounds were detected in the GC mass spectra, seven of which constitutv about 51.5% of the total amount of compounds present in the essential oils were identified. Our data demonstrate that the leek essential oil constituents as well as callus formation can be affected by culture medium condition.Persian shallot, also called leek ( Allium ampeloprasum L.) is one of the widely used vegetables in Iranian diet and is considered as a medicinal plant  were soaked in water for 6 hours and then sterilized in the sterile chamber for 10 mins in a solution containing 1.25 % sodium hypochlorite to which a few drops of 20% tween had been added. Then, the sodium hypochlorite was slowly drained and the seeds were washed five times, each time for two mins, with sterile distilled water. Then, the sterilized seeds were grown in the hormone– free MS medium to produce sterile plant. The sterilized explant samples, including leaf segments obtained from seed culture were evaluated. These seeds had been cultured in the base culture medium MS that contained 30 g/ L of sucrose and 8 g/ L of agar, the hormone kinetin and auxins such as 2, 4– D, kinetin, and BAP at different concentrations. The hormonal treatments using explant– leaf samples used in this study were as follows: the 2, 4–D treatment at two concentrations of 0.1 and 0.5 mg/ L, the Kin treatment at the 0.5 mg/ L, the BAP treatment at two concentrations of 0.1 and 0.5 mg/ L  in the format of randomized complete block design and in three replications. The leaf explants and seed samples transferred into petri dishes containing 25 ml of the culture medium were placed in the dark at 25± 2°C. The percentages of callus formation were measured and the mean callus wet weights were compared to find the best hormonal treatment condition.Seeds of local leek  was performed using the method of distillation with water and the Clevenger apparatus from 30 grams of callus during 180 mins of extraction time.The essential oils of leek were extracted using the method of distillation with water and injected into the GC- mass apparatus under thermal scheduling. After injection, the GC-mass spectra and the mass spectra of the constituents of each essential oil were obtained. Then, the constituents of each essential oil, Kovats retention indices (K1), and the percentage of each constituent were determined by comparing the mass spectra of unknown compounds with those of standard compounds, and also by calculating retention indices. The gas chromatograms show the peaks according to the order of retention times. The greater the area under the peak, the higher the percentage of the constituent in the essential oil.Chemical analysisThe essential oils were identified using the chromatography method by GC mass (model Hewelet 6890 with Detector 5975C). After extracting ther by the Clevenger, the essential oil was dried by dry sodium sulfate and then injected into the GC mass under the thermal scheduling program. Next, the constituents of each essential oil were determined.Statistical analysisThe experimental data were evaluated by employing the SPSS software on the basis of Duncan’s multiple range test at 5% probability level.Leaf segmentsThe leaf segment explants and the seed samples started to swell gradually after they were cultured in the callus formation medium for two weeks, and started to produce calli after 3 weeks. In most experimental treatments, the majority of leaf explant samples and all seed samples turned into calli. Therefore, the weights of the calli formed in the treatments were compared in order to compare different treatments.The comparison of means revealed that the effects of different hormonal compounds and their mutual effects on callus formation of leaf explants  and seedEffects of the 2, 4– D and BAP on the callusComparison of the means of the data indicated that the hormonal treatments containing 0.5 mg/ L of 2, 4– D and 1 mg/ L of BAP , and theThe effects of the type of leaf explant samples on the percentage of callus formationThe results obtained from analysis of variance of the type of leaf explant samples  and 3 shThe results concerning the analysis of variance of the effects of different growth regulators on the percentage of callus formation in Allium sp. leaf explants and seeds are presented in GC- mass spectraThe percentage of the essential oils (w/w) were 0.3% from callus organ by the water extraction method. The peaks of the GC- mass of the essential oil of the plant leek obtained from leaf culture and the essential oil of its callus obtained by water extraction are presented in Constituents of the essential oils of leaves and calli of leek The active constituents in the leaves of leek accounting for 96.5% of compounds found in these leaves shown in  were napSeveral endogenous and exogenous factors on the plant are effective in callus formation and growth. The chemical factors, mineral elements, and growth regulators are the most important growth factors that influence plant differentiation and growth initiation. In cell culture, growth and morphogenesis are similarly controlled by the type and concentration of plant hormones and the mutual relationships between the hormones. In our study, an auxin and a cytokinin were used as hormones to induce callus formation. Auxins are commonly required to induce callus formation in leaf explants and seed samples. The most common auxins used for inducing callus formation is 2, 4–D. However, the use of 2, 4– D must be limited as much as possible, because it can cause mutation and may also prevent photosynthesis. Moreover, The joint use of cytokinins and auxins results in the stimulation of cell division. In this research, the hormones 2, 4– D, BAP, and Kin were used to induce callus formation. In some of the treatments, callus formation was induced due to the presence of auxins; and this shows the importance of the use of auxins in inducing callus formation. In our study, it was found that the mutual effects of these three hormones were significant, and that the highest percentage of callus formation belonged to the T4 and T5 treatments, which included the use of 0.5 mg/ L of 2, 4– D and 1 mg/ L of BAP, and 0.1 mg/ L of 2, 4– D and 0.5 mg/ L of Kin, respectively. The hormone 2, 4– D at the three levels of 0.5, 0.1, and 0.0 mg/ L, and the hormone Kin at the two levels of 0.5 and 0.0 mg/ L were used in separate treatments to evaluate their effects. Comparison of the appearances of the calli obtained in media containing different hormonal combinations showed that in media including 2, 4– D as the only source of inducing callus formation the calli were white, while yellow calli were produced in media containing BAP. The type of leaf explants sample is one of the important factors that greatly influence callus formation. In this experiment, which was conducted to compare the effects of the type of explants sample on the percentage of callus formation, the largest callus wet weight (1.3020 g) belonged to explants leaf sample (as compared to seed samples).Allium (These results show that the hormonal treatments have not had any significant effect on callus formation. The embryogenic callus cultures were friable, nodular and similar in appearance to those obtained leek -16. The Allium , 17-18.The active constituents of essential oil according to in vitro might be manipulated in a way to optimize the bioactive molecules production toward useful medicinal drugs.The obtained results indicate that the leek essential oil constituents as well as callus formation can be affected by culture medium condition. Since the active ingredients of leek have many pharmacological properties, by controlling the culture medium ingredients, the quality and constituents of leek essential oil produced The authors declared no conflict of interest.


Very limited information is available regarding the incidence of cancer in sub-Saharan Africa. We analyzed changes in cancer patterns from 1991 to 2008 in Maputo (Mozambique).We calculated the rates of incidence of different cancer sites by sex in the 5-year age-group of the population of Maputo city as well as age-standardized rates (ASRs) and average annual percentage changes (AAPC).5 in 2003–2008. In females, the most frequent cancers were of the uterine cervix, the breast and KS, with the former increasing along the whole study period  with an ASR of 62.0 per 105 in 2003–2008 as well as breast cancer .Over the 18-year study period a total of 12,674 cases of cancer  were registered with an overall increase in the risk of cancer in both sexes. In males, the most common cancers were those of the prostate, Kaposi sarcoma (KS) and the liver. Prostate cancer showed the most dramatic increase over the whole study period , with an ASR of 61.7 per 10Overall, the risk of cancer rose in both sexes during the study period, particularly among cancers associated with westernization of lifestyles , combined with increasingly rising incidences or limited changes in cancers associated with infection and poverty . Moreover, the burden of AIDS-associated cancers has shown a marked increase. Knowledge of cancer patterns in the different populations is crucial to guide preventive efforts –4. For tAfter the end of the civil war in 1991 the Department of Pathology of the Maputo Central Hospital (MCH) started a cancer registry with the aim of obtaining information on the occurrence of cancer in the population of Maputo city. This lengthy registration of data has provided a unique opportunity to study temporal trends in cancer patterns in an African setting.Interestingly, epidemiological data had previously been obtained by the Department of Pathology of the MCH  from 1956 to 1961 in a cancer survey covering the metropolitan area of 60 square kilometers of Maputo (then known as Lourenço Marques) [More than 50 years later, significant social and epidemiological changes have taken place in Mozambique, with a progressive urbanization of the population and the eruption of the HIV epidemic which has disproportionately affected many sub-Saharan African countries.In the present study we analyzed the changes in cancer patterns in Maputo city, Mozambique from 1991 to 2008.This study was performed in the Department of Pathology of the MCH, a 1500-bed hospital that is the only quaternary care center in Mozambique and is a national referral center. This department primarily serves the Southern Zone of Mozambique including Maputo and its surroundings , with a small proportion of cases derived from other areas of the country due to the national referral nature of the institution. The department receives virtually all specimens from the city of Maputo. The department has a database of all the samples received for pathological diagnosis including specimens of most types of cancers .th version of the ICD[This study was approved by the National Bioethics Committee of Mozambique, and the Mozambican Ministry of Health (Ref. 389/CNBS). The analysis included all the cases of cancer registered in the Department of Pathology of the MCH from January 1, 1991 to December 31, 2008. Data were entered into a Microsoft Access database  which, upon data entry, prevents the use of nonexistent codes and performs checks for internal consistency between variables. The database was carefully reviewed  to confirm that no duplications had been registered. Information about previous analyses in the same patient was also obtained, in order to identify potential duplicate registrations. Cases were identified in the registries as histological studies , cytological specimens, and autopsies. For each case identified basic demographic data were collected, including age, sex and address. Patients from sites other than Maputo were excluded. For each specimen, the date of diagnosis, method of diagnosis, site from which the specimen was taken (topography), and microscopic morphology were recorded. Patient records/information were anonymized and de-identified prior to analysis. All topographic and morphologic classifications were made according to the International Classification of Diseases for Oncology (ICD-O),11 and wf the ICD. In concf the ICD,7. MultiCensuses of the population of Mozambique were performed in 1980, 19971414 and 20The rates of incidence of cancer were calculated for the 5-year age-group by sex, for each year (1991–2008) and for three time periods: 1991–1996, 1997–2002 and 2003–2008. Age-standardized rates (ASRs) were calculated using the world standard population. The ave5 in males and 102.6 per 105 in females, being 182.7 per 105 in males and 186.0 per 105 in females in the period from 2003–2008. 5 in 2003–2008 (5 versus 3.9 per 105)  a total of 12 674 cases  were registered. The rates of the incidence of cancer were shown to have increased in both sexes over the study period with an AAPC of +4.8% (95% CI: 4.0–5.5) for males and +5.1% (95% CI: 4.3–5.9) for females. The ASR for the 1991–1996 period was 102.3 per 10003–2008 . During per 105) . The ageper 105) .5 in 2003–2008 (5 versus 6.3 per 105 for cancer of the uterine cervix and 16.3 per 105 versus 2.1 per 105 for breast cancer)  . The inc cancer) .6 in 1991–1996 to 20 per 106 in 2003–2008, and that of KS rose from 0.4 per 106 in 1991–1996 to 11.0 per 106 in 2003–2008. The mean age of children with Burkitt’s lymphoma remained constant throughout the study period (6.8–6.6 years).Among children (0–14 years) 738 cancers were registered during the 18-year study period. Of the 738 pediatric tumors 420 (56.9%) occurred in males, 311 (42.1%) in females and in 7 cases the sex was not recorded. The most common tumors were non-Hodgkin’s lymphomas (32.5%) and KS (13.4%). Within the group of non-Hodgkin’s lymphoma, 48.9% (133/272) were Burkitt’s lymphoma. The incidence of non-Hodgkin’s lymphoma increased from 3.6 per 10This study is one of the few reports providing solid data on cancer trends in sub-Saharan Africa over a period of 18-years. Moreover, a major strength of this report is that data from the same area had been previously published in Mozambique from the period from 1956 to 1961, thus providing the unique opportunity to compare the trends in cancer for the same area over a long period covering almost 50 years. During this period major socio-economic and behavioral changes have occurred in sub-Saharan Africa,6.5 in 2003–2008). These results are in agreement with reports showing that cancer of the uterine cervix is the most common cancer in women in sub-Saharan Africa, with the highest incidence in eastern and southern Africa[Cancer of the uterine cervix was found to be the most common cancer in women during the whole study period with the incidence increasing at an average of 4.7% per year, being highest at the end of the study period  was one of the highest observed in Africa[Prostate cancer was found to be the most common cancer among men, with a significant increase in its incidence  over the 18-year study period. Prostate cancer was rarely diagnosed in sub-Saharan Africa in the 1950s and 1960s,28, wherin Africa, with thin Africa and some5 in 2003–2008) remains relatively low by global standards[The incidence of breast cancer was very low in Mozambique in the 1950s. Nonethestandards,15; howeAll cancers related to HIV infection  significantly increased during the study period. This differs with the decrease in the incidence of this cancer observed in other geographical areas of sub-Saharan Africa as a result of the availability of anti-retrovirals,8.5 according to the IARC report, which is similar to the 62 per 105 found in the last period of the study. A significant limitation of our study is that cases of leukemia and those with only a clinical diagnosis were not registered during this period. Another limitation is that statistics in Mozambique, as in other African countries, are very sparse because of the lack of or the unreliability of data registration. Thus, the real incidence of cancer might be underestimated.A major strength of our study is that since 1991 the MCH has received virtually all the specimens from the city of Maputo during the study period and registered all the cases of cancer based on pathological verification of the diagnosis. Special efforts have been made to maximize the completeness of registration and ensure the quality of data. However, it cannot be ruled out that improvement in health service coverage in the country might have contributed to the increase of observed incidence of some cancers. In order to indicate the completeness of the registry, we compAs has occurred in much of urban Africa, the lifestyles of the population of Maputo are changing rapidly with the changes in the population from one comprising relatively recent immigrants from villages, to that of second generation inhabitants, engaged in wage-earning or informal economy, and the purchasing of foodstuffs and other necessities, rather than producing themselves,6. This In conclusion, the ongoing surveillance system provided by the cancer registry in this predominantly urban Southern African population has provided a picture of the evolution of cancer in modern sub-Saharan Africa. Interestingly, there is a predominance of cancers of demonstrated infectious etiology, such as HPV-associated cancer of the uterine cervix and other AIDS-related cancers  mirroring the increase in the HIV-AIDS epidemic in the country. To date, there is little evidence of the onset of the epidemic of tobacco-related cancer which has been such an important feature of the cancer profile in economically developed countries. These findings are of relevance to guide cancer prevention policies in Mozambique and countries of similar characteristics.


The objective of this study was to evaluate the association between rest heart rate (HR) and the components of the auditory evoked-related potentials (ERPs) at rest in women. We investigated 21 healthy female university students between 18 and 24 years old. We performed complete audiological evaluation and measurement of heart rate for 10 minutes at rest (heart rate monitor Polar RS800CX) and performed ERPs analysis (discrepancy in frequency and duration). There was a moderate negative correlation of the N1 and P3a with rest HR and a strong positive correlation of the P2 and N2 components with rest HR. Larger components of the ERP are associated with higher rest HR. Cardiovascular diseases are the most frequent causes of morbidity and mortality around the world during the last decades –4. The cSince the CNS actively regulates the cardiac system, auditory evoked-related potentials (ERPs) are an important method used to evaluate it because it captures electrical responses in the cortex due to an acoustic stimulus. Brainstem auditory ERPs are characterized by bioelectric responses of thalamic and cortical activity, measured in milliseconds; it assesses cortical activities involved in the skills of discrimination, integration, memory, and attention in the brain, besides the integrity of central auditory nervous system .Few studies have focused on the cardiac and auditory systems through musical stimuli –10; cardKnowledge of physiological responses involved in the relationship between the central auditory pathways and cardiac autonomic regulation is important for the development of future therapies to propose intervention and prevent the development of disorders of the cardiovascular system. In this sense, it is important to have new studies to help to understand the association between cardiac autonomic control and the electrical activity in the auditory system. Therefore, we aimed to evaluate the association between HR and ERPs at rest in women.This research protocol was approved by the Ethics Committee in Research of the State University of São Paulo (case number 419/2012) and was in accordance with Resolution 196/96 of the National Health Council of 10/10/1996. The sample consisted of 21 apparent healthy female subjects between 18 and 24 years old. All volunteers were informed about the procedures and objectives of the study and, after agreeing, signed a consent form.We excluded smokers, people with any degree of hearing loss or middle ear disorders, subjects with related cardiorespiratory, metabolic, neurological, and/or any condition that prevented the individual to perform the procedures, and those users of drugs that alter cardiac autonomic regulation. For this characterization, the following procedures were performed: medical history and research of hearing threshold (air and bone conduction) and acoustic impedance measurements; resting blood pressure and heart rate measurement.The normal parameters of hearing assessments considered in this study were pure tone audiometry thresholds below or equal to 25 dB and airway below or equal to 15 dB bone conduction , with acData collection was conducted in a quiet room electric and sound-proof in temperatures between 21°C and 25°C and humidity between 50 and 60%. Volunteers were instructed to not ingest alcohol and caffeine in the 24 hours prior to evaluation. After the initial assessment, the heart rate receiver Polar RS800CX  was placed on the chest of the volunteers in the sternum region for analysis. After placing the strap and the monitor, subjects were positioned in a chair and told to remain at rest for 10 minutes. Blood pressure was measured using a sphygmomanometer and stethoscope. Blood pressure and heart rate were measured at rest prior to the auditory ERPs. Only volunteers who showed no significant differences in the values of these two measures participated in the survey.The examination of the auditory ERPs was performed with the individual in a state of alert watching a video (without sound) for distraction and recommended to not direct their attention to sound stimuli in oddball paradigm , unattended test. We used Biologic's Evoked Potential System (PE) for data acquisition and five disposable electrodes placed on Fz and Cz in reference to the right lobe (A2) and left (A1) and ground in Fpz, using the two channels of the equipment record.The parameters used for the ERPs were filter between 0.5 and 30 Hz binaural stimuli in the frequency discrimination, the standard stimulus which was elicited at a frequency of 750 Hz and 1000 Hz for the deviant stimulus , the duration discrimination on, the standard stimulus , deviant stimulus  both in the frequency of 1000 Hz (with a probability of 20%), intervals between stimuli of 1.1 ms, intensity of 70 dB HL, analysis time of 500 ms, prestimulus analyses time of 0 ms, a sensitivity of 100 microvolts alternating polarity, and number samples of 200 stimuli .The wave identification ERPs followed criteria in the literature, including visualization of sequential peaks of negative-positive-negative waves, that is, N1, P2, N2 complex, respectively, between 60 and 300 ms, observed in do twice traces . As the Testing took approximately 50 minutes. In order to maintain a standard quality test, in the volunteers who showed myogenic interference, we suggested change positions and when necessary the examination was repeated.P values were considered statistically significant when P < 0.05 and all confidence intervals were constructed with 95% statistical confidence. For correlation values we considered strong correlation for r > 0.5, moderate correlation for r between 0.3 and 0.49 (0.3 > r > 0.49), and weak correlation for r < 0.3. The statistical software used was GraphPad Software StatMate 2:00 version for Windows, GraphPad Software, San Diego, California USA.The Shapiro-Wilk Test was applied to evaluate distributions; Person and Spearman correlation Tests were applied for parametric and nonparametric distribution, respectively, in order to investigate correlation between variables. The Correlations between latencies and amplitudes of N1, P2, N2, and P3a and N2-P3a interamplitude, measured at Cz and Fz , relatedThe results obtained show correlation between some components of the ERP and HR as shown in Tables The ERP is one of the promising measures used in the research of central auditory processing that reflects cortical activity involving listening skills from the simple to the most complex. HR is mediated by the direct activity of the ANS through sympathetic and parasympathetic branches and is an indicator usually expressed as the number of heartbeats per minute (bpm).The findings of the literature state that the decrease or increase in HR is caused by the selective activation of cardiac neurons in the amygdala and in the reticular formation, structures located in the cerebral cortex. The reticular formation is responsible for the regulation of alertness and subsidizes the attentional process and the amygdala is specifically related to emotions .Anatomically and physiologically the link between cardiovascular and hearing impairment due to structures responsible for cardiovascular regulation and components of ERPs that presents similar functions in the regulation of alertness and sensory attentional process is evident. The N1 component of the ERP is associated with attention and initial decoding of the auditory stimulus. The P2 is related to temporal acoustic stimulus, which brings information to the level of the auditory cortex, the early cortical processing of sound and features. The N2 contributes to physical breakdown of the acoustic characteristics of the stimuli and is also responsible for passive, preattentional, automatic perception, discrimination, and sound recognition , 18 respIt was possible to observe in the Cz scan that when the right and the left ear were aurally stimulated, there was a moderate negative correlation in the amplitude of N1 and P3a, respectively. It was reported that when the electrical activity reaches the cortex in areas associated with attention and initial decoding of the stimulus and during stimulation the stimulus starts to be processed in the auditory pathway through activity updates for automatic allocation of sensory attention, while HR increases the amplitude of N1 and P3a decreases.Some authors , 12 relaMoreover, we observed strong positive correlation of the amplitude of P2 and N2 amplitude and latency with HR in the scan Cz and Fz in the right and left ear. Therefore, the results showed that the lower the electrical activity of the auditory processing is, the lower the HR. P2 and N2 components reflect the acoustic characteristics of the stimuli , which cA study of simulated driving mental fatigue testing associated with another type of auditory cortical potential observed significant reduction in P300 amplitude, indicating that when the individual was exposed to the mental fatigue there was a decrease of attention to the auditory task. The authors suggested that auditory electrophysiological response is related to mental fatigue, which has impact on the function of the CNS, which consequently controls and regulates the cardiovascular system .Finally, regarding the parameters of frequency and duration, we observed statistical correlation only in the frequency protocol in both ears. This fact can be explained because their stimuli were presented in an oddball paradigm and differentiate in intensity, which can make them more discriminating and hence may affect the ANS and interfere with the heartbeat.There was association between cardiovascular parameters and the central auditory pathways, indicating physiological responses of the studied variables and the relationship between them. We suggest further research in this field, in order to clarify and confirm this association.


The authors and editors retract this publication  followinAfter the publication of the article, concerns were raised about Figures 2,4, 5 and 6. The authors stated that the wrong blots were included in several of the published figures and that the p-JNK blots in Figure 4B were inadvertently duplicated from a figure in an earlier publication. A Correction [Figure 2D - B-actin: This panel duplicates the B-actin panel in Figure 2A.Figure 2F- B-actin: The first band from the original blot for this figure was not included in the published panel.Figure 2I - B-actin: Upon evaluation of the raw blot supplied by the author, the editors are concerned that the evidence supplied does not support the results reported in this figure.An institutional inquiry has been undertaken at the University of Campinas (UNICAMP) in São Paulo, Brazil and while the investigation acknowledged errors in the preparation of the figures, they did not find evidence of research misconduct. However, the preparation of the figures falls below the standard of publication and therefore the authors and the editors have agreed that the correct action is to retract the article. The authors apologize to the scientific community and will seek to publish a corrected manuscript version corroborating the findings of this work.


The reliability of experimental findings depends on the rigour of experimental design. Here we show limited reporting of measures to reduce the risk of bias in a random sample of life sciences publications, significantly lower reporting of randomisation in work published in journals of high impact, and very limited reporting of measures to reduce the risk of bias in publications from leading United Kingdom institutions. Ascertainment of differences between institutions might serve both as a measure of research quality and as a tool for institutional efforts to improve research quality. A systematic analysis of in vivo research reveals poor reporting of measures that reduce the risk of bias and an inverse relationship between impact factor and the reporting of randomization. Bias occurs in in vivo research when there is a systematic error, or deviation from the truth, in the results of a study or the conclusions drawn from it. There are a large number of potential sources of bias, and the risks of some of the most important of these (selection bias and measurement bias) may be mitigated through simple study design features (randomisation and blinded assessment of outcome) ,2. WhereMeasuring the impact of scientific work is important for research funders, journals, institutions, and, not least, for researchers themselves. These measures are usually based on journal impact factor or citation counts and are used to inform individual and institutional funding decisions and academic promotions and to establish an informal hierarchy of journals that in turn guides where authors choose to submit their work. A journal’s impact factor is derived from the number of citations to articles published in that journal during the preceding 2 years and as such reflects the extent to which publications in that journal have influenced other work. The use of journal impact factors for the purposes described above rests on an implied assumption that highly cited articles, and by extension journals with a high impact factor, describe high-quality research.PLOS journals (which have been one of the most enthusiastic proponents of the ARRIVE guidelines) remains low  in 1992 and 1.3% [1/77] in 2011) , blinded assessment of outcome in 788 (29.5%), a sample size calculation in 20 (0.7%), and a statement of potential conflict of interest in 308 (11.5%). There was substantial variation between different disease models in the prevalence of reporting of measures to reduce the risk of bias, being lowest for glioma and experimental autoimmune encephalomyelitis and highest for myocardial infarction . Reportiin 2011) .It is not known whether in vivo research published in high-impact journals is at lower risk of bias than that published in journals of lower impact. If scientists could be confident that their work would be judged on its own merits, then the case for expeditious publication in well-indexed online journals with open access, unlimited space, and the lowest publication costs would become unassailable. We therefore examined the relationship between journal impact factor and reporting of risks of bias in these 2,671 publications.p > 0.05). However, there were important differences in the reporting of individual risk-of-bias items. Median journal impact factor was 2.6 higher for studies reporting a potential conflict of interest  but was 0.4 lower in studies reporting randomisation . There was no significant difference for either the blinded assessment of outcome  or sample size calculation .The median impact factor was 3.9 (interquartile range 2.6 to 6.3), and using median regression, there was no relationship between journal impact factor and the number of risk-of-bias items reported . We identified 4,859 publications from these institutions with a publication year of 2009 or 2010. By screening the title and abstract of these—and where necessary, the full text—we identified 1,173 publications that contained primary reports of in vivo research.Finally, we were interested to establish whether formal assessment of research performance is sensitive to these issues of rigour. To do this, we measured the reporting of measures to reduce the risk of bias in in vivo research published from the five UK institutions ranked highest across six units of assessment in biomedical sciences in the 2008 Research Assessment Exercise (RAE) ; 201 of 1,165 reported blinding (17.3%); 101 of 1,169 reported inclusion or exclusion criteria or both (10.4%); and 16 of 1,168 reported a sample size calculation (1.4%). Only one publication reported all four risk-of-bias measures , nine puFurther, there were interesting differences between institutions. The reporting of randomisation ranged from 7.2% (Institution B) to 16.3% (Institution A); the reporting of blinding from 12.4% (A) to 23.6% (C), the reporting of inclusion and exclusion criteria from 4.6% (D) to 11.6% (A), and the reporting of a sample size calculation from 0% (E) to 5.1% (D). There were significant differences between institutions in the reporting of each risk-of-bias item; for the reporting of randomisation, Institution B was significantly worse than all other institutions, and for the reporting of a sample size calculation, Institution D was significantly better than all other institutions . The rigThese datasets were assembled at different times for different purposes, and so comparisons between them have limited validity. However, it is possible to compare across the datasets the reporting of measures to reduce the risks of bias in those publications with a publication year of 2009 or 2010 . SomewhaThere are of course a number of weaknesses to our approach. Foremost among these is that our measures of rigour (the reporting of measures to reduce the risk of bias) and of perceived quality  are of necessity very indirect: we were only able to ascertain whether publications reported measures to reduce the risk of bias, not whether they had actually done so. However, previous studies have shown that such reporting is associated with lower estimates of efficacy ,4, and sSecondly, we have not been able to consider the consequences of any upward drift of journal impact factor over time, although in the CAMARADES dataset, we observed only a small increase in mean impact factor, from 4.28 in 1992 to 4.40 in 2011.Thirdly, we had a low threshold for considering studies to meet the various criteria. Bara et al. have shown  that forFinally, the relationship between institutional esteem and risk-of-bias reporting may be a consequence of the editorial practices of the journals in which high-quality research is reported; that is, they may be more likely to accept manuscripts from institutions of repute. Alternatively, the relationship between journal impact factor and risk of bias may be a consequence of such journals making publishing decisions based on institutional esteem rather than the quality of submitted manuscripts. However, the most parsimonious explanation of our findings is that journal editorial policies and those charged with assessing the quality of published work, including peer reviewers, have given insufficient attention to experimental design and the risk of bias, and that this has led investigators to believe that these factors are not as important as the novelty of their findings.In spite of these weaknesses, we believe we have provided important empirical evidence of the reported rigour of biomedical research. Firstly, we show that reporting of measures to reduce the risk of bias in certain fields of research has increased over time, but there is still substantial room for improvement. Secondly, there appears to be little relationship between journal impact factor and reporting of risks of bias, consistent with previous claims that impact factor is a poor measure of research quality . ThirdlyIt is sobering that of over 1,000 publications from leading UK institutions, over two-thirds did not report even one of four items considered critical to reducing the risk of bias, and only one publication reported all four measures. A number of leading journals have taken steps that should over time improve the quality of the work they publish –10, and Of greater concern is that there still appears to be a lack of engagement with these issues amongst those charged with assessing the quality of published research. In the course of this work, we approached the UK Higher Education Funding Council (HEFC) seeking details of those publications submitted to them in the context of the 2014 REF; they declined a Freedom of Information request on the basis that disclosure would not, in their view, be in the public interest. The REF should of course have a role in championing good science, but it should also be a force for improvement, seeking to provide measures against which institutions and scientists can monitor improvements in the rigour of their research.S1 Data(XLSX)Click here for additional data file.S2 Data(XLSX)Click here for additional data file.S3 Data(XLSX)Click here for additional data file.S4 Data(XLSX)Click here for additional data file.S5 Data(XLSX)Click here for additional data file.S1 Fig(TIF)Click here for additional data file.S1 Text(DOCX)Click here for additional data file.


We study the feasibility of a hybrid Brain-Computer Interface (BCI) combining simultaneous visual oddball and Steady-State Visually Evoked Potential (SSVEP) paradigms, where both types of stimuli are superimposed on a computer screen. Potentially, such a combination could result in a system being able to operate faster than a purely P300-based BCI and encode more targets than a purely SSVEP-based BCI.We analyse the interactions between the brain responses of the two paradigms, and assess the possibility to detect simultaneously the brain activity evoked by both paradigms, in a series of 3 experiments where EEG data are analysed offline.Despite differences in the shape of the P300 response between pure oddball and hybrid condition, we observe that the classification accuracy of this P300 response is not affected by the SSVEP stimulation. We do not observe either any effect of the oddball stimulation on the power of the SSVEP response in the frequency of stimulation. Finally results from the last experiment show the possibility of detecting both types of brain responses simultaneously and suggest not only the feasibility of such hybrid BCI but also a gain over pure oddball- and pure SSVEP-based BCIs in terms of communication rate. In this study, the brain activity is recorded using electroencephalography (EEG), which offers the advantage over other methods  increases.Some of the earliest EEG-BCI systems were based on the P300 component of the event-related potential ERP, , 2). The. The2]).us onset –6 and tous onset ). Howeveie. flickering stimuli). When the latter is at a sufficiently high rate (>6Hz), stable and synchronized neural oscillations at the stimulus frequency and its harmonics are evoked over the visual cortex  the vector representing the EEG data.For each trial we estimated the power of the EEG signal measured by the Oz channel in the first, second and third harmonic of the stimulation frequency of the trial.vs. hybrid) and the interaction between those 2 factors. We considered random intercept for the subject, and for the subject-stimulation frequency and subject-stimulation type interactions. The p-values for the fixed effects were obtained by likelihood ratio tests of the full model with the effect in question against the model without the effect in question. For factors with significant effects, the p-values for the contrasts were obtained via Markov-chain Monte Carlo (MCMC) sampling [We obtained thus for each subject, stimulation frequency and stimulation type, 12 power values  for all three harmonics. The data for each harmonic were analysed with a linear mixed effect regression. The fixed factors considered were the stimulation frequency , the stimulation type , and downsampled to 256Hz. For the detection itself we used a technique proposed by [Minimum Energy Combination method. It results in a set of linear combinations of the original EEG signals for which the noise is minimized at the frequencies of interest (i.e. the 4 stimulation frequencies) and their harmonics (we considered the first three harmonics). In the second step, a scoring function is calculated for each of the 2 stimulation frequencies and the one with the highest score is identified as the winner frequency. A correct detection corresponds to the case where the winner frequency matches the actual target frequency. The scoring function corresponds to the average of the signal-to-noise ratios across harmonics and components of the spatially filtered signals. The signal-to-noise ratio is calculated as the ratio of the estimated signal power and the estimated noise power at the desired frequency . Thncy . For the P300 classification problem, p is estimated by the division of the number of times the correct P300 target was identified by the total number of trials, regardless of the results of the SSVEP classification . This means that the P300 classifier is always applied to data corresponding to the actual target screen portion and therefore, N is the number of possible commands in each screen portion (N = 6). Finally, for the hybrid condition, p represents the division of the number of times the correct symbol was identified (when both P300 and SSVEP classifiers identify the correct target) by the total number of trials, and N the total number of symbols the user can choose from (N = 12). As ITR typically reflects online performance and this study relies on offline analyses of EEG, the ITR values reported should be regarded as estimates of expected online performance.Besides icon detection accuracy, we also measured the Information Transfer Rate  expre.g.[The average responses to target stimuli for each of the 5 experimental conditions, all subjects and a selection of EEG channels covering the scalp from frontal to occipital locations are shown in e.g., 37), thFor example, it can take the form of a time shift as observed for subjects S3 and S5 in the frontal and central channels, where the positive peak observed between 200 and 300ms appears slightly earlier for the pure oddball condition than for the hybrid conditions. It can also take the form of a difference in peak amplitude as one can see in the ERPs of subject S2 where the negative peak observed in the parietal electrodes between 100 and 200ms is characterized by a larger amplitude in the pure oddball condition than in the hybrid conditions. Similarly, the ERPs of subject S6 in the central and parietal channels are characterized by one negative peak at around 150ms and a positive one at around 200ms, both peaks showing a stronger magnitude in the pure oddball condition than in the hybrid ones.One can also observe differences in both amplitude and latency, as for the ERPs recorded for subject S2 in the occipital channels where the positive peak between 200 and 300ms appears earlier with a stronger magnitude in the pure oddball condition than in the hybrid ones. Similarly the ERPs recorded for subject S9 in the frontal and central channels between 100 and 200ms appear earlier and have a stronger magnitude for the pure oddball condition than for the hybrid ones.In In The results from the previous section support the observations made from χ2(40) = 40.1, p = 0.47).The detection accuracy (averaged across subject) with respect to the number of repetitions considered is shown in Therefore, and despite the differences between oddball and hybrid ERPs reported in the previous section, the oddball detection does not appear to be affected by neither the presence of the SSVEP stimulation, nor the frequency of the SSVEP stimulation (at least for the 4 frequencies tested).In In vs. hybrid, χ2(1) = 0.0004, p = 0.98), nor the stimulation frequency (χ2(3) = 2.54, p = 0.47), nor the interaction between them (χ2(3) = 4.4, p = 0.22).For the first harmonic, no significant effect on the signal power was found for neither the stimulation type  = 0.552, p = 0.46), nor the interaction between stimulation type and stimulation frequency (χ2(3) = 5.59, p = 0.13). However, we observed a significant effect of the stimulation frequency on the signal power (χ2(3) = 10.6, p = 0.014). The contrast analysis (MCMC sampling) revealed a significant linear trend between the stimulation frequency and the signal power (pmcmc < 0.001) and no significant quadratic or cubic trend . As the stimulation frequency was treated as a 4 levels factor (and not a continuous variable) by the statistical model, we do not conclude about the existence of a possible linear relationship between the power of the EEG signal and the stimulation frequency as continuous variables. However, the model does identify a monotonic decrease of the power of the EEG response in the second harmonic with increasing frequency.Concerning the second harmonic, no significant effect on the signal power was found for neither the stimulation type  = 3.37, p = 0.07), the stimulation frequency (χ2(3) = 4.09, p = 0.25), or for the interaction between them (χ2(3) = 4.5, p = 0.22).For the third harmonic, as for the first harmonic, no significant effect on the signal power was found for the stimulation type .In versus 6 for the oddball ERP detection and 2 for the SSVEP detection). As shown in Although the target icon detection accuracy is necessarily lower than the ones of oddball ERP and SSVEP detection, it offers a larger choice of items to communicate than the two modalities individually . Unlike for the original method, we did not average the SNRs across harmonics, instead, we kept the SNRs from all three harmonics  and used the data to train a linear SVM for each subject . The linear SVM was then applied on the test data and, for each trial, the frequency for which the SVM output was the highest was identified as the In Thus, while using training data to obtain subject-specific optimized weights for each harmonic results in an SSVEP detection accuracy that is at least as good as with the original method without training, the improvements realized on the tested population are rather limited.The results from our first two experiments suggest that a hybrid visual P300-SSVEP BCI with both stimulus paradigm physically overlapping would not compromise the detection of any of the 2 types of evoked responses.Our third experiment confirmed those results showing the possibility of detecting both P300 component and SSVEP activity simultaneously, and the results obtained suggest not only the feasibility of such a hybrid BCI system but also a possible advantage over pure P300- and SSVEP-based ones in terms of communication rate.All the results obtained in our study were obtained from offline analyses of the EEG data. Therefore, further work should report on the feasibility of such hybrid P300-SSVEP BCI in an online setting and focus on comparing the performance obtained with respect to the online performance of equivalent systems purely based on P300 and SSVEP. As our proof-of-concept experiment was performed with only 2 SSVEP stimuli, future online studies should also investigate designs with larger number of SSVEP stimuli and report on the influence of the amount of SSVEP stimuli on both performance and user comfort. If successful, such systems are expected to increase the number of choices an P300-based BCI offers without decreasing the communication speed as well as to overcome the limitation in the number of stimuli that can be displayed on-screen for SSVEP-based BCIs.


To evaluate the hematological, cytogenetic, and molecular responses in Colombian patients with CML chronic myeloid leukemia (CML) treated with imatinib.  Two groups of patients, one with the novo diagnostic and another in state of complete cytogenetic remission were followed for 12 months with quantitative PCR evaluations every three months and with chromosomal analysis every 6 months.  The group with the novo diagnosis showed 50% of complete cytogenetic remission at 12 months while the other 50% were considered to have primary resistance. Respect the molecular analysis, 10.5% of the patients reached undetectable BCR-ABL transcripts at 12 months. In the complete cytogenetic remission group, 10.6% lost the state of complete cytogenetic remission at 12 months, 50% reached undetectable BCR-ABL transcripts but 10% showed levels higher than 10%, which in our standardization was equal to no molecular response.  Despite having received the conventional dosages of 400 mg/day of imatinib, the cytogenetic and molecular responses obtained in our group of Colombian patients with CML, were lower than those in other international studies. This improvement is due to the fact that Imatinib induces significant remission of the Philadelphia clone, which prevents the disease from progressing into acute phases in 97% of the patients that get to complete cytogenetic remission and 100% for those who also reduce the level of the BCR/ABL transcription in 3 logarithms or more within the first 12 monthsThis investigation, descriptive in nature, was carried out in Colombia between January of 2008 and December of 2009 with the participation of 6 hematologic centers, and was authorized by a local ethics committee. The therapeutic conducts adopted during the follow-up period depended exclusively and freely on the treating physician according to his/her experience and on the protocols of each hematologic center. The patients were divided in two groups: de novo and complete cytogenetic remission (CCR). The criterion for inclusion in the first group was the possibility of BCR/ABL fusion proven by PCR in peripheral blood in patients who were clinically suspected to have CML, and for the second, confirmation of the complete cytogenetic remission state in the bone marrow; additionally patients had to be over 18 years old and have written consent of their participation. The follow-up was done over one year of medical visits with hemogram and molecular assessments every three months and chromosomes every six months: visit 0 (inclusion), visit 1 (90 days), visit 2 (180 days), visit 3 (270 days) and visit 4 (360 days), with some visits made at intermediate periods if the hematologist considered it necessary: the drug was administered by the Health Sponsor Company (EPS in Spanish) to which the patient was affiliated. For the de novo group the Sokal Index was calculated resorting to www.leukemia-net.org at the time of inclusion and the hematological response estimated by each physician according to conventional criterionsthese analyses were performed on bone marrow samples according to published protocols-1 a 10-6) of an mRNA (1-5 ug/uL) with Ct < 20. These same curves for BCR-ABL and GUS were used as a reference to calculate the relative concentration of the samples with the software that came with the Light Cycler 480.The quantification of the BCR-ABL transcription was carried out with a real time PCR (RQ-PCR) using TaqMan method over a Light Cycler 480 platform from Roche. The expression of the beta-glucuronidase (GUS) gene was taken as reference for the normalization of the BCR/ABL p210 transcription expression. The oligonucleotide ENF501 (sense), ENR561 (antisense) and the TaqMan ENP541 probe were used to detect the amplification BCR-ABLFor the descriptive component, percentages were used in the case of discrete and median variables along with their corresponding statistical dispersion (standard deviation) for the case of the continuous variables. In order to illustrate and analyze the behavior of the laboratory variables throughout the follow-up, two-way charts were used. The relation between the quantitative measures of the tumor load in peripheral blood and bone marrow was done with Pearson product-moment correlation coefficient. All of the statistical analysis procedures were done with a Stata 5(r) program. The percentages on each table correspond to the rate of patients that were evaluated at that moment.Twenty-five patients were included in the de novo group and 27 in the CCR group, for a total of 52. At the end of the follow-up, 18 patients remained assessables in the de novo group and 19 in the CCR. The losses of the first group were due to three disease-related deaths, one follow-up loss, two to voluntary withdrawals from the study and one to change to another drug; in the second group, one death after an angioedema not related to the illness, three due to therapy failure, two follow-up losses and two voluntary withdrawals. The median dose of imatinib was 400 mg/day, un patient went up to 600 mg and six to 800 mg; when the study was concluded, 4 patients switched to a second generation tyrosine kinase inhibitor (3 dasatinib y 1 nilotinib). After 12 months, 50% of the patients from the de novo group had treatment failure while in the CCR group 11% relapsed. In The novo group responded quickly to the imatinib with complete responses after 3 months of 79.1%, which increased to 84.2% by the end of the study. In the CCR group after 9 months, 13.6% had lost the hematologic response, although at 12 months all the patients that were evaluated remain in CHR. Figure 1 shows the evolution of the white blood cell and platelets count in the novo group.In the de novo group, 23.8% remained with low risk, 47.6% remained in medium and 28.6% in high. Following the 2010 European LeukemiaNet (ELN) criterion, at 6 months 55.5% of the de novo group responded optimally , 11% sub-optimal (>35% Ph+) and 33.4% failed (> 95% Ph+). At 12 months, 50% reached an optimal response (complete cytogenetic remission), 5.5% suboptimal (less than complete cytogenetic remission) and 44.5% failed (>35% Ph+) . One patIn the de novo group a decrement in the transcript BCR-ABL levels during the year of treatment was observed, reaching complete molecular responses (undetectable transcripts) in 5.6% and 11.8% in the bone marrow at 6 and 12 months respectively. These percentages were co-related with the molecular responses obtained in peripheral blood . MeanwhiThe complete molecular response in the CCR group was consolidated from 15.4% at inclusion to 50% at 12 months. Three cases (11.5%) showed expressions of the BCR/ABL transcript above 10% (-1 log) at the time of the inclusion, one fell out of follow-up, another exhibited a cytogenetic relapse at 6 months and maintained it until 12 months, and the last held the complete cytogenetic response with decrease of tumor load . The cor, , This investigation evaluated the hematological, cytogenetic, and molecular responses in patients with chronic myelogenous leukemia (CML) treated with Imatinib in the context of a developing country where socioeconomic conditions could influence the outcome of biological responses. Thanks to tyrosine kinase inhibitors, which efficiently inhibit the Ph+ clone, the chromosomal and molecular analysis becomes the cornerstone for the monitoring of the tumor load. The biological responses have been synthesized in international guidelines such as ELN which make clinical decision-making much easier,,, ,The optimal cytogenetic response at 6 and 12 months was of 55.5% and 50% respectively, inferior to that obtained in clinical trials, which vary from 62% to 69% ,,,,We implemented a previous local standard for the molecular monitoring establishing a baseline obtained from the Ph+ of 30 de novo patients, which served as a reference in order to transform the percentage of the relation (BCR-ABL)X100/(GUS) of each patient into the respective logarithm. ,Of the long-term follow-ups it has been concluded that in order to reach the state of complete cytogenetic remission at 18 months it is necessary to respond optimally with minor cytogenetic remission at 3 months and with partial cytogenetic remission at 6 months, as opposed to patients classified with failure or suboptimal response in these same time periods, which have a low probability of reaching complete cytogenetic remission, and if they do, have a high probability of losing it later on,Although the majority of the patients continue with the course of treatment for a long period after reaching the state of complete cytogenetic remission, there is still a subgroup that loses it, (with accumulative incidence of 20% to 26% on 4 years) (between 20% and 26%Today there is evidence that one of the main causes of relapse is the emergence of genetically unstable clones refractory to imatinib due to mutations in the BCR-ABL kinase domain or carries of additional cytogenetic anomalies to Ph+; the first was not evaluated in this study and the second was not observed in our patients. Contrariwise, the molecular response observed in the CCR group at the time of inclusion was heterogeneous  with 11.This investigation intended to evaluate the tumor load in a series of patients undergoing treatment with Imatinib without interfering with the therapeutic decisions. The important limitation of this study derive from this observational design, such as being unable to control the variables that influence in the responses like the adherence to treatment, undefined criteria used to escalate the dosage of Imatinib, among other things. Also, the small and restricted size of the group and loss of patients limited our use of statistics in order to further inquire about significant differences between categories. In conclusion, the investigation showed that the optimal cytogenetic responses expected in the novo group and the losses in hematological and cytogenetic responses in the CCR group are not similar to those obtained in clinical trials and reported in developed countries. It is important to know whether similar findings occur in other developing countries and we must call attention to search social, biological and clinical-management reasons that may hinder better outcomes. One measure to be taken would be to adhere to international response criteria with greater rigor and we hope the approval of second-generation tyrosine kinase inhibitors as a first line treatment may improve response rates in our medium.


Applying sub-lethal levels of high-pressure homogenization (HPH) to lactic acid bacteria has been proposed as a method of enhancing some of their functional properties. Because the principal targets of HPH are the cell-surface structures, the aim of this study was to examine the effect of sub-lethal HPH treatment on the outermost cellular structures and the proteomic profiles of two known probiotic bacterial strains. Moreover, the effect of HPH treatment on the metabolism of probiotic cells within a dairy product during its refrigerated storage was investigated using SPME-GC-MS. Transmission electron microscopy was used to examine the microstructural changes in the outermost cellular structures due to HPH treatment. These alterations may be involved in the changes in some of the technological and functional properties of the strains that were observed after pressure treatment. Moreover, the proteomic profiles of the probiotic strains treated with HPH and incubated at 37°C for various periods showed different peptide patterns compared with those of the untreated cells. In addition, there were differences in the peaks that were observed in the low-mass spectral region (2000–3000 Da) of the spectral profiles of the control and treated samples. Due to pressure treatment, the volatile-molecule profiles of buttermilk inoculated with treated or control cells and stored at 4°C for 30 days exhibited overall changes in the aroma profile and in the production of molecules that improved its sensory profile, although the two different species imparted specific fingerprints to the product. The results of this study will contribute to understanding the changes that occur in the outermost cellular structures and the metabolism of LAB in response to HPH treatment. The findings of this investigation may contribute to elucidating the relationships between these changes and the alterations of the technological and functional properties of LAB induced by pressure treatment. Recently, there has been increasing interest in the potential of applying techniques such as pulsed electric field (PEF), high-hydrostatic pressure (HHP), or high-pressure homogenization (HPH) to enhance the survival rate of probiotic strains or to modify their overall functionality in a positive manner. Among these processes, HPH has been proposed for the treatment of raw materials or the sub-lethal treatment of starters or non-starters and probiotic cells for use in the production of probiotic fermented milks or cheeses with improved sensorial, technological, or functional properties  using transmission electron microscopy (TEM). These strains were chosen based on the results of previous studies that demonstrated their ability to increase certain functional properties in response to HPH treatment .The cells in overnight cultures were harvested by centrifugation . The pellets were washed twice using a solution of 9 g NaCl/l and they were re-suspended in sterile phosphate-buffered saline (PBS) (pH 7.4) at a final concentration of approximately 8 log CFU/ml. The cells were subjected to high-pressure homogenization (HPH) at 50 MPa using a PANDA high-pressure homogenizer . The inlet temperature of the samples was 20°C and the temperature was increased during the treatment at a rate of 3°C/10 MPa. To prepare control samples, the suspended cells were homogenized at 0.1 MPa. Immediately after the treatment, the samples were rapidly cooled to 10°C in a water bath.2HPO4/KH2PO4 buffer, pH 7). These samples were stored at 4°C for 2 h. After aldehyde fixation, the samples were prepared according to Bury et al. (Transmission electron microscopy (TEM) was used to investigate the morphological changes caused by the HPH treatment. Ten milliliters of the control samples and the HPH-treated samples were centrifuged  and the pelleted cells were fixed by suspending them in 2.5% glutaraldehyde  no incubation (50 MPa T0); (ii) incubation at 37°C for 30 min (50 MPa T30); (iii) incubation at 37°C for 60 min (50 MPa T60); (iv) incubation at 37°C for 120 min (50 MPa T120). To prepare control samples, suspended cells were treated using the homogenizer at 0.1 MPa (0.1 MPa C).After being subjected to HPH treatment at 50 MPa, the suspended cells were incubated as follows: (2O/CH3CH2OH (300/600 μl) and were treated according to the method of Putignani et al.  and then re-suspended in buttermilk, previously acidified or not to pH 4.6 using lactic acid , at a level of approximately 8 log CFU/ml. These preparations were HPH treated as described above (paragraph 2.2) at 0.1 MPa (control samples) or at 50 MPa and were stored for 30 days at 4°C. The buttermilk used was reconstituted (77 g/l) from lyophilized buttermilk purchased from a local dairy and was sterilized at 115°C for 30 min. Buttermilk was chosen as the dairy medium for this study because previous studies had shown that it supported the growth and the survival of adequate numbers of LAB cells during storage  were used to detect the aroma compounds as reported by Patrignani et al. . SamplesThe results of volatile profile analysis of each sample were expressed as the mean values of six independent replicate analyses (conducted on different days) and the data were analyzed using Principal Component Analysis (PCA) using Statistica 6.1 software . Eight replicate MALDI TOF MS Biotyper analyses of each sample were conducted and each replica was considered independently. Prior to performing principal component analysis (PCA)-based hierarchical clustering, each spectrum was subjected to mass adjustment, smoothing, baseline subtraction, Normalization and peak picking. The dendrogram for a single organism was created using with distance measurements (correlation), linkage (average) and a 300-score threshold values using MALDI Biotyper 3.1 software . The Pearson's correlation coefficients were calculated using spectral row data using R-Bioconductor to establish the reproducibility of the intra- (replicates) and inter-strain conditional data.L. paracasei A13 and L. acidophilus DRU cells did not significantly affect their viability, as observed in other studies of LAB strains . Increasing the incubation period to 60 min after HPH treatment increased the signals in the region between 3500 and 5200 Da. However, the 50 MPa T30 samples showed characteristic peaks at 6367.97, 7818.22, and 7818.22 Da that were absent in cells under the other conditions. Further increasing the incubation period to 120 min decreased the intensity of the peaks in the region between 3000 and 5500 Da.Figure L. acidophilus DRU cells. The spectral profiles of the 0.1 MPa C cells and the 50 MPa T0 and 50 MPa T30 cells included more peptide peaks, particularly in the 2000–5000 Da region, compared with the spectral profiles of 50 MPa T60 and 50 MPa T120 samples. In contrast, the profiles of treated cells had peaks of higher intensity in the mass range of 7000–10300 Da compared with those in the profile of untreated cells. In addition, the profiles of the treated cells were characterized by the presence of peaks  that were absent in the profiles of the control cells. Moreover, the intensity of the peaks in the 6951.51 and 7641.54 Da region of the spectra of the treated cells differed from that of the control-cell spectra.Figure L. paracasei A13 cells showed two major clusters  or at 50 MPa (HPH treated). All of the samples were stored at 4°C for 30 days. The aroma profiles and cell viability rates were monitored throughout the storage period. The viability results confirmed that buttermilk supported the survival of adequate numbers of probiotic LAB cells during refrigerated storage, independently of the level of HPH treatment, as demonstrated previously , buttermilk was inoculated with L. paracasei A13 and L. acidophilus DRU cells were expressed as the % of the total peak area, as shown in Tables The content of the compounds detected in the aroma profile of uninoculated buttermilk and buttermilk samples that were inoculated with The most abundant molecules detected in the non-inoculated and untreated buttermilk samples were ketones, such as 2-propanone, 2-butanone, 2-pentanone, and 2-heptanone, aldehydes, such as hexanal, furfural, and benzaldehyde, alcohols, such as hexanol and 2-furanmethanol, and acetic acid Table .L. paracasei A13 cells, the differences between the samples at 15 and 30 days of storage were not pronounced. All the samples at pH 7 were characterized by a higher content of ketones, mainly 2-nonanone, 2-pentanone, and 2-heptanone. Significant levels of 2,3-butanedione and 3 hydroxy-2-butanone were also detected in the HPH-treated samples at pH 7 at 30 days. Alcohols, mainly furanmethanol, were more abundant in buttermilk samples at pH 7, whereas acids (mainly acetic acid) predominated at pH 4.6, reaching relative proportions that were always greater than 45%. The content of esters in the samples containing L. paracasei A13 cells was always negligible, unlike that of samples containing L. acidophilus DRU cells.As expected, the aroma profiles of the buttermilk samples containing cells of the two strains studied were quite different from the profiles of the control samples due to microbial activity. However, In the case of the samples containing L. acidophilus DRU-containing samples after 15 days of refrigerated storage, with no significant differences due to the pH value or HPH treatment, and the most prominent of these compounds were hexanal, 4-methyl-benzaldehyde, benzaldehyde and furfural. However, the levels of these compounds in the non-treated samples remained stable during 30 days of storage, whereas their levels in the HPH-treated buttermilk samples fell down below 2% during this period. Ketones were the most prominent chemical group present under all of the conditions, but significant differences were observed in relation to HPH treatment and the pH level. At 15 days of storage, the total ketones represented approximately 29 and 45% of the total peak area at pH 4.6 and 7, respectively, with no differences related to HPH treatment. The higher level of total ketones in buttermilk at pH 7 was mainly due to the higher concentration of 2-heptanone compared to the buttermilk at pH 4.6. The content of 2-butanone was higher in the treated samples, whereas the level of 2-propanone was higher in the control samples. 2-pentanone and 2-nonanone were detected in significant and constant concentrations in all of the samples. At 30 days of storage, the ketone concentrations in the non-treated control buttermilk were little changed, but those of the HPH-treated buttermilks had drastically increased, largely due to increases in the relative concentrations of 2-pentanone, 2-heptanone, and 2-nonanone. The alcohols present were mainly represented by furanmethanol, the concentration of which was higher in the non-treated buttermilk sample than in the HPH-treated buttermilk samples at 15 days of storage. This difference was significantly greater at 30 days of storage regardless of the pH value. In contrast, the pH value strongly affected the relative percentage of acids, the levels of which were higher at pH 4.6 than at pH 7, independently of HPH treatment and the storage period. Finally, a significantly higher concentration of ethyl acetate was observed in HPH-treated buttermilk at pH 7 at 15 days of storage. The level of this ester was increased at 30 days of storage and was accompanied by an increased level of ethyl hexanoate.Aldehydes accounted for approximately 15% of the total peak area of the volatile molecule profile of the L. paracasei A13 cells or L. acidophilus DRU cells, respectively, which demonstrated that the first two principal components (PC1 and PC2) explained more than 85% of the total variability. In both cases, PC1 accounted for the greater part of the variability , and the samples could be grouped into four clusters according to whether they were HPH treated and the pH of the medium. The control buttermilk samples were grouped in the upper part of the plot, whereas the HPH-treated samples were grouped in the lower part. Moreover, the acidified samples were grouped on the left side of the plot, and the pH 7 buttermilk samples were grouped on the right side.To better evaluate the effect of HPH treatment on the aroma profile of buttermilk, a Principal Component Analysis (PCA) was conducted using the % of the peak area of the volatile compounds listed in Tables Figure In previous studies, it was demonstrated that HPH treatment at 50 MPa enhanced certain probiotic properties of LAB and changed the fatty acid composition of the cell membrane as response to the sub-lethal stress applied . These changes could be responsible for the increased hydrophobicity of the cells observed by Tabanelli et al.  followinAlthough analysis of MALDI-TOF spectra did not permit the identification of the MS/MS peptides, specific peptide fingerprints associated with the strain, the HPH treatment and the incubation period were obtained using this technique. The proteomic profiles of cells of the probiotic strains L. paracasei A13 and L. acidophilus DRU that were treated using HPH and were incubated at 37°C for different periods showed peptide patterns different from those of untreated cells. These differences can most likely be attributed to the effect of HPH on the cell-surface proteins and the cellular response to the HPH treatment. However, L. paracasei A13 and L. acidophilus DRU cells showed different behaviors independently of the HPH treatment and the incubation period that appeared to be related to their differential responses to the applied stress.L. paracasei A13 cells demonstrated that the peptide profiles of treated cells obtained immediately after the hyperbaric treatment were significantly different from those of the control cells, whereas the differences diminished during the incubation period. In fact, the peptide profiles of treated cells incubated for 60 and 120 min were more similar to that of the control cells. The changes in the peptide profiles of treated cells after 30 min of incubation with respect to the peptide profiles of the control cells can be attributed to the presence of specific enzymes and stress proteins that are involved in the restoration of the status quo ante.In particular, the dendrograms associated with L. acidophilus DRU cells showed that the peptide profiles of these cells obtained immediately after pressure treatment were more similar to those of the control cells than were the profiles obtained later, showing that the HPH treatment had less effect on this strain than on L. paracasei A13, most likely due to the higher resistance to physical stresses conferred by the S-layer. However, the hyperbaric treatment caused significant modifications of the peptide profile that became evident during the incubation period, indicating that the HPH treatment induced persistent metabolic changes. The lack of peptide identification did not allow distinguishing among the released and novel peptides. However, in the case of L. paracasei A13, the similarity of the peptide spectrum of the control cells and that of the treated cells immediately after hyperbaric treatment, as well as the appearance of characteristic peaks during the incubation of the treated cells, suggested that both released and novel peptides contributed to the profile changes that were observed over time. In contrast, in the case of L. acidophilus DRU, the similarity of the spectra of the control cells and those of the cells immediately following the HPH treatment suggested that the synthesis of novel proteins was a key mechanism in the stress response of this S-layer-endowed strain.In contrast, the analysis of the dendrograms associated with It is well-known that exposure to physico-chemical stresses results in increased levels of synthesis of stress-response proteins. Jofré et al.  showed tL. paracasei samples). Moreover, the pH value of the medium affected the volatile profiles through affecting the metabolic pathways of the bacteria and the level of activity of their enzymes. In addition, the pH value was shown to alter the volatility of compounds such as acids through affecting their interactions with the buttermilk matrix as well as the water-binding capacity of the proteins present  was detected at 15 and 30 days of storage. Lanciotti et al. (Lactobacillus species that had been directly treated using a sub-lethal HPH level. Additionally, Patrignani et al. (The analysis of the aroma profiles of the two different species showed that they had specific fingerprints (i.e., acetic acid was more pronounced in the profile of the i et al.  showed ai et al.  reportedThe significant differences in the volatile-molecule profiles of these samples could be due to the effects of the HPH stress on the microbial cells. In particular, the increase in the content of ketones and esters observed after pressure treatment could be associated with higher levels of activity of lipases and esterases. There is much evidence showing that sub-lethal HPH treatment affects the membrane fatty-acid desaturase enzymes that are involved in the active response of cells to high-pressure stress Somero, . In addiThe results of the present study demonstrated overall changes in the aroma profile and the production of molecules that positively affected the sensory profile of probiotic cell-containing buttermilk samples that were pressure treated. Therefore, because probiotic products manufactured using only probiotic strains are often characterized by the lack of desirable sensory features or a homogeneous aroma profile, treating probiotic cells with HPH might differentiate the products and enhance their positive sensory properties.In addition, the results obtained suggested that HPH has several biotechnological applications, including modulating the volatile-molecule profiles of dairy products, improving specific enzymatic activities of cells and enhancing the probiotic properties of bacterial strains. Finally, the proteomic approach used in this study has contributed to add another dowel to the understanding of the mechanisms underlying the stress responses of probiotic strains by demonstrating the involvement of the peptide profile in the response to HPH, which is one of the most promising technologies for application at the industrial level, particularly in the dairy-product sector.However, the promising aspects of HPH treatment indicated by the results obtained in this study must be further investigated to better understand the relationships among the genomic, volatilomic and peptide-metabolic profiles.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.