N-(3-aminopropyl)-N-dodecylpropane-1,3-diamine were effective even at 0.02%–2%. Subsequently, eight microbiocides containing the selected active ingredients were chosen and applied three times on the surface of wood samples colonized by bacteria and moulds. ABM-1 and ABM-2—6% solution; Rocima 101—8%; Preventol R 80—12%; Acticide 706 LV—15% and Boramon—30% were the most effective disinfectants. Under laboratory conditions, ABM-1, Boramon and Rocima 101 ensured antimicrobial protection of new wood samples for six months. In situ, 30% Boramon and 8% Rocima 101 applied by spraying effectively protected the historical wood from bacterial and mould growth for 12 and 3 months, respectively. Colour and luminance of the new wood were not altered after exposure to the biocides. Boramon and Rocima 101, applied by the spraying method, caused no significant change in the colour of the historical wood. Results from this study were used to develop a procedure for the protection of wood in historical buildings against biodeterioration.The aim of this study was to select effective and safe microbiocides for the disinfection and protection of historical wooden surfaces at the former Auschwitz II-Birkenau concentration and extermination camp. We tested seven active compounds against bacteria and moulds, of which didecyldimethylammonium chloride and  For centuries, wood has been used in the construction of houses, ships, weapons and various tools, among other things. Due to its chemical composition, this natural organic material is easily colonized by microorganisms and insects, leading to its deterioration under conditions favourable for their growth ,2,3,4. Wood damage by bacteria and fungi involves degradation of cellulose, hemicellulose and lignin. This leads to aesthetic deterioration of the surface  and, above all, structural and mechanical changes  ,5,6,7. HDifferent chemical compounds have been regularly used, for about 200 years, in order to protect wood from damaging factors . InitialChemical wood preservatives can be divided into impregnates and preservatives, which are used to protect wood against biological agents and fire, and biocides, which are applied to remove and neutralize biological agents already present in the wood ,15. BiocCurrently, preservation of wood  is often a complex and multistage process. The following factors must be taken into account: structural ; biological; and chemical . Furthermore, both environmental and economic aspects must be considered when using biocides. In the case of historical wood, the impact of chemicals on the material is extremely important, as it can lead to its discoloration ,17. ImprSelection of active biocidal substances against the microflora isolated from historical wood at the former Auschwitz II-Birkenau concentration and extermination camp;Selection of biocides containing the selected active compounds;Checking the activity and assessing the effectiveness of the biocides against microbes colonizing the wood surface;Protecting the wood samples from further contamination with microorganisms, under laboratory and in situ conditions;Evaluating the impact of biocides on new and historical wood colour and luminance;Developing a procedure for the protection of historical buildings from biodeterioration.The aim of this study was to select safe and effective microbiocides, for disinfection and protection of historical wooden surfaces at the former Auschwitz II-Birkenau concentration and extermination camp; this camp was part of the Auschwitz-Birkenau in Oświęcim, Poland. The scope of the work included:N-(3-aminopropyl)-N-dodecylpropane-1,3-diamine (APDA), hydrogen peroxide (HP), glutaraldehyde (GA), sodium hypochlorite (SH), boric acid (BA), and lactic acid (LA). The susceptibility of bacteria to five of the seven compounds tested was similar (Pseudomonas fluorescens showed low sensitivity to DDAC (MIC 7%).The quantitative and qualitative analysis of microorganisms colonizing wooden surfaces, such as doors, floors, bunk beds, door frames, beams and structural walls in the barracks at the former Auschwitz II-Birkenau concentration and extermination camp, was carried out in a previous study . The res similar . The onlEngyodontium album was considered the most sensitive of all moulds tested (s tested . DDAC anN-(3-aminopropyl)-N-dodecylpropane-1,3-diamine (APDA) > didecyldimethylammonium chloride (DDAC) > sodium hypochlorite > hydrogen peroxide > glutaraldehyde > boric acid > lactic acid. Based on these results, two active ingredients were selected for further study: didecyldimethylammonium chloride, belonging to quaternary ammonium salts, and N-(3-aminopropyl)-N-dodecylpropane-1,3-diamine, a polyamine. These compounds are active ingredients in a variety of formulations available on the market.The compounds evaluated are ranked by their decreasing antimicrobial activity: Based on data from the Office for Registration of Medicinal Products, Medical Devices and Biocidal Products , six biIn addition to concentration, the number of biocide applications is a very important factor in the disinfection process. In the present study, each disinfectant was sprayed onto the surface three times at two different concentrations. In order to determine the activity of the biocides, a five-level scale was used see . These sABM-1 and ABM-2 at a concentration of 6%; Rocima 101 at 8%; Preventol R80 at 12%; Acticide LV 706 at 15% and Boramon at 30% were the most effective against mixed populations of bacteria and moulds on the wood. Each of these disinfectants must be applied three times for effective disinfection.2 of surface in a single spraying procedure. Preventol R80 was the most expensive (€22.8 for a solution at a concentration of 6%). ABM-1 and ABM-2 were the cheapest, not exceeding €2.5, which is nine times less than the cost of other disinfectants.In addition to biocidal activity, the economic calculation was also considered i.e., the average cost of disinfectant consumption per 100 mNext, the biocides were evaluated for their effectiveness in wood preservation, against mixed cultures of bacteria and moulds. The results obtained under model conditions show that Rocima 101 and ABM-1 at concentrations of 8% applied by the fogging method, as well as Boramon at 30% and Rocima 101 at 8% applied by the spraying method, are highly effective in protecting non-historical wood against bacteria and moulds. No growth of microorganisms was recorded on the samples protected with these biocides, after six months of incubation. Differences between methods of biocides application are described in In order to verify the laboratory tests, the same disinfectants were applied under in situ conditions. They were checked for effectiveness in preserving historical wood; the treated wood was then exposed in one of the barracks at the former Auschwitz II-Birkenau concentration and extermination camp, for 12 months .Under in situ conditions, spraying with 30% Boramon and 8% Rocima 101, effectively protected the historical wood samples against bacteria for up to 12 months . Mould gTwo disinfectants, Rocima 101 and ABM-1, at a concentration of 8% were applied by fogging. Both of them effectively protected the historical wood samples from bacterial growth for 12 months . The mouAs with the new material, Boramon was not fogged on the historical material, since its effective concentration of 30% would adversely affect the environment. The methods of application onto wood surfaces were also compared under in situ conditions utilizing Rocima 101. We found that spraying was more effective for protecting against the development of microorganisms on the historical samples.E) and change in luminance (ΔL) were evaluated by visual and spectrophotometric methods. Values ΔE < 2 and ΔL < 1 determined by the instrumental method, were considered the levels at which the observer using the visual method does not notice any changes in the colour and luminance of the sample [The appropriate disinfectant must not only be selected based on its antimicrobial activity, but the colour and luminance of the treated wood should also be checked after application. Samples of new and historical wood were disinfected by spraying and fogging. Post disinfection treatments, the colour difference (Δe sample .p < 0.05) colour differences expressed as ΔE. The values of ΔE < 1 means that the observer did not notice any colour difference. The observed changes in the luminance of the material ΔL did not exceed 0.5, which also means that these changes were unnoticeable to the observer (E = 2.82) was observed in the case of the historical material fogged with Rocima 101, where the wood slightly darkened. At the same time, its application by spraying caused no significant change in the colour of the historical wood. In the case of the new wood samples disinfected by spraying Boramon and Rocima 101, and by fogging ABM-1 and Rocima 101, there were no statistically significant (observer . The higp < 0.05) differences of luminance and colour between the historical material and the new material after fogging may be connected with the inhomogeneous surface of the historical material. The statistically significant (N-(3-aminopropyl)-N-dodecylpropane-1,3-diamine, didecyldimethylammonium chloride, sodium hypochlorite, hydrogen peroxide, glutaraldehyde, boric acid and lactic acid) were evaluated to determine their activity on microorganisms isolated from the tested wood surfaces. This allowed us to select the compounds with the highest activity. Quaternary ammonium compounds (QACs) inhibited the growth of bacteria and moulds at the lowest concentrations, compared to other substances. The high effectiveness of QACs against moulds, decay fungi and insects that attack wood has been previously described [Various types of microbiocides are used for wood decontamination and preservation. Modern disinfectants are no longer monocomponent solutions, but mixtures of compounds with multidirectional mechanisms of action. It is unlikely that one synthetic or natural compound will eliminate biological factors adversely affecting wood ,21. In tescribed ,10,15. Tescribed ,22.In the next phase of the study, Boramon, which additionally contains boric acid, was used in addition to QAC-based preservatives. According to the European Chemicals Agency (ECHA) and the Biocidal Regulation 528/2012, due to its harmful effects on reproduction, this substance is currently used as a biocidal product only for wood preservation . Boron compounds, however, are often used as wood preservatives ,10,15,23After evaluating the disinfectant properties, experiments were performed to protect the wood surface against re-infection by bacteria and moulds. In laboratory conditions, preservatives containing QACs: Rocima 101 and ABM-1 at concentrations of 8%, and Boramon at 30%, effectively protected the wood samples against microbial growth despite the high relative humidity of 80% and temperature of 28 °C. However, these results were not reproducible under variable in situ conditions. Similar observations were made by Young et al.  who alsoE and luminance ΔL. The results are lower than those obtained by Tomak et al. [E = 2.29 to ΔE = 3.48 under the influence of boric acid at concentrations of 1% and 5%. The authors concluded that the values obtained indicate a slight colour difference. In contrast, Ozgen and Yildiz [In studies on wood preservatives, it is important to check the impact of these compounds on the material. A change in the colour of wood is a measurable parameter and, at the same time, an essential visual element. In the case of new materials, colour differences are readily noticed by consumers and often result in lowering its value. A change in the colour of historical wood caused by the action of chemical compounds may deteriorate its aesthetic value and, above all, contribute to the total destruction of the historical object ,25,26. Id Yildiz , who useAfter removal of the organisms responsible for biodeterioration, it is necessary to provide appropriate environmental conditions. Moisture is one of the main factors contributing to the development of not only mould and bacteria, but also algae and insects. Excess moisture can result from faulty construction of a building, poor site drainage, a leaking roof or a leaking plumbing system, insufficient insulation, or inadequate ventilation. All this may cause rainwater to leak into the interior of the building. Improving the structural condition of the building will significantly reduce the growth of microorganisms on historical materials ,28,29. In this study, we developed a procedure for the protection of historical wooden buildings from biodeterioration . Both, bPseudomonas fluorescens, Staphylococcus equorum, and Bacillus cereus; and moulds Alternaria alternata, Chaetomium globosum, Cladosporium cladosporioides, Engyodontium album, and Penicillium citreonigrum. The nucleotide sequences of the 16S rRNA gene of the bacteria used in the study were deposited in GenBank, the National Centre for Biotechnology Information . Mould strains were deposited in the Culture Collection ŁOCK 105 under the collection numbers Alternaria alternata ŁOCK 0594, Chaetomium globosum ŁOCK 0591, Cladosporium cladosporioides ŁOCK 0592, Engyodontium album ŁOCK 0590, Penicillium citreonigrum ŁOCK 0597. The bacteria were maintained on tryptic soy agar slants  and the moulds were stored on malt extract agar slants  at 4 °C. In order to activate the strains, the biomass was collected from slants and sub-cultured: the bacteria into tryptic soy broth  and the moulds onto MEA slants. The cultures were incubated at 30 °C for 24–48 h (bacteria), and 28 °C for 5 days (moulds).The effectiveness of active compounds and biocides was tested for three selected strains of bacteria and five strains of moulds, isolated from the wooden surfaces of the historical barracks at the Auschwitz II-Birkenau State Museum in Oświęcim. These include the bacteria http://bip.urpl.gov.pl/pl/biuletyny-i-wykazy/produkty-biob%C3%B3jcze). These included didecyldimethylammonium chloride , N-(3-aminopropyl)-N-dodecylpropane-1,3-diamine , hydrogen peroxide , glutaraldehyde , sodium hypochlorite , boric acid  and l-lactic acid . The concentrations of these active compounds were determined based on the concentrations of these ingredients in various commercially available biocides in Poland , which were then uniformly spread on the surfaces of the media. Sterile paper disks  were soaked with 15 mL solutions of the compounds, at the test concentrations , and the biomass was suspended in saline solutions (0.85% NaCl). Bacterial strains were combined in equal volumes to obtain mixed cultures. The density of the suspension was adjusted to 108 CFU/mL. The MEA slants of five-day fungal cultures were washed with sterile distilled water supplemented with 0.01% of Tween80. The density of the mould inoculum in the mixed cultures was determined using a haemocytometer, and adjusted to 106 conidia/mL.Antimicrobial activity of biocides was tested by the disc diffusion method, as described in Samples of new material, in the form of white poplar wood fragments measuring 50 × 20 × 10 mm, were used in the study. The samples were sterilized twice at 121 °C for 15 min., and then stabilized in a constant climate chamber  for seven days, at 28 °C and relative humidity (RH) 80%. However, historical wood fragments, collected from the Auschwitz II-Birkenau State Museum in Oświęcim, were used under in situ conditions.8 CFU/mL) and moulds (106 conidia/mL), activated in the media ((NH4)2SO4 0.075%, K2HPO4 0.025%, MgSO4·7H2O 0.125%, yeast extract 0.125%, glucose 0.5% and agar 0.1% pH 6.0), were applied onto the surface of new wood samples [The mixed cultures (1 mm each) of bacteria (10 samples . The sam3 of air with the addition of 5% of MIST-60 containing polyols). Twenty-four hours after the application of biocides, the sample surfaces were inoculated with mixed cultures of bacteria (108 CFU/mL) and moulds (106 conidia/mL). The samples were incubated in a constant climate chamber at 28 °C and 80% RH for seven days, one month, three months and six months. The effectiveness of antimicrobial activity was determined by the contact plate method, as described above. Control samples were materials not subjected to the biocide treatment.Selected biocides  were applied by the spraying method to the surface of each sterile, conditioned sample of new wood  or fogging method  were applied onto the surface of each sterile, conditioned sample of historical wood by the spraying method and by the fogging method. The samples were placed on metal shelves in the washroom located in the western part of the brick barracks B-65 at the Auschwitz II-Birkenau State Museum . The effIn order to determine the activity of the biocides against bacteria and moulds, a calibration scale was used according to L—luminance ranging from 0 (black) to 100 (white).a—the scope from red to green.b—the scope from yellow to blue.The change in colour and luminance of wood samples was quantified by the spectrophotometric method ,33. The 00E model was used to describe colour differences. In addition to the trichromatic components, it takes into account four characteristics: saturation, hue, brightness and blue colour. The following correlation was used to develop the results. It is the Euclidean distance between two points in a three-dimensional space of colour:The ΔTrichromatic components were measured at three points of each tested area, and the results were averaged. Samples were analysed by a spectrophotometer (Konica Minolta CM-2500d).Measurements were performed on the new and historical wood samples before biocide application, after two applications of Boramon and Rocima 101 by the spraying method, and after one application of ABM 1 and Rocima 101 by the fogging method. Control samples consisted of historical wood not subjected to the biocide treatment. E and ΔL represent means from three independent samples ± SD. Differences between means were tested by variance analysis (one-way ANOVA) with the post-hoc Tukey test. Probability (p) values of <0.05 were considered significant. Statistica v.10.0  was used for calculations.The results of ΔN-(3-aminopropyl)-N-dodecylpropane-1,3-diamine. The majority of the microbiocides (six of eight tested) contain these compounds and in laboratory conditions they effectively inhibit the growth of microorganisms after triple application by spraying. In addition, in situ Boramon 30% and Rocima 101 8% applied by spraying, effectively protected the historical wood from growth of bacteria for 12 months and moulds for 3 months. In the article, a monitoring scheme for the protection of historical wooden buildings from biodeterioration was suggested. It covers all stages of comprehensive recognition of the same object from disinfection to conservation. The presented procedure may help to perform similar analyses by other researchers.In this work, effective and safe microbiocides for the disinfection and protection of historical wooden surfaces at the former Auschwitz II-Birkenau concentration and extermination camp were indicated. Their concentration, times and method of application has been taken into account and the impact of microbiocides on historical material was examined. The most effective were didecyldimethylammonium chloride and


Portuguese freshwater reservoirs are important socio-economic resources, namely for recreational use. National legislation concerning bathing waters does not include mandatory levels or guidelines for cyanobacteria and cyanotoxins. This is an issue of concern since cyanotoxin-based evidence is insufficient to change the law, and the collection of scientific evidence has been hampered by the lack of regulatory levels for cyanotoxins in bathing waters. In this work, we evaluate the profile of cyanobacteria and microcystins (MC) in eight freshwater reservoirs from the center of Portugal, used for bathing/recreation, in order to determine the risk levels concerning toxic cyanobacteria occurrence. Three of the reservoirs did not pose a risk of MC contamination. However, two reservoirs presented a high risk in 7% of the samples according to the World Health Organization (WHO) guidelines for MC in bathing waters (above 20 µg/L). In the remaining three reservoirs, the risk concerning microcystins occurrence was low. However, they exhibited recurrent blooms and persistent contamination with MC up to 4 µg/L. Thus, the risk of exposure to MC and potential acute and/or chronic health outcomes should not be disregarded in these reservoirs. These results contribute to characterize the cyanobacterial blooms profile and to map the risk of toxic cyanobacteria and microcystins occurrence in Portuguese inland waters. Cyanobacterial blooms are becoming more frequent and widespread in freshwater reservoirs worldwide, mainly due to anthropogenic activities and to climate changes . Some toPortuguese surface freshwater resources are largely used for energy production, irrigation, recreational activities and as a source of drinking water for human consumption . An impoThe Portuguese legislation for drinking water, Decreto-Lei nº306/2007 , transpoAccording to national specificities and circumstances, some European countries complemented the European Bathing Water Directive  and implThe lack of a national database compiling the results of cyanobacteria and cyanotoxin monitoring hinders the characterization of Portuguese inland waters concerning toxic cyanobacterial blooms and the associated risks for public health and, in consequence, the implementation of guidelines.Furthermore, we must take into account that the use of those reservoirs is increasing, not only for the production of drinking water, but also for ludic activities, such as water sports, fishing, sailing, swimming and bathing. In addition, the vicinities of those reservoirs host camping, fairs, national and international summer music festivals, natural parks and hunting reserves. Thus, the potential risks associated with these reservoirs must be evaluated and managed in order to protect the health of their users.In this work, we compiled the results obtained during 14 years of monitoring of cyanobacteria and microcystins in eight freshwater reservoirs located in the centre of Portugal used for bathing and recreational activities. Based on these data, we profiled cyanobacterial blooms and determined the risk levels of toxic cyanobacteria occurrence and putative human exposure scenarios in these reservoirs.The variation of cyanobacterial density and microcystin concentration in the monitored freshwater reservoirs is presented in Microcystis aeruginosa (16–28% of the samples), their bloom frequency was below 10% and microcystins were never detected in the samples  and, in A6, blooms were detected during the entire two-year monitoring period , Table 1iborskii , were of6 cells/mL, particularly in A8 . It was the case of reservoirs A1, A2 and A3. However, blooms were also detected in October  and occasionally in November , December (A4 in 2007) and January (A8 in 2002/2003). Except for A4, where rix spp. , Table 2The risk levels concerning cyanobacteria density and microcystin concentrations, based on the WHO Guidelines for bathing waters , were determined for each reservoir as shown in The minimum water volumes required to reach the tolerable daily intake (TDI) through accidental swallowing in the reservoirs are shown in The study of the occurrence of cyanobacteria in Portuguese freshwater reservoirs dates back to the 1930s, but it was only in the 1990s that research on the toxicity and distribution of toxic species gained special emphasis, with the works of Vasconcelos and collaborators, particularly in the north of the country (revised in ). In theWith this work, we intended to evaluate the trend of cyanobacterial blooms and microcystins in eight freshwater reservoirs used for recreation and determine the risk levels and putative human exposure scenarios associated with those blooms.Microcystis aeruginosa, microcystins were never detected in water samples. These reservoirs are classified as mesotrophic (A1 and A3) and oligotrophic (A2), which might explain the low cyanobacteria density along all the monitoring period (from 9 to 14 years). Besides, non-toxic Microcystis aeruginosa strains might have been predominant within the cyanobacterial communities in these reservoirs. In three reservoirs , the occurrence of microcystins represented a low risk of potential acute effects because toxin levels were always below 4 μg/L and the water volumes required to produce such effects in adults were quite improbable. However an accidental swallowing of 100 mL of water in reservoirs A5 and A6 could already represent a health risk in children weighing up to 10 Kg. In addition, the risks resulting from repeated exposures should not be disregarded, particularly in the eutrophic reservoir A6, where the risk level based on cyanobacteria density was high and where blooms were quite persistent. Potential toxic genus  were dominant and, inclusively, two strains of M. aeruginosa isolated from reservoir A4 in October 2003 and from reservoir A5 in September 2002 were previously reported as producers of several microcystin variants  [M. aeruginosa. Although no cyanobacterial strains were isolated from reservoir A6 during the monitoring period, this reservoir has a previous history concerning the occurrence of toxic strains of M. aeruginosa, producers of several variants of microcystins [M. aeruginosa strains isolated from reservoir A8 [The results show that the studied freshwater reservoirs have distinct profiles of cyanobacteria proliferation and microcystins occurrence and, consequently, different risk scenarios concerning the potential human exposure to cyanotoxins. Three reservoirs , did not present a potential risk of human exposure to microcystins. Although a low risk based on cyanobacteria density was observed in more than 50% of the samples and despite potentially toxic cyanobacteria occurred, mainly nd MCYR) ,24,25. Tocystins ,24,25. Iocystins  was excervoir A8 ,24,25. Microcystis, Dolichospermum, Aphanizomenon and Oscillatoria. Previously, other authors also reported the prevalence and dominance of Microcystis spp. in freshwaters from the north [Dolichospermum (formerly named as Anabaena) [Aphanizomenon [Planktothrix [Cylindrospermopsis raciborskii [β-N-methylamino-l-alanine (BMAA) [C. raciborskii also occurred sporadically, but its toxicity was not evaluated. This points out that the risk levels in the studied reservoirs may be underestimated considering that cyanobacterial species that are potentially producers of other toxins also occur frequently and at high cell densities.Overall, we found a great diversity of species, being the most prevalent, among the potential toxic genera, he north ,28 and she north ,23,24 ofnabaena) ,23,28, Aizomenon ,30 and Pktothrix ,23,31,32ktothrix ,25,33. Iktothrix ,25,33. Aktothrix ,29,34,35ktothrix . Besidesiborskii  and the e (BMAA)  that havWe should also emphasize that, besides the acute cases of illness in humans exposed to microcystins during recreational activities ,40, contOn the other hand, it has been reported that cyanotoxins may accumulate in horticultural crops  that migMicrocystis, Aphanizomenon and Dolichospermum, but it also contained high levels of aluminum [The available epidemiologic data concerning human exposure to cyanotoxins in Portuguese freshwaters is restricted to two situations. The first occurred in 1993 in a dialysis unit in Évora Hospital , where twenty dialysis patients died ,46. Thisaluminum ,46. Unfoaluminum . A more aluminum  alerted aluminum . Unfortualuminum . There ialuminum ,48,49,50It was not our goal with the present paper to draw conclusions about the causes of the blooms in the studied reservoirs. Indeed, the monitoring period was not the same among them and, consequently, the seasonality of blooms in the reservoirs can hardly be compared. Nevertheless, we observed that, in a general way, the periods of 2000–2002 and 2007–2009 corresponded to the highest bloom frequencies. It is interesting to note that these dates were preceded by periods of extreme droughts, namely in 1998–1999 and 2004–2006 ; the latAphanizomenon spp., Dolichospermum spp. and even Cylindrospermopsis raciborskii, probably due to their highly successful adaptive strategies.On the other hand, although the blooms were detected mainly in spring and summer seasons, the reservoirs A4 to A8 also presented blooms in autumn and winter. These blooms occurred when the average air temperature was above normal for that period  or in rainy months that were preceded by heat waves and/or dry summers  ,52. ThisMany countries that regulate cyanotoxins in drinking water use a parametric value based on the WHO Guideline Value for MCLR of 1 μg/L . HoweverIn summary, we identified potential risk scenarios of human exposure to cyanobacteria and microcystins in five of the eight monitored recreational freshwater reservoirs located in central Portugal. Considering the cyanobacterial diversity observed in those reservoirs, we hypothesize that other cyanotoxins might occur and, consequently, that these risks might be underestimated. Therefore, the inclusion of other cyanotoxins in monitoring plans will be essential to clearly identify the risks associated with cyanobacteria in such reservoirs, particularly, the risks arising from exposure during recreational activities. Additionally, the systematization of monitoring data at a national scale would enable to obtain scientific-based evidence that can contribute to establish regulatory or guideline values for cyanobacteria and cyanotoxins in recreational freshwaters and, thus, to prevent health risks in such important socio-environmental water resources.For this study, a retrospective approach was used in which data of cyanobacteria and microcystins obtained between the years 2000 and 2015 was compiled. These analyses were performed in our laboratory, in the scope of the water quality monitoring programs implemented by the local water management entities. The sampling plan and the collection of water samples were the sole responsibility of the local authorities. Notice that, due to confidential obligations, we cannot identify the reservoirs. Sampling was performed in eight freshwater reservoirs located in the center of Portugal , betweenAliquots of 100 mL were preserved with Lugol’s solution (1 drop/10 mL) for phytoplankton identification and quantification. Two aliquots of 5 mL were kept frozen (−20 °C) until microcystin analysis. Phytoplankton species were analyzed according to the Utermöhl technique . Briefly®, Helsinki, Finland) and acceptable sample variance was <15%. Results were obtained from two replicate samples and are given in μg MCLR equivalents per L water. This method provides a detection and quantification limit of 0.10 and 0.15 μg/L, respectively.Total microcystins in water samples were analyzed by enzyme-linked immunosorbent-assay (ELISA), using a specific plate kit , according to the manufacturer instructions. Previously frozen 5 mL aliquots were defrosted at room temperature (one freeze/thaw cycle) and sonicated  for 2 min (10 s sonication/2 s rest cycle) to release intracellular microcystins. Absorbance was measured at 450 nm using a microplate ELISA photometer (Thermo, Labsystems Multiskan AscentThe risk levels of each reservoir were calculated according to the WHO Guidelines in bathing waters , which c


Background: Swallowing and swallowing-related quality of life studies following stroke were almost always performed by including both patients with brainstem and cortical involvement. It was aimed in this study to show the presence of dysphagia in patients with only cortical ischemic stroke and to investigate the interaction between dysphagia and quality of life as well as to evaluate the effect of a rehabilitation program in the acute phase.Methods: Seventy-two patients with cortical stroke (between 0 and 30 days) and dysphagia were included. Swallowing function of patients was assessed by dysphagia screen questionnaire and fiberoptic endoscopic assessment. Also, functional impairment and swallowing quality of life were assessed. The swallowing rehabilitation program for 4 weeks was given to all patients.Results: All patients demonstrated disorders related to oral phase , pharyngeal phase  or both phases. The swallowing function, swallowing quality of life and functional impairment were improved at the end of therapy.Conclusion: Swallowing quality of life is severely affected in cortical hemispheric stroke patients and can be improved with an early rehabilitation program. Severe dysphagia is usually observed during the first 2-4 weeks, with a prevalence ranging from 29%-81%.1-3 Aspiration pneumonia is seen in patients with dysphagia during the first year, with a mortality rate as up to 45%.5 Studies have been reported that if it is early diagnosed and treated, the complications may be reduced or even prevented.6 Moreover, it has been reported that when a person has a dysphagia, the ability to enjoy almost all of life is affected. A minor or intermittent dysphagia can lead to stress in both psychological and social situations. Episodes of choking can lead to a fear of eating that can result in malnutrition and social withdrawal.7,8Dysphagia may cause to important complications such as hydrational and nutritional deficiency, aspiration pneumonia and even death.9 However, unilateral cerebral hemispheric infarction is seen more often than brainstem events.10 Despite the frequency of dysphagia is typical in the brainstem or bilateral infarct, it is often seen subsequent to one-sided hemispheric infarct in our daily clinical practice. Swallowing studies have been generally concentrated on the brainstem, ischemic and hemorrhagic types of stroke, but patients with brainstem and cortical involvement were evaluated in combination.11,12 In these studies, most of the hemispheric strokes involve middle cerebral artery (MCA) and perfusion areas. Elaborating more on demographic characteristics of these studied populations indicated that severe dysphagia has been observed in these patients. However, studies have not been performed on ischemic stroke patients with only hemispheric cortical involvement and there is also no study investigating the dysphagia-related quality of life among these patients. Therefore, we aimed to reveal dysphagia in cortical stroke and to demonstrate its impact on quality of life.Since central control mechanism of swallowing is located in the brainstem, severe dysphagia is more likely to occur when stroke involves this part.This study was performed on 72 patients who referred to our Physical Medicine and Rehabilitation (PMR) clinic between 2015 and 2016. The subjects, ranged from 50 to 75 years and applied for rehabilitation of some problems such as functional impairments in the first 30 days after ischemic hemispheric MCA stroke, approved by magnetic resonance imaging, and had dysphagia which was shown by dysphagia questionnaire, were included. This dysphagia questionnaire that our standard used in clinic includes a neurologic examination test and a water drinking test as well as oxygen saturation measurement by pulse oximetry.The neurological examination test included some abilities such as head lifting, independent seating balance and cranial nerves related to deglutition. On the basis of this, neurological measure outcome (NMO) was created. Based on monitoring and documentation of the water drinking test, swallowing outcome (SO) was calculated. Dysphagia outcome (DO) was calculated by combining the scores for NMO and SO. Accordingly, patients with a score of 3 and below were evaluated as normal.Seventy-two patients were included between 4 to 15 scores according to DO score in this study. Patients with a history of tumor, head-neck operation, past stroke, known swallowing disorders, the presence of gastroesophageal reflux disease, dementia  or psychiatric diseases, brainstem, hemorrhagic, subcortical and/or bilaterality, and smoking were excluded. In addition, exclusion criteria for flexible fiberoptic endoscopic evaluation of swallowing (FEES) method were the severe infective disease, bleeding risk and/or decompensated cardiac disease.Patients and their caregivers were given information regarding the study and their inscriptive approvals were obtained before starting the study. The confirmation of the Ethics Committee of the hospital was taken. The study was carried out in conformity with the standards of the Helsinki Declaration.Characteristics of 72 patients including age, gender, education, dominant side, additive diseases and disorders associated with respiratory and dental, infarct area, and passing time following stroke were documented. Educational status was as illiterate, under 5-year, 5-year, 8-year, 11-year and over 11-year education.The motor available condition was evaluated by Brunnstrom motor level for upper and lower limbs as well as hand, separately and graded between 1 and 6. The assessment of swallowing function: Swallowing disorders were evaluated using dysphagia screen questionnaire and FEES.Mann assessment swallowing ability (MASA) test: MASA was applied to a screen test. Twenty-four areas were assessed such as vigilance, communication, hearing, speech and respiration disorders, movement limitation, weakness and incoordination in swallowing muscles, the presence of reflexes associated with swallowing. The score was calculated between 38 and 200 points.FEES: The test was conducted using a 3.4 mm-diameter fiberoptic nasopharyngoscope. Evaluations were applied in a seated situation or a vertical posture to the utmost. Local anesthetics were not applied to prevent its side effects. To evaluate penetration, aspiration and presence of residue, water was used as the fluid, yogurt and a biscuit as the consistency food. The function of the pharyngeal stage was assessed with these foods and findings were saved as the video images. Swallowing status was determined between 1 and 6 according to the endoscopic measurement scale generated by Warnecke, et al.13 According to this, 1 point was defined as normal swallowing, while 2-6 points as dysphagia. The swallowing abnormalities  were detected and noted with respect to results of swallowing assessment procedures.Other evaluation parameters: The functional status was measured with functional independence measure (FIM), which evaluates two important parts of functions as a motor and cognitive status. This scale includes 18 items and 6 parts comprised of personal care, continence, mobility, transfer, interaction and social cognition; and each item has score between 1 and 7. The total score is between 18 and 126.14 to evaluate the quality of life in patients with oropharyngeal dysphagia. SWAL-QOL contains 44 questions on domains of eating disorder, duration of eating, desire to eat, choice of meal, communication, anxiety, mental health, social functioning, fatigue, and sleep. Each question is evaluated by a score ranging between 1 (the worst) and 5 (the best) points. Each domain can be evaluated separately. In our study total scoring was used.Swallowing-related quality of life scale (SWAL-QOL) was used to evaluate the impact of swallowing disorders on quality of life. It was developed by McHorney, et al.Study evaluation protocol: The study was performed by specialists that composed of swallowing team members in our hospital as blinded to therapy distribution. The dysphagia screen questionnaire, FIM and SWAL-QOL were applied by the 1st PMR expert on the 1st day of hospital admission. Afterwards, patients were evaluated with the endoscopic method by a blinded otolaryngology specialist and were sent to the 2nd PMR specialist.Rehabilitation methods: Daily care for oral hygiene training and the required swallow maneuvers, head and trunk positioning and diet modification were given according to the condition of swallowing disorder of all patients. Furthermore, oral motor-strengthening exercises for lips, tongue and jaw, cold-tactile stimulation as well as intermittently or alternatively galvanic stimulation to bilateral masseter or submental muscles, according to the presence of an oral or pharyngeal disorder or both, were received by the same physiotherapist. This program was performed for 4 weeks, 20 sessions in total (1 hour a day and 5 hours per week). Apart from these, cognitive, respiratory, sensorial and motor rehabilitation therapies were given to all subjects.Comparisons: Dysphagia severity level defined by the FEES and MASA, as well as FIM and SWAL-QOL scores were reevaluated after therapy. The results of therapy and changes within the group were compared.Statistical analysis: SPPS software  version for Windows was used for statistical analysis. Shapiro-Wilk test was used to know whether the quantitative data are normally distributed or not. Descriptive statistics were presented as the mean ± standard deviation (SD) or median (minimum-maximum) for quantitative data and frequencies and percentages (%) for qualitative data. Statistically critical differentiations in recurrent evaluations within the group were shown with the Wilcoxon signed-rank test. The Bonferroni correction was performed to avoid potential Type I mistakes in within-group comparison (P < 0.025).A total of 72 patients with hemispheric ischemic patients aged between 50-75 years, who were admitted to hospital as an inpatient, treated in our center during the first 30 days following stroke and were enclosed in the study.The mean DO which defined the patients as dysphagia was 9.32 ± 2.45. At hospital admission, 8 patients (11.1%) received regimen-3  diet comprising liquid, semi-solid, and solid foods, 42 patients (58.3%) received regimen-2 diet consisting of semi-solid foods supplemented with intravenous infusion of fluids, 18 patients (25.0%) were fed with nasogastric catheter , and 4 patients (5.6%) received gastrostomy catheter. The mean age of the 72 patients was 63.32 ± 11.17 years. Among 72 patients, 25 (34.7%) were female, while 47 (65.3%) were male. The patients had right , and left  hand dominance. In all patients, ischemic stroke involved MCA region (100%). The mean passing time after stroke was 16.51 ± 8.32 days. Demographic and disease characteristics of subjects are presented in According to Brunnstrom staging of the motor functions of the patients at admission, median motor function stage of the upper and lower extremities, and hands of the patients were 2.00 (2.19 ± 1.34), 2.00 (2.53 ± 1.42), and 1.00 (2.12 ± 1.09), respectively.Distribution of bedside screening test and FEES levels results which demonstrated pre-treatment swallowing functions, functional disability and SWAL-QOL scores are shown in Distribution of bedside screening test and FEES levels results which demonstrated post-treatment swallowing functions, functional disability and SWAL-QOL scores are shown in The significant improvement was detected after treatment swallowing functions of the patients . A significant improvement was detected in cognitive, and total disability scores of functional disability scale using FIM , while the change in motor function scores was not significant (P = 0.467). Also, a significant improvement was found in SWAL-QOL score (P = 0.001).9-11,15-17 In previous studies, it has been shown that brainstem stroke is a primary risk factor for persistent dysphagia. Because, the swallow response is generated in the brainstem swallowing center located in the medulla oblongata which combined knowledge directed from the oral, pharyngeal and suprabulbar areas. These centers were considered to be autonomous central pattern generators largely controlling the synchronization and timing of swallowing. However, a complex array of cortical representation, including motor, premotor, and sensorimotor cortices, appears to be crucial in its effective coordination.18 These cortical areas provide volitional deglutition and supply primarily to trigger swallowing and control the of swallow motor response.9,18,19 Normal swallowing is generally divided into four stages. However, this division is not as simple as it is said to be. The normal swallow is a complex, fast, continuous sequence of coordinated muscle movements and there is some overlap between the phases. Cortical stroke has been shown to have impact on the pharyngeal phase of the swallow, with impairment to initiation and duration and increased frequency of penetration and aspiration as well as with impairment in pharyngeal transit and longer oral transit.20,21Dysphagia in patients with stroke commonly occurs after ischemia of the cerebral cortex. In recent studies, it has been reported that most of these patients had regained their swallowing functions, while in 11-15% of them dysphagia which led to complications as aspiration pneumonia had persisted.6,22 Moreover, in patients with severe dysphagia who require feeding via a gastrostomy tube, no difference has not been detected between brainstem involvements and cortical hemispheric strokes.23,24 The other two studies have been demonstrated that the presence of dysphagia is related to MCA involvement in patients with cortical stroke.25Brainstem strokes may account for up to 15% of all strokes. In two recently performed studies, in which cortical hemispheric involvement was reported in 90% of the patients who were diagnosed, and followed up with dysphagia at early stroke period, spontaneous return of swallowing functions was indicated in only 9.5% and 37% of the patients, 1 month later in the first, and 3 months later in the second study.Because of these reasons, in our study, we included 72 patients with ischemic stroke involving perfusion area of MCA. We evaluated our patients for a mean period of 16.51 ± 8.32 days after stroke, which was somewhat longer than conventionally reported recovery time, and detected disorders related to oral , pharyngeal , and oropharyngeal  phases. These patients (n = 72) also represented the whole spectrum of mild to very severe dysphagia. Patients’ SWAL-QOL scores were nearly half of maximum well-being index scores, and general functional impairment levels were one-third of normal scores. We applied a combination treatment also including electrical stimulation on our patients for 4 weeks. 26 while in some studies it was advocated that it would provide beneficial effects if applied at an early stage.6 However according to the guidelines on rehabilitation of stroke lack of adequate data have been stated.27Generally, spontaneous recovery of swallowing function occurs within the first 2-4 weeks, so in previous studies initiation of a rehabilitation program for dysphagia was postponed after that period. In guidelines for management of dysphagia, treatment of dysphagia is absolutely advised,3,10 Especially early phase was reported as a window of opportunity.6,28 In a study, in patients who received classical treatment at an early period in stroke within the first 2 weeks 100% improvement was achieved in oral phase problems, and 75-90% recovery in pharyngeal phase disorders with a lesser number of treatment sessions than in patients that same treatment initiated in one month later.In recent years, the presence of a cortical inhibition in both intact and damaged hemisphere, and functional recovery induced by compensatory cortical re-organization have been indicated.6Also, in patients applied treatment after 4 weeks, oral and pharyngeal phase problems were detected which were regressed in 15%, and 45% of the cases, respectively. Aspiration detected video fluoroscopically was persisted in 60% of these patients.29-32 Similar to our study, Bulow, et al. reported that dysphagia treatment was effective even in their subacute phase of patients with hemispheric stroke.32In the light of this information, we also applied combined rehabilitation program for our patients at a considerably early stroke period. Indeed, in studies performed using both traditional methods, and new techniques which involve electrical stimulations, it has been reported that especially combination treatments decreased dysphagic complications and increased rate of oral feeding.33,34The medical complications of dysphagia include aspiration pneumonia and malnutrition. Other complications of dysphagia in stroke patients are psychological and social effects because eating is an enjoyable social activity, and inability to eat usually may affect patient morale and quality-of-life.33,34 These meta-analyses have been reported that fears of these patients, and their reflections on the social environment to be adverse parameters affecting their quality of life. Since they are most frequently seen especially during the acute phase, quality of life is most affected during this early period. However, these studies have been most frequently performed in cases with brainstem strokes.16,34 We considered that hemispheric strokes are more frequent, and low quality of life may be more prevalent in these patients. Therefore, in our study SWAL-QOL scale was used. The items on the SWAL-QOL address desire for eating, dysphagia symptom frequency, mental health, social concerns related to swallowing problems, food selection, fear related to eating, and the burden of dysphagia. While the quality of life of nearly 50 of our studied patients was deteriorated before treatment, this rate dropped down to 30% after treatment.Swallowing-related quality of life has been evaluated using different scales, and different studies have reported that fear from choking during eating, and inability to control dysphagic symptoms, physical, and social insecurity secondary to anxiety and fear are the most frequently encountered problems. In meta-analyses performed, especially evaluation of the quality of life of dysphagic patients has been indicated.Despite lack of similar studies in the literature, our result suggests that dysphagia related to hemispheric strokes is as important as those associated with brainstem strokes with respect to quality of life.As a result, cortical strokes are frequently encountered in our clinical practice, and contrary to our classical information they can induce dysphagia which will be able to affect the quality of life. We think that rehabilitation programs that applied these patients in early stroke period will decrease both medical and psychosocial complication rates related to dysphagia.


Ruditapes decussatus, also known as the European clam. Ruditapes decussatus is morphologically and ecologically similar to the Manila clam Ruditapes philippinarum, which has been recently introduced for aquaculture in the very same habitats of Ruditapes decussatus, and that is replacing the native species. Currently the production of the European clam is almost insignificant, nonetheless it is considered a high value product, and therefore it is an economically important species, especially in Portugal, Spain and Italy. In this work we: (i) assembled Ruditapes decussatus mitochondrial genome from RNA-Seq data, and validated it by Sanger sequencing; (ii) analyzed and characterized the Ruditapes decussatus mitochondrial genome, comparing its features with those of other venerid bivalves; (iii) assessed mitochondrial sequence polymorphism (SP) and copy number variation (CNV) of tandem repeats across 26 samples. Despite using high-throughput approaches we did not find evidence for the presence of two sex-linked mitochondrial genomes, typical of the doubly uniparental inheritance of mitochondria, a phenomenon known in ∼100 bivalve species. According to our analyses, Ruditapes decussatus is more genetically similar to species of the Genus Paphia than to the congeneric Ruditapes philippinarum, a finding that bolsters the already-proposed need of a taxonomic revision. We also found a quite low genetic variability across the examined samples, with few SPs and little variability of the sequences flanking the control region (Largest Unassigned Regions (LURs). Strikingly, although we found low nucleotide variability along the entire mitochondrial genome, we observed high levels of length polymorphism in the LUR due to CNV of tandem repeats, and even a LUR length heteroplasmy in two samples. It is not clear if the lack of genetic variability in the mitochondrial genome of Ruditapes decussatus is a cause or an effect of the ongoing replacement of Ruditapes decussatus with the invasive Ruditapes philippinarum, and more analyses, especially on nuclear sequences, are required to assess this point.Despite the large number of animal complete mitochondrial genomes currently available in public databases, knowledge about mitochondrial genomics in invertebrates is uneven. This paper reports, for the first time, the complete mitochondrial genome of the grooved carpet shell,  Caenorhabditis elegans)—is uneven. To better understand invertebrate mitochondrial biology—and, most importantly, mitochondrial biology and evolution in general—it is necessary to adopt a more widespread approach in gathering and analyzing data. Failing to do so would bias our knowledge toward a few taxonomic groups, with the risk of losing a big part of the molecular and functional diversity of mitochondria. Actually, despite maintaining its core features in terms of genetic content, mtDNA in Metazoa shows a wide range of variation in some other traits such as, for example, genome architecture, abundance of unassigned regions (URs)—namely regions with no assigned product —repeat content, gene duplications, introns, UTRs, and even additional coding genes  being available in public databases , up to now sequencing has been focused mostly on vertebrates , and the current knowledge about mitochondrial genomics in invertebrates—with the notable exception of few model organisms . Ruditapes decussatus—also known as the European clam—is distributed all over the Mediterranean coasts, as well as on the Atlantic shores, from Lofoten Islands (Norway) to Mauritania, including the British Isles. Ruditapes decussatus lives in warm coastal waters, especially in lagoons, and it is morphologically and ecologically similar to the Manila clam Ruditapes philippinarum, which has been recently introduced for aquaculture in the very same habitats of Ruditapes decussatus. Ruditapes philippinarum, native from the Philippines, Korea, and Japan, was accidentally introduced into North America in the 1930s, and from there it was purposely introduced in France (1972), UK (1980), and Ireland (1982) for aquaculture purposes . Upon introduction of the more robust Ruditapes philippinarum, Ruditapes decussatus suffered a population decline in the Southwestern Europe  analyzed and characterized Ruditapes decussatus mitochondrial genome, comparing its features with those of other venerid bivalves; (iii) assessed mitochondrial sequence polymorphism (SP) and structural variants—copy number variation (CNV) of tandem repeats—among the sampled animals.Molluscs in general, and bivalves in particular, exhibit an extraordinary degree of mtDNA variability and unusual features, such as: large mitochondrial genomes (up to ∼47Kb), high proportion of URs , novel protein coding genes with unknown function, frequent and extensive gene rearrangement, and differences in strand usage . MoreoveRuditapes decussatus specimens used in this study were collected from the Northern Adriatic Sea, in the river Po Delta region  at the end of July 2011, during the spawning season. Each individual was dissected, and gonadal liquid collected with a glass capillary tube. All the samples showed ripe gonads, consistently with the time of the year when the sampling occurred. The gonadal liquid was checked under a light microscope to assess the sex of the individual, and to make sure that the sample consisted of mature gametes. Both the gamete samples and the clam bodies were flash-frozen in liquid nitrogen, and stored at −80 °C, until nucleic acid extraction. The 26 In total, 12 samples  were used for RNA-Seq. Total RNA extraction and library preparation were performed following the protocol described in Ruditapes decussatus was not available in the databases, so we used the transcriptome data to generate a draft to be used as a guide for Sanger sequencing. Illumina reads from all 12 samples were pooled and compared to a set of 20 Bivalvia mitochondrial genomes to identify reads with mitochondrial origin. Alignment was done using BLASTN. All reads with similarity yielding E-value < 1E-5 were then assembled into contigs using the A5 pipeline  of 13 females to assess its variability (see Results and Discussion). The list of the primers and their sequences are reported in In total, 14  Primer3  and testhttp://www.macrogen.com).Sanger sequencing was performed by Macrogen Inc. . tRNAs and other secondary structures were drawn with the software Varna GUI (rrnS) and large subunit (rrnL) were identified with BLASTN, and annotated considering the start and the end of the adjacent genes as the boundaries of the rRNA genes. Non-genic regions were annotated as URs. In order to identify the putative D-loop/control region (CR), we analyzed the LUR with the MEME suite , Meretrix lamarckii (M-type), Meretrix lusoria, Meretrix lyrata, Meretrix meretrix, Meretrix petechialis, Moerella iridescens, Nuttallia olivacea, Paphia amabilis, Paphia euglypta, Paphia textile, Paphia undulata, Ruditapes philippinarum (F-type), Ruditapes philippinarum (M-type), Semele scabra, Sinonovacula constricta, Solecurtus divaricatus, Solen grandis, Solen strictus, Soletellina diphos and the sea urchin Strongylocentrotus purpuratus . The list of the species used in the phylogenetic analysis and in the comparative analyses of DNA motifs, sequence similarity, and gene order are available in Open reading frames (ORFs) were identified with ORF finder . Alternaarna GUI . RibosomME suite  to find http://tandem.bu.edu/trf/trf.html), since the complete LUR sequence was available . In the other cases, in which the LUR could not be sequenced without gaps, the number of repeats was inferred from agarose gel electrophoresis.The number of repeats in the LUR of the reference sample (F4) was calculated with tandem repeat finder   and EasyrProScan .https://ndownloader.figshare.com/files/8906839). In four cases  a clear polyadenylation signal was present, in other four cases  it was not. Contig 4, the only one including a single gene (cox3), ends with just 8 As, so it is not clear if a polyadenylation signal is present in this case. In Contig 7  there is a polyadenylation signal (56 As) after the cox2 gene.Despite using HTS on extracts of ripe gonads , and multiple assembly strategies (see Discussion for details) we could not find evidence for DUI. The de novo assembly process produced 9 contigs, of which 8 included multiple genes, and one included a single gene see . The seqde novo assembly. We first tried to connect the contigs designing primers close to the 5′ and 3′ ends of each contig and pairing them following the gene order of Paphia, because the sequence of genes in the contigs suggested that Ruditapes decussatus gene order might have been similar. During such process, Contig 1 turned out to be a chimeric assembly between two non-contiguous portions of the mtDNA, one including atp6, nd3, and nd5, the other including cox1, tRNA-Leu1, nd1, nd2, and nd4L. Once we amplified and sequenced the portions of mtDNA between the contigs, we proceeded with the Sanger resequencing of the remaining parts.The nine contigs were used as a scaffold for the primer walking procedure used for Sanger validation of the cox1, nd1, nd4L, cox2, cob, atp8, nd4), and ATG . The stop codons found are TAG  and TAA . The nd4 gene has an incomplete stop codon (TA-). 22 tRNA genes were identified, including two tRNAs for leucine, tRNA-Leu1(TAG) and tRNA-Leu2(TAA), and two for serine, tRNA-Ser1(TCT) and tRNA-Ser2(TGA), both showing degenerate D-arm branches. tRNA structures are shown in rrnS and rrnL, were both identified: the rrnS is located between cox3 and cox1, while rrnL is between cytb and atp6. URs were identified on the basis of unannotated spaces between different genes; we found 24 URs  and nd4 (61%). The nucleotide composition of every gene is shown in The analysis of the nucleotide composition points out that the mitochondrial genome of this bivalve species exhibits high A+T content, totaling 63% vs 37% G+C. The minimum values of A+T are found in atp8 and nd5 (atp8 gene (https://ndownloader.figshare.com/files/8387672), with a change in Gibbs free energy (ΔG) of −71.38 Kcal/mol. We also amplified and sequenced the LUR of 13 more females. We were not able to completely sequence LURs longer than 2,110 bp, because of the known difficulties in Sanger sequencing of regions including multiple repeats. The sequence alignment of the 13 LURs is available for download from figshare (https://ndownloader.figshare.com/files/8360789). LUR lengths, inferred from gel electrophoresis, are reported in S. purpuratus. The sea urchin was included in the analysis because Mytilus edulis and Mytilus galloprovincialis with regulatory elements of the sea urchin CR. Accordingly, the search with GOMo assigned a series of GO terms related to transcription to the two motifs (The UR11 is the LUR and is located between  and nd5  and 2A. tp8 gene . DNA seco motifs .https://ndownloader.figshare.com/files/8902537), of which 18 are represented by indels, 6 of which are located in 4 different coding genes: one each in cox1, cytb, and nd5, plus 3 in cox3 . cox3 see . A file Ruditapes decussatus mtDNA map , and the BLASTN identity (colored inner circles) with complete mtDNAs of other 10 venerid species (see list in https://ndownloader.figshare.com/files/8360792). Ruditapes decussatus and P. euglypta  resulted to be a chimeric sequence obtained by the misassembly of two smaller contigs. Most of the contigs (eight out of nine) contained more than one gene, and most of the tRNA genes were included in the de novo assembly. Except for tRNA-Pro, tRNA-Ile, and tRNA-Leu1, all the other tRNA genes are organized in two big clusters: a 13-gene cluster positioned between cox3 and nd6, and a 6-gene cluster between nd6 and nd4. The assembly retrieved 6 out of 13 tRNAs from the first cluster , and 4 out of 6 tRNAs from the second cluster (missing tRNA-Met and tRNA-Asp). All the tRNA genes not located in these two clusters  were included in the contigs. The presence of a clear polyadenylation signal in four of the assembled contigs  is of 18,995 bp, and it includes 13 protein-coding genes, 22 tRNAs and 2 rRNAs. Our data support the presence of the atp8 gene in the mtDNA of Ruditapes decussatus; atp8 has been reported as missing in several bivalve species, however, more accurate searches often led to the identification of the gene, so, in most cases, the alleged lack of atp8 is likely ascribable to annotation inaccuracies due to the extreme variability and the small size of the gene , a common feature in bivalve mtDNAs; moreover, T is the most common nucleotide at the third codon base (64.6%). The most common codon is UUU (Phe), which is also the most commonly used in bivalves, as well as in other invertebrates , as observed in other bivalve species , and by analysis of the LUR . The CR and its flanking regions are known to be hypervariable, so they are commonly used to assess polymorphism at low taxonomic levels. Our data strongly support a very low genetic variability: the number of SPs in CDS is 145, of which 103 are private of a single individual (mRDI01)—thus reducing the number to 42—while the number of variable sites in the analyzed LURs is 98 over 3,095 aligned positions. Considering the known variability of mtDNA in bivalves . This means that randomly sampled individuals of Ruditapes decussatus used in this work showed a much lower mtDNA variability than Ruditapes philippinarum siblings. A previous analysis on the cox1 gene of Ruditapes decussatus reported a nucleotide diversity (π) of 0.15 for a population from the Northern Adriatic Sea , and are present at relatively low frequencies in the specimens carrying them, except for SP_6364 which has a frequency of 80% in fRDI04. The cox3 gene shows three SPs: the first one, SP_17619, does not cause a frameshift, and results in the deletion of one alanine residue, and its frequency in mRDI01 is 97%. The second one, SP_17621, consists of a deletion of a G with respect to the reference sequence, which is the Sanger-sequenced mtDNA of sample F4; all the individuals analyzed with RNA-Seq carry this deletion except for mRDI01 which, at that position, has the same sequence of the reference mtDNA . The third indel, SP_17624, consists of an insertion of two nucleotides, and its frequency in mRDI01 is 99%. So, basically, for cox3 we have three types of sequences: (i) the Sanger-sequenced reference, which yields a 966 bp (321 aa) ORF; (ii) a sequence found in 11/12 of samples analyzed with RNA-Seq (except mRDI01) that carries a single-nucleotide deletion (SP_17621), and yields a 963 bp (320 aa) ORF; (iii) a sequence, private of mRDI01, which is obtained by combining SP_17624 and SP_17621 , which produces a 963 bp (320 aa) ORF. Interestingly, the ORFs obtained from the sequences described in (ii) and (iii), are almost identical, namely the sequence obtained by RNA-seq in 11/12 samples and the sequence obtained by RNA-Seq in mRDI01 are basically the same, and differ from the Sanger-sequenced reference, yielding an amino acid sequence that differs in the last 35 residues . Given this consistent difference between the sequence obtained by Sanger-sequencing of DNA, and those obtained by RNA-Seq, it is tempting to speculate that this difference might be caused by RNA editing, a mechanism observed in mtDNA of some animals , and because it overcomes the problem of primer specificity, a limitation of the classical approach. One possible concern about using HTS approaches based on short reads in presence of DUI is about the ability of softwares to detect divergent reads and assembly them correctly. More specifically, one could ask what is the divergence threshold under which the assemblers are not able to partition the contigs into two sex-linked groups. We do not know such a threshold, but we used different assembly strategies trying to retrieve sex-specific mtDNA sequences from our data. Other than the approach reported in Materials and Methods (which is the one that produced the data reported here), we tried other techniques. After identifying reads that blasted to bivalve mitochondrial sequences present in GenBank and discarding all the other reads, we generated A5+CAP3 assemblies: (i) for each of the samples (obtaining 12 separate assemblies), and (ii) pooling the six males together and the six females together, and assembling the two sex-specific pools. Both these approaches did not show evidence of sex-specific mtDNAs. Then we took the assembly obtained from the females and removed the reads from each of the samples that mapped (<8 mismatches) to these sequences. We then used the remaining reads as A5 input. The program could not assemble anything. Lastly, we tried the software MetaVelvet —that assRuditapes decussatus is characterized by SMI of mitochondria, so a male-transmitted mtDNA is not present in this species.DUI is very young in this species, so the two sex-linked mtDNAs did not have the time to diverge.Ruditapes decussatus, so this hypothesis must be taken into consideration.A role-reversal event occurred recently. Role reversal  is a process—observed so far only in species of the Mytilus complex—by which F-type genomes invades the male germ line becoming sperm-transmitted, thus turning into M-type mtDNAs . This evThe divergence between the two sex-specific mtDNAs is too low to be detected. This could be the outcome of two different situations.Ruditapes decussatus populations, but considered the variability usually observed in bivalves, we find the difference unsurprising. On the contrary, the lack of variability among the other 11 samples is remarkable. For these reasons, we are inclined to believe that mRDI01 divergence is compatible with hypotheses (1) and (2). That said, there still could be a third, quite conjectural, hypothesis by which these data might indicate an incipient DUI, not yet fixed in the population.In our data, even if there is no clear evidence of a male-specific mtDNA, a male sample (mRDI01) clearly stood out from the others, both males and females see . OverallAll in all, we have a preference for the first explanation, but the present data are not sufficient to exclude the others, and a more thorough investigation is necessary to assess this point.Cyclina sinensis, Ruditapes philippinarum, and Meretrix lamarckii (Up to now DUI was identified in only three Veneridae species: amarckii . The sta10.7717/peerj.3692/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.3692/supp-2Supplemental Information 2Click here for additional data file.10.7717/peerj.3692/supp-3Supplemental Information 3Click here for additional data file.10.7717/peerj.3692/supp-4Supplemental Information 4Click here for additional data file.10.7717/peerj.3692/supp-5Supplemental Information 5Click here for additional data file.10.7717/peerj.3692/supp-6Supplemental Information 6Click here for additional data file.10.7717/peerj.3692/supp-7Supplemental Information 7BP, Biological Process; CC, Cellular Component; MF, Molecular Function.Click here for additional data file.10.7717/peerj.3692/supp-8Supplemental Information 8Click here for additional data file.


Normal coronary flow velocity reserve (CFVR) (≥ 2) obtained in theleft anterior descending coronary artery (LAD) from transthoracicechocardiography is associated with a good prognosis, but there is no studycorrelating CFVR with submaximal target heart rate (HR).To evaluate the prognostic value of CFVR obtained in the LAD of patients withpreserved (>50%) left ventricular ejection fraction (LVEF) who completeda dobutamine stress echocardiography (DSE), considering target HR.Prospective study of patients with preserved LVEF and CFVR obtained in theLAD who completed DSE. In Group I (GI = 31), normal CFVR was obtained beforeachieving target HR, and, in Group II (GII = 28), after that. Group III (GIII=24) reached target HR, but CFVR was abnormal. Death, acute coronaryinsufficiency, coronary intervention, coronary angiography without furtherintervention, and hospitalization were considered events.In 28 ± 4 months, there were 18 (21.6%) events: 6% (2/31) in GI, 18%(5/28) in GII, and 46% (11/24) in GIII. There were 4 (4.8%) deaths, 6 (7.2%)coronary interventions and 8 (9.6%) coronary angiographies without furtherintervention. In event-free survival by regression analysis, GIII had moreevents than GI (p < 0.001) and GII (p < 0.045), with no differencebetween GI and GII (p = 0.160). After adjustment, the only difference wasbetween GIII and GI (p = 0.012).In patients with preserved LVEF and who completed their DSE, normal CFVRobtained before achieving target HR was associated with betterprognosis. Thus, this study aimed at assessing theprognostic value of CFVR obtained in the LAD of patients with preserved LVEF(>50%) who completed DSE after reaching submaximal target HR.Normal CFVR in the LAD can be obtained before (early) or after (late) submaximaltarget heart rate (HR) is reached.23 which has assessed CFVR during DSE.This is a prospective observational study performed during two years in a populationselected from the previous study by Abreu et al.,The decision to refer patients with known or probable CAD for assessment with DSE wasexclusively up to their attending physicians. After collecting the clinical history,risk factors for CAD were assessed and transthoracic echocardiography was performed.When not contraindicated, patients underwent DSE. The exclusion criteria were asfollows: uncontrolled arterial hypertension; unstable angina; congestive heartfailure; recent myocardial infarction (within one month from DSE); important heartvalvular disease; prostate disease or glaucoma with contraindication for atropineuse; and non-sinus rhythm.The present study included patients with preserved LVEF (>50%) on transthoracicechocardiography and who completed DSE after attaining submaximal target HR. NormalCFVR (≥ 2) was classified into early or late, based on being obtained beforeor only after reaching submaximal HR, respectively. In all cases with abnormal CFVRvalues, CFVR recording was obtained at the end of DSE. The DSE protocols and CFVRrecordings are described below.µg/kg/min and 3-minute intervals. The images wereobtained at rest, low-dose, peak and recovery phases, and compared on aquadruple screen. Atropine could be added after the second stage . The DSE wascompleted after submaximal target HR [(220 − age) x 85%] was attained and/ormyocardial ischemia was found.At our service, the DSE protocol instructs patients to suspend beta-blockers 72hours before the exam, and to resume their use after the procedure. The otherdrugs should be maintained. All patients were informed about the risks andobjectives of the exam, which was only initiated after the patient's verbalconsent. For the DSE, the Vivid 7 echocardiography device  withsecond harmonic image and the M4S multifrequency transducer with frequencyranging from 2 to 4 MHz were used. The left ventricle was visualized in theapical (4- and 2-chamber) and parasternal (long and short axes) views at restand during dobutamine use at the doses of 10 (low dose), 20, 30 up to 4023Ischemia was considered as the report of typical angina, new contractileabnormality or worsening of a preexisting one (except from akinesia todyskinesia). The exam would be interrupted in the presence of intolerance tomedication, hypertensive peak (blood pressure > 230/120 mm Hg), or cardiacarrhythmia. The left ventricle was divided into 16 segments, and a numericalscore was given to each segment depending on contractility as follows: normal =1; hypokinetic = 2; akinetic = 3; or dyskinetic = 4. The segmental contractionscore index was calculated by dividing the points obtained by 16.Pulsed color Doppler of the LAD was recorded in the left lateral decubitus, thesame position used for the DSE. The LAD was visualized in its mid-distal regionwith a pre-established specific preset, based on acquisition from the lowparasternal long-axis, 2-chamber or modified 3-chamber view, concomitant withlittle adjustments of angulation or rotation of the transducer. Using a smallbox of color Doppler with Nyquist limit of approximately 20 cm/s, LAD appearedas a tubular image, in which the greatest possible stretching and extension weredetermined, as well as the smallest angulation, with the Doppler cursor, whosesample volume was 2 mm. By use of pulsed Doppler, the flow assessed wascharacterized by biphasic spectrum with diastolic predominance, and anterogradecurves above baseline were recorded. Initially, the Doppler velocity scale waslimited to 80 cm/s and could be widened during DSE, allowing the capture ofsubsequent velocity increases of the Doppler curves.24By using Doppler assessment of LAD synchronized with electrocardiography, peakdiastolic velocities (PDV) were recorded, selecting three spectral curves atrest and during stress, not necessarily continuous, but with good quality andhigher velocities. The CFVR was obtained by dividing the PDV (mean of threepeaks) occurring during DSE by the baseline PDV (mean of three peaks) recordedat rest. By use of the same transducer, visualization of the two-dimensionalleft ventricular image and of Doppler of the LAD was alternated. Thus, thequadruple screen of the DSE was filled in the different stages, concomitantlywith PDV recordings until the end of the exam. Right after completing the exam,the DSE result was defined, and CFVR, calculated.During the study, the patients' management was determined exclusively by theirattending physicians. Independently of their groups, the patients were followedup to assess the occurrence of events, which were established as follows:cardiovascular death; acute coronary insufficiency; coronary intervention; coronary angiography (without further interventionduring follow-up); and hospitalization . Cardiovascular death was considered as death secondaryto any of the events cited or any other condition with acute cardiac impairment.Because of the different intensities and possible gradation of events, in theabsence of death, the follow-up of all patients was maintained.Information on all patients' clinical outcome was obtained through contact withthe patients, their guardians or attending physicians, and through medicalrecord or death certificate review.Continuous variables were expressed as mean ± standard deviation, whilecategorical variables were expressed as absolute number and percentage.Data descriptive analysis per group was performed by use of contingency tablesand descriptive measures. The homogeneity of the groups in regard to thecategorized variables was tested by use of Fisher exact test. The normality ofthe distribution of quantitative variables per group was assessed by use ofShapiro-Wilk test. The homogeneity of the groups regarding variances wasassessed with Levene test. The homogeneity of the groups regarding quantitativevariables was analyzed with ANOVA  for variables withnormal distribution, or with nonparametric Kruskal-Wallis test of independentsamples for variables with non-normal distribution. The variables whose groupsdiffered significantly underwent sub-hypothesis tests by use of the minimumsignificant difference test. Overall survival for the event in the groups wasanalyzed by use of Kaplan-Meier regression. The groups were adjusted by use ofCox regression and Wald statistics, and underwent pairwise comparison. Theanalyses were performed with the SPSS software 20.0 . For all analyses, p < 0.05 was adopted as statisticallysignificant.Of the 100 patients with LAD flow obtained at rest, 92 could have their LAD flowobtained during stress. However, 5 patients could not complete their DSE.Therefore, this study population consisted of an initial sample of 87patients.The assessment lasted 28 ± 4 months, and follow-up was performed in 83patients of the 87 (95.4%), because 1 patient with early CFVR and 3 withabnormal CFVR were lost to follow-up. Of the 59 patients with normal CFVR, 31had early CFVR (Group I) and 28 had late CFVR (Group II). Group III consisted of24 patients with abnormal CFVR. The clinical data of the 83 patients studiedwere as follows: mean age, 63 ± 11 years; men, 48 (57.8%); hypertensive,58 (70%); dyslipidemic, 53 (64%); diabetic, 12 (14.5%); and known CAD, 24 (29%)patients. Of the echocardiographic variables assessed only at baseline, LVEF was preserved,while left ventricular mass index evidenced ventricular hypertrophy and did notdiffer between the groups. Regarding the echocardiographic and hemodynamicvariables recorded at rest and during stress, the segmental contraction scoreindex did not differ between the groups, and the frequency of DSE compatiblewith myocardial ischemia was low. Heart rate and double product did not differ;however, the groups differed in the number of patients attaining maximal HRpredicted for age .On Doppler assessment of LAD, the groups differed regarding the HR during PDVrecording at rest, as well as the PDV values at rest. However, during stress,PDV did not differ, resulting in different CFVR values in the groups . When coµg.kg-1.min-1, when HR was farbelow the submaximal HR calculated for the case  were positive for myocardial ischemia, 1 in GroupI, 2 in Group II and 3 in Group III, and events were observed in 4 of those 6patients, with 1 coronary angiography without further intervention and 1 stentimplantation in Group II and Group III. Considering all 83 patients studied, themean time for occurrence of events was 17 ± 8 months. During thefollow-up of 28 ± 4 months, events were observed in 18 (21.6%) patientsas follows: 4 deaths (4.8%); 6 coronary interventions (7.2%); and 8 coronaryangiographies without further intervention (9.6%). Considering all events, 6%(2/31) occurred in Group I, 18% (5/28) in Group II, and 46% (11/24) in Group III.Of the 8 coronary angiographies without further intervention, only 3 wereperformed within 1 year of follow-up (1 in Group II and 2 in Group III), whileall interventions (stent implantation) were performed after 1 year of follow-up.Of the 4 deaths, 1 occurred in Group I (26.5 months after DSE) and wasattributed to complications after myocardial revascularization surgery. Thedeath in Group II (3 months after DSE) occurred during heart surgery to treatexacerbated mitral insufficiency  and CAD. Theother 2 deaths were observed in Group III: 1 simultaneous with pulmonaryembolism; and 1 occurred 20 months after DSE during heart surgery for heartvalve replacement in a patient with calcified coronary arteries. During thefollow-up, we obtained no information allowing us to infer the diagnosis of theacute coronary insufficiency or of the hospitalization due to a cause other thanthose already cited .Regarding the Kaplan-Meier regression analysis of event-free survival, Groups Iand II did not differ, and had a better outcome than Group III. However, afteradjusting for age and LVEF, Group II did not differ from Group III, and the bestevent-free survival was maintained only in Group I when compared to Group III.26A negative pharmacologic stress echocardiography for ischemia associates with goodprognosis and less need for myocardial revascularization. However, in both micro-and macrocirculation contexts, CFVR obtained in the LAD adds value to theinformation provided by stress echocardiography. Patients with exams showing normalmyocardial contractility and normal CFVR in the LAD have mortality lower than 1% peryear, while those with impaired contractility and abnormal CFVR have mortalitygreater than 10% per year. Even for octogenarians, RFVC is a strong and independentpredictor of death and of myocardial infarction, mainly when contractility ispreserved. Those results support the measurement of CFVR during pharmacologic stressechocardiography, favoring its incorporation into routine practice.27In several studies assessing myocardial ischemia or risk stratification by use oftransthoracic echocardiography, CFVR and contractile abnormality are evaluated byuse of adenosine or dipyridamole, or patients are submitted to an additional stresswith dobutamine to assess the induction of contractile abnormality. However, thevasodilator effect of dobutamine in the presence of preserved LVEF is comparable tothat of adenosine, which is similar to that of dipyridamole. Because dobutamine isone of the most used drugs in stress echocardiography, it is worth noting thepossibility of, in the same exam, having a consistent positive inotropic effect onthe cardiac muscle and a proper coronary vasodilating effect to calculateCFVR.22 However, the prognostic value of early CFVR hasnot been established in the literature, and this has motivated the presentstudy.The CFVR obtained during DSE can anticipate the probable result of the exam regardingcardiac muscle contractility, so that a better reserve associates with bettercontractile response, regardless of whether CFVR is obtained early orlate.28 However, to which extentpathological conditions, such as hypertension, diabetes and dyslipidemia, affecteach individual cannot be established. Thus, Group III had higher baseline PDV,which could express predominance of an abnormal microvascular component over apossible epicardial coronary artery stenosis. However, regardless of which component(micro- or macrovascular) is more important, both can be related to worseprognosis.27The groups assessed in this study did not differ regarding age, sex, presence ofhypertension, dyslipidemia, diabetes and known CAD, use of medications, and not evenleft ventricular mass index and baseline double product and HR, factors that couldinfluence the measurement of baseline PDV, and, consequently, CFVR.29The recording of a slightly altered segmental contraction score index at rest andduring stress might have resulted from the preserved LVEF and lack of ischemicresponse in a greater number of exams. During the study, 4 of the 6 patients withpositive DSE for ischemia had events, which might have been expected by theirattending physicians or might partially represent a bias. However, although 77 ofthe 83 patients (92.8%) could be considered of low risk because of their negativeDSE, determining the expectation of good prognosis, the CFVR measure provided betterrisk stratification. Regarding the patients with negative DSE for ischemia and latersubmitted to intervention, progression of preexisting CAD might have occurred orthere might have been a false-negative DSE for ischemia. An explanation for thatcould be the fact that maximal HR was not achieved.The hemodynamic studies were requested by the attending cardiologists, as were thefurther interventions, which were indicated based on the importance of the coronaryartery stenosis. Most hemodynamic studies (11/14 - 79%) were performed after 1 yearof follow-up. However, it is worth noting that, considering only patients withnegative DSE for ischemia, in 80% (8/10) of those with hemodynamic study, that studywas performed more than 1 year after the DSE, possibly expressing rather a clinicaldecision than a bias of the CFVR result previously informed. Those observations cansuggest that the disclosure and recognition of the importance of the CFVR obtainedon stress echocardiography is still limited, which could determine decisions ratherrelated to the presence or absence of ischemia. Based on the research protocol, wedo not interfere with the management of the attending cardiologists, but it is worthnoting that only 1 hemodynamic study was requested for Group I patients.The 4 deaths in this study occurred among patients with negative DSE for myocardialischemia. In Group I, that event occurred after myocardial revascularization, whichwas performed 2 years after the exam. In Group II, the death occurred 3 months afterthe DSE, resulting from complications during surgery to repair acute mitralinsufficiency in a patient with mitral valve prolapse. However, the deathcertificate available did not provide further information on the relevance of theCAD reported. In Group III, 1 death was attributed to pulmonary embolism, and theother death occurred in a patient who met no criteria for severe heart valve diseaseon DSE. The patient died 20 months after the exam, during heart surgery for heartvalve replacement, when calcified coronary arteries were identified.On the Kaplan-Meier regression analysis of event-free survival, the groups ofpatients with normal CFVR did not differ between themselves, and had better outcomethan those with abnormal CFVR. However, after adjusting for age and LVEF, Group IIdid not differ from Group III, and the better event-free survival was maintainedonly for Group I.The literature shows that the prognosis of patients with normal CFVR is better thanthat of patients with abnormal CFVR. However, in our study, the patients withpreserved LVEF only had better prognosis in the presence of normal early CFVR.µg.kg-1.min-1). 21 who have reported that patients with normal CFVR recordedin the intermediate stage of DSE (20µg.kg-1.min-1) belonged to thegroup that had no myocardial contractile abnormality next to the coronary arteryassessed. In addition, Ahmari et al.22 have reported that patients who developed no ischemia had abetter CFVR with the intermediate dose of dobutamine.20 have reported that, during DSE, 96% of the patients achievednormal CFVR before reaching submaximal target HR, and all of them had a negativeexam for ischemia. In our study, the findings of Group I could result from a smallerimpairment of the micro- and macrocirculation, which favors the attainment of normalearly CFVR during DSE. However, further studies are necessary to confirm thishypothesis.In our study, all patients with normal early CFVR maintained that normal conditionduring all stages of DSE, and none of them showed contractile impairment of theanterior wall. This suggests that, from the time normal early CFVR is achieved,continuing its recording is no longer necessary. In a study including only patientsat low risk for CAD, Forte et al.29Attaining normal early CFVR in the LAD identifies patients with better prognosis.In addition, in that condition, the occurrence of contractile abnormality in theanterior wall during DSE is unlikely. In the exclusive context of contractileabnormality, normal early CFVR is particularly useful when the visualization ofthe anterior wall is hindered during a stage with higher HR, or even whenmaximal HR is not achieved, because the accuracy of DSE is lower in thatcircumstance.Despite the prospective nature of this study, some limitations apply. A largersample and a longer follow-up could add more information. However, this studysample size and follow-up duration were similar to those of some studies herecited. To assess prognostic value, the CFVR was obtained through PDV recordedonly in the LAD, but that condition has been validated and used in severalstudies cited in the present study. The most important limitation of this studyis that we neither had complete access to the therapy used by the attendingcardiologists, nor knew the reasons for choosing each patient's management,mainly regarding the coronary angiographies without further intervention.In patients with preserved LVEF and who completed the DSE, the normal CFVR obtainedbefore achieving submaximal target HR associated with better prognosis. This studysuggests that, after attaining normal early CFVR, continuing its recording is nolonger necessary.