We describe a model of a hexameric p7 complex, which forms a transiently-open channel capable of conducting ions in simulation. We investigate the ability of the hexameric model to flexibly rearrange to adapt to the local lipid environment, and demonstrate how this model can be reconciled with low-resolution electron microscopy data. In the light of these results, a view of p7 oligomerization is proposed, wherein hexameric and heptameric complexes may coexist, forming minimalist, yet robust functional ion channels. In the absence of a high-resolution p7 structure, the models presented in this paper can prove valuable as a substitute structure in future studies of p7 function, or in the search for p7-inhibiting drugs.Hepatitis C virus (HCV) p7 is a membrane-associated oligomeric protein harboring ion channel activity. It is essential for effective assembly and release of infectious HCV particles and an attractive target for antiviral intervention. Yet, the self-assembly and molecular mechanism of p7 ion channelling are currently only partially understood. Using molecular dynamics simulations (aggregate time 1.2  Hepatitis C remains a serious global health problem affecting more than 2% of the world's population, and current therapies are effective in only a subset of patients, necessitating an ongoing search for new treatments. The p7 viroporin is considered to be an attractive possible drug target, but rational drug design is hampered by the absence of a high-resolution p7 structure. In this paper, we explore possible structures of oligomeric p7 channels, and discuss the strengths and shortcomings of these models with respect to experimentally determined properties, such as pore-lining residues, ion conductance, and compatibility with low-resolution electron microscopy images. Our results present an image of p7 as a rudimentary, minimalistic ion channel, capable of existing in multiple oligomeric states but exhibiting a bias towards hexamers and heptamers. We believe that the work presented here will be valuable for future research by providing plausible 3-dimensional atomic-resolution models for the visualization of the p7 viroporin and serve as a basis for future computational studies. Hepatitis C virus (HCV) infection is a global health problem affecting approximately 2% of the world's population Flaviviridae, which is a class of small enveloped RNA viruses. The HCV genome consists of a 9.6-kb positive-stranded RNA molecule encoding a single polyprotein precursor, later processed by both host and viral proteases into ten separate proteins. These are: the Core protein, which composes the nucleocapsid; the viral envelope E1 and E2 glycoproteins; the p7 viroporin; NS2, required for virus assembly; and the replication machinery consisting of NS3, NS4A, NS4B, NS5A, and NS5B  segments connected by a conserved, cytosolic, positively charged loop region, with both the N– and C–termini facing the lumen of the endoplasmic reticulum (ER) p7 oligomers have been found in both heptameric While the basic structural features of p7 are becoming gradually better understood, the conditions that lead to the assembly of a functional channel and the mechanism of ion channelling in terms of gating and selectivity remain in large measure unknown. In the work presented here, we used the monomeric p7 structure from Montserret et al. The coexistence of multiple oligomeric forms, including those with even and odd numbers of subunits, would not be unique to p7. For example, the Vpu viroporin of HIV-1 exhibits such polymorphic behavior Inter-subunit interactions were evaluated for each of the oligomeric models. The Hexamer and Heptamer B models appear to have the best inter-subunit contact, as illustrated from the solvent-accessible surface area (SASA) per subunit, which is seen to be minimized in the Hexamer B and Heptamer B models . The penThe Hexamer B, Heptamer B, pentamer and tetramer models  were thought to be promising in terms of the residues that point towards the center of the pore, in particular, H17 and F25, which had already been proposed to face the pore, but also the hydrophilic S21  were constructed by applying the appropriate symmetry operations to the model of the p7 monomer determined by NMR and MD, as described by Montserret et al. Insofar as the hexamer and the heptamer are concerned, two types of oligomeric states were constructed — one in which the monomers extend radially away from the center and have minimal contact with one another Click here for additional data file.Figure S2Electrostatic potential maps of (A) Heptamer B (B) Hexamer B before opening of pore (C) Hexamer B after opening of pore. The potential map of Hexamer B before the opening of the central pore shows a barrier between the interior of the pore and the solvent; the maps of Heptamer B and of Hexamer B after the opening of the pore show that the interior of the pore is accessible to solvent.(TIF)Click here for additional data file.Figure S3Radial profiles of each of the p7 models, calculated using HOLE. The green and yellow lines mark the positions of F25 and I32, respectively. In the Hexamer C plot, L20 and Y31 are also marked in purple and blue, respectively. In the Hexamer B and Hexamer C plots, the solid black profile represents the model before the opening of the pore, and the dashed gray profile represents the model afterwards.(TIF)Click here for additional data file.Figure S4A variety of models obtained by driving the upright Hexamer B model into the 16 Å EM map, as determined by differences in starting conditions  and force constants (f and g) used in the MDFF algorithm. It was clear that the model fit best into the map at (TIF)Click here for additional data file.Figure S51 to the plane of the first aromatic ring and the vector u12 connecting the centroid of the two interacting aromatic rings, and by the normal n2 to the plane of the second aromatic ring and vector u12. Combinations of Angular distribution for the (TIF)Click here for additional data file.Video S1Video showing the upright Hexamer B model being driven into the EM density using the MDFF method.(MPG)Click here for additional data file.Video S2Video showing the simulation trajectory for the Hexamer B model; the pore opens during the simulation when the mutual interactions of the F25 side chains and I32 side chains are broken.(MPG)Click here for additional data file.Video S3Video showing the trajectory for the EM-fitted Hexamer B model in a POPC bilayer; over the course of the trajectory, the helices begin to straighten as a result of hydrophobic mismatch.(MPG)Click here for additional data file.Video S4Video showing the trajectory for an additional conductance simulation done with a K33Q-R35Q mutated Hexamer B model. The applied voltage was 1500 mV, and the system was simulated for 84 ns. Without the positively charged K33 and R35 blocking the entrance to the channel, potassium ions were able to enter and pass through the channel, and a cation selectivity, as seen in experiments, was observed. However, only a few ions passed through the channel over the course of the simulation, leading to a very small measured conductance value. This suggests that either our simulations are too short to reproduce the order of magnitude of the conductances measured in experiments, or that the ion parametrization is suboptimal.(MPG)Click here for additional data file.


The present study has been focused on two types of the disease, namely, LQTS1 and LQTS2. The examination of two appropriate genes expression  at the transcription level by QRT-PCR in a group of LQTS patients and a healthy control group showed different transcriptional activities of KCNH2 gene in LQTS2 patients compared to the control individuals. KCNQ1 gene expression study did not reveal such differences between both groups. The results indicate that QRT-PCR may serve as a complimentary method to the identification of molecular alterations in genetic determinants of LQTS2 only, but it cannot be used as a sole diagnostic criterion.Congenital long-QT syndrome is a genetic disorder associated with abnormalities in the function and/or structure of cardiac ion channels. Up to the present, 13 types of the disease have been described (LQTS1-13) which result from the fact that 13 genes of which mutations can have an influence on the occurrence of the disease have been identified. Characteristic symptoms of the disease include the changes in the ECG , “ Prolonged episodes of TdP may turn into a ventricular fibrillation (VF) [Long-QTS (long-QT syndrome) is a hereditary disorder associated with myocyte ion channel abnormalities. The disease is characterized by prolonged QT interval and unique morphology of the T wave on ECG. The syndrome is also manifested by the occurrence of ion (VF) , 2. The ion (VF) , 3–5.KCNQ1, a gene encoding the IKs subunit of the slow potassium channel, KCNH2 (HERG), that encodes the IKr subunit of the rapid potassium channel and SCN5A encoding subunit of the sodium channel [Until recently, the mutations in the thirteen genes have been identified to be genetic causes of  long-QT syndrome –10. Most channel   is a common test tube method used for amplifying a selected DNA sequence that is used in genetic testing. It is a modification of the classical polymerase chain reaction (PCR). The method includes reverse transcriptase, an enzyme that uses RNA as a template to rewrite it into cDNA (a strand of DNA complementary to mRNA) –15. ThisThe overarching aim of the study was to demonstrate whether genetic testing performed by QRT-PCR technique can be used as an alternative approach in the diagnosis of the long-QT syndrome and whether QRT-PCR is a suitable diagnostic method for determining the number of mRNA transcripts of genes responsible for types 1 and 2 of long-QT syndrome manifestation.KCNQ1 (LQTS1) and KCNH2 (LQTS2) was supposed to confirm the presence of the long-QT syndrome in the group of patients from the families in whom the syndrome has been previously diagnosed on the basis of supraventricular arrhythmia by ECG.Determination of the number of mRNA copies of KCNQ1 mRNA copies (expression) in patients diagnosed with type 1 LQT syndrome is different in comparison to the healthy individuals.The number of KCNH2 (HERG) mRNA copies (expression) in patients diagnosed with type 2 LQT syndrome is different compared to the healthy individuals.The number of Two research hypotheses were formulated and tested in this study.The studies included six families whose 34 members have been examined for the long-QT syndrome at the Department of Pediatric Cardiology, Medical University of Silesia in Katowice-Ligota. Types and subtypes of the disease were diagnosed with the use of clinical tests, including ECG.The long-QT syndrome was recognized in 21 individuals, 18 of whom were diagnosed with type 1 LQTS  and the other 3 with type 2 LQTS (LQTS2b). Thirteen members of the families showed no clinical symptoms of the disease and they were healthy .The consent to use blood samples taken from patients was obtained from the Bioethics Commission of the Medical University of Silesia in Katowice. The experimental group was set up based on specific inclusion and exclusion criteria. The inclusion criteria involved the first-degree relatives with known long-QT syndrome (based on diagnostic criteria by Schwarz and Moss >1p).The exclusion criteria for healthy individuals included enrollment of all patients according to the scale of Schwarz and Moss <1p and outside the sinus rhythm, patients with AV block or right bundle branch block with sick sinus syndrome, and structural heart disease and inflammation of the heart muscle, patients with electrolyte disturbances , patients with endocrine disorders and neurological diseases, and patients during drug therapies that may affect the repolarization period.There has been phenotypic classification of patients with long-QT syndrome within the two most common types of long-QT syndrome, LQTS1 and LQTS2, that is, having intelligence and are different compared to control durations of individual parameters repolarization QT QTo, QTP, and TpTe and morphological evaluation of the T wave in the ECG standard and stress. Division of LQTS subtypes was made based on the morphology of repolarization (QT period) and the morphology of the T wave as Ia: “infant type” of T-wave, Ib: T wave with a characteristic wide base, Ic: proper morphology of the T wave, Id: the late beginning of the T wave, IIa:two-humped T wave, IIb: T wave slightly two-humped in the peak region, and IIc: T wave slightly two-humped in the descending portion.As a material for the study, RNA isolated from peripheral blood mononuclear cells was used . Qualitative evaluation of the extracts was made via agarose gel electrophoresis (1%) with the addition of ethidium bromide. Quantitative assessment of  RNA extracts was performed with the use of the Gene Quant Pro spectrophotometer produced by Amersham Biosiences.KCNQ1 and KCNH2 mRNA molecules was determined based on QRT-PCR reaction kinetics using ABI  PRISM  7000 sequence detection system (Applied Biosystems) and a kit containing fluorescent dye .Transcriptional activity of the examined genes was determined using a commercially available kit . The number of μL 2x QuantiTect Probe RT-PCR Master Mix 2SO4, 8 mM MgCl2, pH = 8.7, dNTP mix, ROX reference dye), 0.5 μL QuantiTect RT Mix , and 1 μL of starters and T Gene Expression Assay probes (Applied Biosystems), RNA template, and apyrogenic water. Together with the examined genes, commercially available DNA standard  was amplified as internal control in each single QRT-PCR for all samples.QRT-PCR was performed in one step using a reaction mix containing 25 Reverse transcription reaction was performed at 50°C for 30 min. Following initial activation of HotStar Taq DNA Polymerase at 95°C for 15 min, a two-stage reaction was carried out with denaturation at 94°C for 15 sec and starter ligation at 60°C for 60 sec. Final elongation of amplificated products was achieved at 72°C for 10 min.μg of total RNA. Statistical significance of KCNQ1 and KCNH2 expression values obtained for experimental group was measured using Mann-Whitney U test. The results were judged significant if P < 0.05. The expression level of the examined genes was inferred on the basis of number of their mRNA copies per 1 Examined families are shown in  KCNQ1 (LQTS1), KCNH2 (HERG-LQTS2), and β-actin was analyzed for patients diagnosed with LQTS and healthy individuals , which are called “genetic noise”, can be obtained using genetic tests. For LQTS patients the type of  “genetic noise” is detected on the level of 4% by genetic testing , 17.Recent literature reports on the level of knowledge and application of genetic tests for inherited heart diseases diagnosis have been published in a novel publication of HRS and EHRA experts , 18. HowMutations in 13 different genes are responsible for the congenital, genetically determined LQT syndrome, and on this basis, 13 types of the disease (LQTS1-13) were described. Mutations in those genes encoding ion channel proteins  located in the myocardium, result in their structural abnormalities and/or loss of function and incorrect cardiac repolarization , 20–23.The analysis was intended to verify whether the use of genetic test—QRT-PCR—carries a diagnostic weight for detecting two major types of disease LQTS 1 and 2.KCNQ1 gene is expressed in pancreas, kidneys, placenta, and lungs. The highest mRNA transcript levels of KCNQ1 gene were observed in heart muscle cells; no expression was found in brain, skeletal muscles, or liver. The highest mRNA transcript levels of KCNH2 gene were observed in heart muscle and brain [In humans nd brain . In the KCNH2 mRNA copies is higher in patients with LQTS2 than in healthy individuals (Mann-Whitney U test P = 0.014).In this study, the members of six families clinically diagnosed with LQT1 or LQT2 syndrome were examined. The results of analysis confirmed one of the hypotheses indicating the presence of a statistical trend that the number of HERG is known to have implications in the construction of the ion channel. The protein product of HERG forms α subunit of rapid potassium channel (IKr) which is responsible for the final phase of repolarization in cardiomyocytes and for the prevention of premature myocardial arousal. As a result of increased HERG copy number, an intensified potassium channel protein translation in patients occurs, which results in an improper construction of IKr channel (too much protein creating α subunit). In view of the presence on the N-terminal fragment of the protein, which is encoded by this gene, a conservative PAS region (Per-Arnt-Sim), its overexpression may have a negative effect. Since the role of the PAS region is to regulate channel inactivation process, the altered amount of a protein can have a negative influence on channel's function [Overexpression of function , 25.KCNQ1 gene expressions in people diagnosed with LQTS1 compared to healthy individuals was not statistically confirmed, probably due to the study limitations.The hypothesis concerning different KCNQ1 and KCNH2 gene with the use of QRT-PCR method was one of the first which used human whole blood samples. The study showed age- and sex-dependent differences in the level of expression of both genes. Healthy women were characterized by a much lower number of KCNQ1 and KCNH2 mRNA copies in comparison to healthy male subjects. In addition, healthy adults showed a much higher number of KCNQ1 mRNA copies than healthy children, while healthy children had significantly lower KCNH2 mRNA copy number in comparison to healthy adults. The level of KCNQ1 and KCNH2 expression was also compared in a group of adult patients under and over 55 years of age. Among adult patients under 55 years of age a very high number of KCNQ1 mRNA copies and underestimated number of KCNH2 mRNA copies were demonstrated compared to patients above 55 years of age [Our former study on age- and sex-dependent level of expression of s of age .KCNQ1 and KCNH2 expression in the group of female patients relative to the group of male patients was analyzed. Comparative analysis of the average mRNA copy number of both genes showed much higher number of KCNQ1 and KCNH2 mRNA transcripts in women than in men with the disease.In the present study, the level of A lot of factors are believed to influence the tendency to cardiac arrhythmias occurrence and QT prolongation on the ECG in women. One of them is the very low level of androgens in relation to men. It is known that androgens have a protective effect on the heart and help to shorten the QT interval. Another causative factor may be genetically determined difference in the density of potassium channels located in the cell membrane of cardiomyocytes. Estrogen effects on the kinetics and the function of potassium and calcium channels and regulation of ion channels expression as well as the dominance of the parasympathetic system also are not without significance. The differences in the length of the QT interval are also caused by lower myocardial weight in women compared to men, different chest configuration, and the different ionic structure of the cell membrane in cardiomyocytes. Age and gender have a significant impact on the clinical course of the disease. It has been shown that the risk of dangerous cardiac events is increased in boys before adolescence, while it is increased in women in adolescence and later , 26, 27.KCNQ1 and KCNH2 expression in patients diagnosed with LQTS2 or LQTS1 and in healthy subjects with the use of QRT-PCR method showed that the length of the QT interval in LQTS1 patients was independent on the gene expression level. However, a relationship between the amount of KCNQ1 mRNA copy number and the length of the QT interval in LQTS2 patients was found. The correlation showed that the lower KCNQ1 mRNA copy number was, the longer QT interval in patients with type 2 LQTS was observed. Surprisingly, in the case of KCNH2 gene of which mutation affects type 2 of LQTS, the expression level of the gene showed no effect on QT interval in patients with LQT2. This result could possibly be caused by an insufficient number of both healthy and sick individuals in the study group [KCNQ1 expression level in LQTS1 patients may be an additional distinctive criterion in the diagnosis of LQTS1. Moreover, it was concluded that it is not possible to confirm or exclude the presence of LQTS2 on a basis of KCNH2 expression. Furthermore β-actin has been shown to be a proper endogenous control in the analysis of KCNQ1 and KCNH2 expression level in patients with LQTS [Another study on dy group . Based oith LQTS .KCNH2 expression (expressed in mRNA copy number) compared to healthy individuals and that on a basis of KCNQ1 expression profile analysis it is not possible to confirm or exclude the presence LQTS1. Certainly, this issue requires further study. The present study also confirmed that β-actin control gene was properly chosen, as there was no statistically significant difference P = 0.620, P = 0.624 (Mann-Whitney U test) in its expression between both groups.The above-described results of our study  do not cKCNQ1 and KCNH2 mRNA copy number and the type and subtype of the disease have been analyzed for the first time. The analysis showed the relationship between the amount of mRNA copies of of KCNQ1 gene and the clinical type of the disease as follows: LQTS1c > LQTS1d > LQTS2b > LQTS1b. The lowest average transcript molecules number was observed in the group of LQTS1b patients, while its highest number was in patients with LQTS1c. For KCNH2 gene the relationship between the amount of mRNA copies and the clinical type of the disease was as follows: LQTS1d > LQTS2b > LQTS1c > LQTS1b. The lowest expression level of this gene was observed in the group of LQTS1b patients, while the highest was in patients with LQTS1d. Data concerning such relationship analysis have not been published until now.In this study the relationship between KCNQ1 expression comparison in 1–6 families revealed the highest average mRNA copy number in the LQTS1 families 1  and 2 . The lowest average mRNA copy number of this gene was found in LQTS1 family 4 . KCNH2 expression analysis in 1–6 families indicated the highest average mRNA copy number in family 2  and the lowest expression of this gene in family 1 . In family 5, the only family, diagnosed with LQTS2 the transcriptional activity of this gene was low .The results of this study also indicate different levels of gene expression among studied families. These results may be explained by the existence of microRNA (miRNA), which in many conditions can have an influence on the occurrence of arrhythmias or an important role in the context of myocardial excitability through the regulation of ion channels, transporters, and cellular protein. A single miRNA can modulate the expression of many different genes with similar functions and affect the phenotype and disease activity. The excess of miRNA may also play a role in a paradoxical increase of target gene expression , 29.Moreover, epigenetic mechanisms of gene regulation may cause many single-gene, as well as poly-gene, genetic diseases. Epigenetics is one of the fastest growing fields in studies of genetic variation which are independent on the primary sequence of  DNA but is a result of specific regulatory mechanisms activity, such as DNA methylation, modification of histone proteins, and the expression of antisense RNA or interference RNA. Phenotype diversity of LQTS may suggest epigenetic modifications of the corresponding genes , 30.In 2007, Miller et al.  decided Until now, the use of mRNA for genetic tests was recommended only in the case of searching the mutations in large genes. mRNA used for genetic testing was derived from the purified lymphocytes (collected from max. 50 mL of blood), skin biopsy, or fibroblasts culture. In addition, there was another limitation to the use of mRNA in the diagnosis of LQTS, namely, the fact that the expression of genes related to the occurrence of this disease is strictly limited to the myocardial cells and that mRNA transcripts could not be successfully extracted from lymphocytes or fibroblasts culture , 32.Using observations from the microarray analysis, Miller et al. noted that the quaternary ammonium surfactants could greatly enhance the yield of rare peripheral blood mRNAs and increase the detection of tissue-restricted gene transcripts. The researchers proved that the cardiac restricted mRNA transcripts can be successfully detected in the peripheral blood without additional procedures required with cell culture, and so forth. Transcripts isolated from the whole blood can be used in the molecular methods .Researches from the Medical University of Silesia, involved in the study of molecular diagnosis of LQTS families, have shown that due to the fact that gene expression is regulated by a number of nonlinear interactions between proteins, RNA and DNA and metabolites derived from different pathways. Due to the facts mentioned above the tested QRT-PCR method is limited in some extent .μg). This method can only suggest possible changes in the number and/or function of the protein; however, the mRNA expression may not be correlated with the protein concentration [KCNQ1 and KCNH2 mRNA copy number on the etiology of LQTS. Some limitation of QRT-PCR technique is also an appropriate level of transcription of the tested gene in the available tissue samples. The optimal material for such analysis could be a myocardium specimen. Unfortunately, myocardial biopsy is not routinely used in the diagnosis of LQTS, and it carries a high risk of complications [First of all, the assessment of mRNA copy number of studied genes using QRT-PCR technology can determine the mRNA expression level  and from healthy individuals (control group) for the analysis of ried out .The level of gene expression is different within studied families and specific subtypes of the disease.Two types of the disease (LQTS1 and LQTS2) cannot be clearly differentiated using Taq-Man QRT-PCR method.KCNQ1 and KCNH2 expression studies.The results can be useful in the next steps of In subsequent work related to this research issue an increase of the study groups size should be considered, as well as the impact of blood storage time, the presence of thrombus, and the degree of total RNA extracts purification on the final result of the experiment.


Sepsis is a major disease entity with important clinical implications. Critical illness survivors present long-term cognitive impairment, including problems with memory and learning. Chemokines are important to the recruitment of leukocytes to infectious tissue, but a few studies described the role of the CC-chemokine receptor 2 (CCL2) in the cognitive process. In this study, we analyze the involvement of CCR2 in the physiopathology of sepsis, especially in development of cognitive dysfunction.-/-) were submitted to a CLP model and we analyzed the survival rate, the severity score of the animals during 144 hours and 15 days after the CLP, and we analyzed the memory of the animals. To analyze the contextual memory, the mice were submitted to the open field method and the water maze procedure. To evaluate the aversive memory, the passive avoidance test was performed.The CCR2-deficient mice (CCR2-/- group submitted to CLP had more severe cognitive impairment in comparison with the WT-CLP group. Interesting, the CCR2-/- Sham group presented cognitive impairment, suggesting that CCR2 is important to the physiological process of cognition. We then submitted CCR2-/- naive mice to water maze and passive avoidance tests. We found that CCR2-/- naive mice have an impairment of aversive and contextual memory. The cognitive impairment was associated with a decrease of BDNF expression in the hippocampus. When we analyze the expression of β-amyloid protein in the brain of CCR2-/- naive mice, we observed the increased in β-amyloid protein expression in the cortex and hippocampus of these animals, accompanied by increased cell proliferation in the dentate girus, and increased caspase-3 and caspase-12 expression in the hippocampus and cortex. We did not observe a difference in the numbers of neurons in the brain from CCR2-/- naïve mice, as well the numbers of microglial cells. But, surprisingly, there was an increase of astrocytes in the hippocampus of CCR2-/- mice.First, we observed that the CLP group had cognitive impairment, but the CCR2CCR2 is involved with the physiology of cognition, with the important role arising in the amyloid accumulation in the brain and induction of the caspase-3 pathway.


Poor adherence to non-pharmacological treatment of hypertension represents a serious challenge for public health policies in several countries. This study was conducted to compare two intervention strategies regarding the adherence of adult women to dietary changes recommended for the treatment of hypertension in a community covered by Primary Health Care Unit.This study is a randomized controlled trial of a sample composed of 28 women with hypertension enrolled in the Primary Health Care Unit located in the urban area of southeastern Brazil. The participants were already undergoing treatment for hypertension but devoid of nutritional care; and were divided into two groups, each composed of 14 individuals, who received interventions that consisted of two different strategies of nutritional guidance: monthly health education workshops alone (Group 1) and combined with family orientation through home visits (Group 2). Adherence to nutritional guidelines was evaluated by dietary, anthropometric, clinical and serum biochemical parameters, measured before and after the interventions. Knowledge on control and risk of hypertension was also investigated. The study lasted five months.2; p = 0.01); waist circumference , systolic blood pressure  and glycemia . In group 1 only waist circumference  changed significantly.Mean age was 55.6 (± 2.8) and 50.7 (± 6.5) in the groups 1 and 2, respectively. The home orientation strategy promoted greater adherence to dietary changes, leading to a statistically significant improvement in the clinical, anthropometric, biochemical and dietary parameters. The group 2 reduced the consumption of risk foods (p = 0.01), oil (p = 0.002) and sugar (p = 0.02), and decreased body mass index (-0.7 kg/mNutritional orientations at the household level were more effective with regard to the adherence of individuals to non-pharmacological treatment of hypertension, regarding the reduction of clinical and behavioral risk parameters. Cardiovascular disease accounts for one third of deaths worldwide. In the Americas, the World Health Organization reports about 140 million people suffer from hypertension (1). Hypertension is one of the most important risk factors of cardiovascular and kidney disease and is a serious public health problem. The estimated prevalence in Brazil is 35% of the population over the age of 40 years. There is a growing prevalence among the young population, representing approximately 17 million affected individuals (2).Changes in lifestyle are fundamental to the prevention and control of hypertension, with diet figuring prominently. Since the 1970s, different countries have introduced programs of health promotion and the prevention of cardiovascular risk factors related mainly to diet, physical activity and quitting smoking (3). In Brazil, there has been an emphasis in recent decades on the importance of public policies directed at nutrition in the prevention of cardiovascular disease. The National Diet and Nutrition Policy (4) and the adoption of the Global Strategy on Healthy Eating, Physical Exercise and Health of the World Health Organization (WHO) (5) are examples of public policies that include recommendations regarding healthy eating as a way to control and prevent cardiovascular disease on the primary care level.In Brazil, the Family Health Program (FHP) is a priority strategy of the Ministry of Health to organize primary care. It has proven to be an adequate model for addressing cardiovascular disease by means of prevention and health promotion aimed at changing the behavior and living habits of individuals without losing sight of the interactions in collective and social spheres .Health education practices in primary health care have traditionally been guided by discourse focused on biological factors and hygiene, addressing disease and verticalized, prescriptive, information . This traditional education model has limits, as access to information alone does not determine a greater commitment on the part of hypertensive patients to their treatment (9). The adherence of such individuals to treatment is a complex, involving a multidimensional process influenced by multiple factors such as related to the individual, health status, therapy, socioeconomic aspects, the health care system and relationships with health care professionals (10).The Family Health Program (FHP) is a health education model that is more coherent with the principles of integrality, respect and the autonomy of individuals. Its objectives are the humanization of health practices through the formation of ties between professionals and users, the democratization of knowledge on the health-illness process, the development of civic inclusion and the encouragement of the organization of the community (8). In this context, the home visit, which is part of the FHP routine, constitutes one form of holistic care, allowing an understanding of the psychological, emotional, social and biological aspects of users of the public health care system (11).The aim of the present study was to compare two nutritional intervention modalities with regard to the adherence of individuals to dietary orientation indicated in the treatment of hypertension and to investigate their knowledge on the disease in a community covered by the FHP. The first intervention was based on group education activities held at the Primary Health Care Unit. The second modality combined the group education activities with home visits that followed a systematic nutritional education program and family follow up.This study was carried out at the Primary Health Care Unit in an urban area of the city, in Porto Firme, Minas Gerais State, Brazil, an area where, two Family Health teams cover a total of 1361 families and 626 individuals with hypertension. The choice of the city was due to the high FHP coverage, which is over 90%. Moreover, the FHP teams hold monthly group activities with individuals with hypertension, but devoid of guidance by nutritionists.The study is a randomized (by lots), non-blinded trial involving 28 participants divided into two groups. Group 1 was made up of women who received orientation regarding the dietary treatment of hypertension through monthly dialogic education workshops, as mentioned above. Group 2 participated in the educative workshops and also received personalized orientation through periodic home visits.The final sample was made up of 28 women (14 in each group) covered by the Primary Health Care Unit who met the eligibility criteria, agreed to participate and attended at least two of the five nutritional education workshops . Of a total of 276 individuals, 238 had a diagnosis of hypertension recorded in their medical profile. Of these, 159 were female, among which 60 were between 45 and 60 years, and 20 fell into some of the exclusion criteria.After screening, there had been selected 40 participants who fulfill the inclusion criteria. The women have been approached in their homes and were informed about the objectives of the study. The women who were absent in more than three visits and the ones who refused to participate were excluded from the sample, totalizing nine women. After the randomization, three women have been excluded from the study for being present in less than 50% of the workshops. Finally, 28 women were selected. They have been allocated, randomly, in one of the two comparison groups.The females were chosen because women, in the context of public health policies, stand out in their abilities of caregivers and also because they are more health aware, either for themselves, as for their relatives. The woman at home has a central role in the care of relatives in relation to hygiene, nutrition, treatment of diseased and self-care. Thus, they are the protagonist in the maintenance and transformation of dietary habits of families .The choice of this particularly age group is justified by the scientific and epidemiological knowledge accumulated, showing consistently higher prevalence of hypertension and its complications in individuals from middle age (14), setting a defining moment for delaying/preventing complications of diseases, increasing the chances of a healthy and active aging (15).Universidade Federal de Viçosa (process number: 030/2009). All participants signed terms of informed consent.The study received approval from the Ethics Committee of the The intervention lasted five months and involved the following strategies and activities as described in Figure Five nutritional education workshops were held in different locations of the city in order to facilitate the access of the population. The workshops addressed topics on hypertension and dietary treatment measures through dialogic, dynamic, interactive lectures using posters, figures, videos and practical demonstrations.Five home visits to each participant in Group 2 were carried out in order to establish systematic, but flexible orientation in accordance with the needs and conditions of each family. The visits lasted from 30 minutes to one hour. Thematic content of the workshops on health education and goals of home visits are outlined in Table The analysis of adherence was carried out through the evaluation of the following clinical and nutritional parameters before and after the intervention: weight, height, body mass index (BMI) and waist circumference, fasting glycemia, triglycerides, total cholesterol and cholesterol fractions, habitual food intake and blood pressure. The data on control and risk of hypertension were collected through a semi-structured questionnaire previously validated.2). Waist circumference was determined using a non-extendable tape and measured in centimeters at the point of the smallest circumference.The anthropometric variables were measured during the nutritional education workshops (T1/T2), as proposed by Jellife (16). BMI was calculated as weight divided by height squared (Kg/mBlood pressure (BP) was determined in accordance with the procedures recommended by the American Heart Association (17) using the routine nursing methods employed at the Primary Health Care Unit. Biochemical variables were analyzed using routine clinical methods and the participants were previously instructed to remain fasting for 12 hours prior to the blood collection. Habitual food consumption was assessed using a food intake frequency questionnaire validated in Brazil (18). Consumption of oil, salt and sugar were estimated from the monthly availability of these products divided by the days of the month and number of diners to home.Excel for Windows 2007 and SPSS for Windows  were used for the statistical analysis. Anthropometric, clinical, biochemical and food intake variables before and after the interventions were analyzed using the non-parametric Wilcoxon test, with the level of significance set at p < 0.05. The choice of this statistical test is appropriate for studies with small sample size and when the variables of interest do not show normal distribution (19). Data on knowledge and grasp of hypertension and per capita/day intake were analyzed using frequency distribution.et al. (20). The scores were obtained for six different intake frequencies for each food: f1 - non-consumed foods; f2 - foods consumed one to three times a month; f3 - foods consumed once a week; f4 - foods consumed two to four times a week; f5 - foods consumed five to six times a week; f6 - foods consumed daily. The highest score was attributed to foods consumed on a daily basis (S6 = 1). Foods were categorized into two groups: Class 1, with foods considered of risk ; and Class 2, with protective or neutral foods, such as rice, beans, whole-grain cereals, fresh fish, olive oil, low-fat milk and byproducts, fruit, vegetables and legumes.Regarding to the data on food intake, individual intake scores were calculated using the method proposed by Fornés In table Mean age was 55.6 (± 2.8) years in Group 1 and 50.7 (± 6.5) years in Group 2. Mean schooling was four years of study [3.9 (± 3.3) in Group 1 and 4.4 (± 3.2) in Group 2]. All participants made use of anti-hypertension therapy. The participants used an average 2.3 different types of anti-hypertension drugs (1.9 ± 0.6 in Group 1 and 2.8 ± 1.1 in Group 2), with a mean intake of 2.8 ± 1.5 and 4.5 ± 2.4 pills/day in Groups 1 and 2, respectively. In both groups, mean BMI characterized obesity (31.3 ± 6.9 in Group 1 and 33.4 ± 5.6 in Group 2). Alcohol and cigarette intake was not common; only one woman in Group 1 used to smoke. A large portion of the participants were classified as active (57.1% in both groups), using the international physical activity questionnaire summary report (21).When asked about the care necessary to control hypertension, 100% of the participants cited the use of anti-hypertension therapy at T0 and T3. Healthy eating was the second most often cited item . The practice of physical activity was cited by 50% of the participants in both groups at T0 and 84.6 and 92.9% in Groups 1 and 2, respectively, at T3. Moderation in the consumption of alcohol and quitting smoking were cited by only one participant at T0 and by 69.2 and 85.7% in Groups 1 and 2, respectively, at T3. Controlling nervousness/anger (leading a more tranquil life) was cited by 32.1% in both groups of the participants at T0 and by 69.2 and 85.7% in Groups 1 and 2, respectively, at T3 .risk foods (n = 14) before and after the intervention for either of the food groups analyzed  , however, there was a significant increase in the intake of protective or neutral foods . Analyzing the foods separately, there was a significant increase in no-fat and low-fat milk intake  and whole-grain bread  and a reduction in the consumption of pig skin/scraps  and sweetened artificial juice  in Group 1. In Group 2, there was a significant increase in the consumption of whole-grain cereals .There were no statistically significant differences in intake scores in Class 1- ods n = 1 before aRegarding the consumption of oil, sugar and salt, there was a statistically significant difference between T0 and T3 in Group 1 only for the per capita/day consumption of oil ; at T0, 100% of Group 1 had values greater than those recommended by the Brazilian food pyramid (16 ml/day), whereas 36% had values below the maximal recommended value at T3 (22). In Group 2, there were significant differences between T0 and T3 in the per capita/day consumption of oil  and sugar , with a mean reduction of 14 mL in per capita/day consumption of oil , and a mean reduction of 27 g of sugar in the per capita/day consumption; however, the mean consumption was 75 g at T3, which is above the maximal recommended value of 56 g/day (23). The Wilcoxon test revealed no significant difference in salt intake in either group; however, there was a significant reduction in per capita/day intake among individuals who consumed more than 15 g of salt/day (n = 4) at T0 , with a mean reduction of 9 ± 1.3 g in daily salt intake by T3.2 , mean reduction in waist circumference of 4.2 cm  , mean reduction in systolic BP of 13 mmHg  and mean reduction in fasting glycemia of 18.9 mg/dl . Regarding fasting glycemia in Group 2, 46% of the women had high initial values (> 99 mg/dl), which were reduced to values within the ideal range at T3 according to recommendations of ADA (24) , with a mean reduction of 2 cm. In Group 2, there were statistically significant differences between evaluations with regard to weight, BMI, waist circumference, systolic BP and glucose, with a mean weight reduction of 1.7 kg  , mean reduction in BMI of 0.7 Kg/mThe educative interventions - health education workshops and home visits - achieved positive results in the knowledge and grasp of individuals with hypertension regarding the disease and risk factors. There was an increase in knowledge on the disease in both intervention groups, especially in Group 2, thereby demonstrating the greater effectiveness of home orientation. These results underscore the importance of follow up and dialogic, participative health education, considering the low level of schooling of the participants and unsatisfactory knowledge of this population regarding their own health status prior to the intervention.The acquisition of knowledge about the disease, and ways to control it, favors adoption of attitudes that can influence health at family and community level (25). Health awareness is preceded by full access to information and education as a way to make individuals and communities capable of greater control over their own wellness, which is an essential goal in public health, especially on the primary health care level .In Group 2, the home orientation strategy - privileged intervention locus - led to beneficial changes in diet, with an increase in the consumption of foods considered either protective or without health risk, especially the consumption of whole-grain cereals. In Group 1, which only underwent the health education workshops, there was also a tendency toward better eating habits, as there was a reduction in the intake of risk foods and an increase in the intake of protective foods, even though there was no statistically significant difference in the consumption of the different food groups before and after the intervention.The home orientation strategy had a more significant effect on the reduction in oil and sugar intake. As the intake estimates of these foods were calculated based on monthly availability in the residence, one may suppose that the home orientation led to an important alteration in intake not only for the women studied, but also for their families, which demonstrates the importance of home visits in the context of the FHP.The reduction in salt intake was not statistically significant in either intervention group. A number of studies in the literature indicate that, despite the understanding of individuals regarding the association between salt intake and high blood pressure, this dietary change is not achieved easily . However, in the group that received home orientation, those individuals who had an initial salt intake greater than 15 g/day achieved a significant reduction in this parameter by the end of five-month period, thereby demonstrating the positive impact on the home intervention to correct large deviations in salt intake.The greater adherence of group 2 was confirmed by changes in clinical and nutritional variables. Group 2 achieved a significant reduction in weight, BMI, waist circumference, systolic BP and glucose, whereas Group 1 only achieved a reduction in waist circumference. However, the reduction in waist circumference in Group 1 indicates significant and important dietary modifications, since the abdominal obesity is related to an increased risk of myocardial infarction, stroke and premature death (29). It should be stressed that the educative workshops Group 1 underwent were held with the participants actively engaged in a participative, dialogic, interactive process.It was not possible to establish a statistical association between dietary changes and alterations in the clinical and biochemical parameters assessed in the different intervention groups. The increase in protective foods - rich in fiber, vitamins and minerals and poor in fat and salt - may at least partially explain the positive results in the clinical, anthropometric and biochemical variables in the group that received home orientation, as demonstrated in studies relating eating patterns and risk factors for cardiovascular disease .The home visit, in the context of health education, is an important tool in the consolidation of new practices encouraging the adoption of healthy lifestyles through health promotion and prevention of diseases and disorders. In this sense, the visit allows the understanding of psycho-affective-social and biological characteristics of individuals and families, recognizing the family activity as a privileged locus for interventions. The diagnosis of the home visit prioritizes the individual's reality and education initiatives. It is a key instrument of intervention in family health and continuity of care, being programmed and used in order to support interventions and to provide comprehensive care for individuals and families. It brings a new meaning in the practices of the professionals of the FHP allowing that the socio-economic and cultural surrounding habits, customs and beliefs are considered in their approach. The challenge is to incorporate the home visit as an individual, family and community activity aimed at solving real life problems. In addition, home visit is an important strategy to motivate the individual, family and community to participate in the planning, organization and control of the therapeutic project.However, the management of home visits in a compulsory manner by health professionals presents serious operational problems. The first concerns the optimization of resources available for health care. The high number of registered families and the high demand for services in the Unit of Primary Health Care makes it impossible to respond to all families through the home visit. Associated with that, are also the work schedules and chores, difficulties related to time and way of locomotion of the team to the households , and fear and ignorance of the population about the purpose of home visit that can disable and/or impair this kind of visit.Despite the individuals in Group I have demonstrated knowledge acquisition on hypertension and their ways of controlling this alone was not sufficient to promote changes and adoption of the dietary modifications recommended for hypertension. It has been demonstrated that knowledge regarding hypertension does not necessarily imply a change in behavior .While reinforcing this in the literature, that points out to the advantages of home visits, especially in regard to the approximation of the health service, represented by the technician, with the reality of family life. It is noteworthy that these results are preliminary and punctual, based on a limited sample, which has major limitations for further generalization of data. This limitation relates mainly to large cities, given the disproportion between the number of cities participating in the program and the enrolled population, generated by the difficulty to expand the Units of Primary Health Care in large cities that face the challenges inherent to the health sector, in addition to the problems posed by urban complexity, such as violence.The positive changes in clinical and dietary parameters, statistically higher in the group receiving guidance at home, indicates better adherence to dietary treatment. The results indicate the potential of this strategy in health education as an important tool to improve adherence to non-pharmacological treatment of patients with hypertension. Due to the complexity of dietary behavior, guidance in the household was the most effective strategy in health education and nutrition. It enabled a deeper knowledge of social and cultural dynamics of feeding and interrelation of families, favoring a more appropriate, in the home, and a consequent assimilation of the guidelines by women and families in the actual environment.Further studies are needed to explore the advantages and limitations of this strategy in contexts of primary health care where the prevalence of hypertension is high.The authors declare that they have no competing interests.AGR - conceived the study, participated in the research literature, methodology, data collection, analysis, discussion of data, drafting the article, and read and approved the final manuscript; RMMC - conceived the study, participated in the process of literature review, methodology, data collection, analysis and discussion of data, drafting the article, directed and coordinated the work, and read and approved the final manuscript.SMR, CMGCD, AQR, FAFC, MMSV- participated in the analysis, discussion of the data, correcting of the article, and read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2458/11/637/prepub


Finally, I collect all these ideas, starting at the origin of the first reaction center proteins and ending with the emergence of the water-oxidizing cluster, to hypothesize that the complex and well-organized process of assembly and photoactivation of Photosystem II recapitulate evolutionary transitions in the path to oxygenic photosynthesis.Due to the great abundance of genomes and protein structures that today span a broad diversity of organisms, now more than ever before, it is possible to reconstruct the molecular evolution of protein complexes at an incredible level of detail. Here, I recount the story of oxygenic photosynthesis or how an ancestral reaction center was transformed into a sophisticated photochemical machine capable of water oxidation. First, I review the evolution of all reaction center proteins in order to highlight that Photosystem II and Photosystem I, today only found in the phylum Cyanobacteria, branched out very early in the history of photosynthesis. Therefore, it is very unlikely that they were acquired via horizontal gene transfer from any of the described phyla of anoxygenic phototrophic bacteria. Second, I present a new evolutionary scenario for the origin of the CP43 and CP47 antenna of Photosystem II. I suggest that the antenna proteins originated from the remodeling of an entire Type I reaction center protein and not from the partial gene duplication of a Type I reaction center gene. Third, I highlight how Photosystem II and Photosystem I reaction center proteins interact with small peripheral subunits in remarkably similar patterns and hypothesize that some of this complexity may be traced back to the most ancestral reaction center. Fourth, I outline the sequence of events that led to the origin of the Mn Photochemical reaction centers are thought to have originated only once in the domain Bacteria. This is because currently there are no described strains in the domain Archaea with photosynthesis based on protein complexes containing chlorophyll or bacteriochlorophyll  and mediate excitation energy transfer from the CP43 and CP47 antenna to the reaction center core  together with PsbJ, which in total make three transmembrane helices, are located perpendicular to the QA-Fe2+-QB axis. At an equivalent position in Photosystem I the PsaL subunit binds the core proteins perpendicular to A1A-FX-A1B: PsaL has three transmembrane helices. At the opposite side of the QA-Fe2+-QB axis in Photosystem II, there are three single-helix subunits PsbL, PsbM, and PsbT making a three-helix bundle. In Photosystem I, PsaJ, and PsaF are found at an equivalent position. While PsaJ has a single transmembrane helix, PsaF has two helices. Although, the second helix of PsaF does not span the entirety of the membrane, because it is bent in the middle  that interacts with PsaL and PsaFJ, see Figure Chlorobaculum tepidum cross-linked with the Cytochrome c551 via a lysine also found in between the 7th and 8th helices  D170 and E189, which are located in the CD loop between the 3rd and 4th helix; and (2) the ligands located in the C-terminal lumenal extension beyond the 5th transmembrane helix, H332, E333, H337, D342, and A344. Then, how did the ligand sphere around the Mn4CaO5 cluster evolve? The first ligand to have appeared was a glutamate at position equivalent to aspartate 170 (D170) of the crystal structures from Thermosynechococcus vulcanus  oxidation. The presence of glutamate near the tyrosine implies that upon oxidation of Mn(II) to Mn(III), the carboxyl side-chain of E170 could have coordinated and partially stabilized the metal. In this scenario, aqueous Mn(II) seems like a plausible electron donor to photosynthesis in an ancestral bacterium prior to the evolution of proper water oxidation. What is more, the initial oxidation and binding of Mn could have pushed the potential of P•+/P even further up, allowing additional oxidation steps or the binding of extra Mn ions. From this evolutionary perspective, it is therefore unnecessary to invoke a catalase or naturally occurring inorganic Mn oxide precipitates as precursors to the Mn4CaO5 cluster. It is also unnecessary to suggest that bicarbonate was a transitional direct electron donor before water. Aqueous Mn(II) might have been the direct electron donor once tyrosine oxidation was possible, followed in time, by water.Ishikita et al.  calculata et al.  for a fuGloeobacter kilaueensis, only has a glutamate at position 189 and lacks all other ligands. This sequence is unique among D1 proteins because it has retained numerous ancestral traits  to Mn(III) occurs for several turnovers without the trapping of any Mn(IV); however, in mutants where D170 was changed to E170, as in the ancestral photosystem, Mn(II) was trapped and oxidized to Mn(IV) and possibly even as a dinuclear Mn2 complex  is oxidized to Mn(III) and bound to the high-affinity Mn binding site, known to be in part provided by D170  then moves to a position similar to that of Mn4 in the crystal structure, where it is coordinated by D170 and E333. This is also consistent with the work by Cohen et al.  is oxidized to Mn(III), which quickly leads to a fully assembled Mn4CaO5 cluster. Unfortunately, the fast events after the binding of the second Mn remain to be characterized; however, the evidence for an intermediary photoactivation step made of two Mn and involving at least D170 and E333 seems to be strong ; then, the D1 and D2 divergence may have started more than a billion years before it. Even such early dates require rates of D1 and D2 evolution almost two orders of magnitude higher than those observed since the last common ancestor to the phylum Cyanobacteria.How long did it take for the ancestral Photosystem II to evolve the MnGloeobacter violaceus and Arabidopsis thaliana share 81.6% sequence identity. Gloeobacter and Arabidopsis are separated by more than two billion years of evolution. In contrast, the D1 and D2 proteins of Gloeobacter share between each other only 33.3% identity over the entire length of the sequences, while the D1 and D2 of Arabidopsis share 25.7% identity. Gloeobacter is the earliest branching genus of Cyanobacteria and it is reasonable to think that it branched out before the Great Oxygenation Event (Criscuolo and Gribaldo, Gloeobacter branched out D1 and D2 were already highly divergent. If we assume a constant rate of evolution, the divergence time for D1 and D2 would be placed well before the formation of the planet, which makes no sense (Figure Chloroflexus aurantiacus share 24.7% identity between each other and those of Blastochloris viridis share 21.9% and are also evolving at a slow rate, albeit just slightly faster than D1 and D2. This is assuming that the phylum Chloroflexi and the phylum Proteobacteria had already appeared, at the very least, around the time of the Great Oxygenation Event. Thus, at that time, they should have carried highly divergent L and M characteristic of their own phylum. If we take into account the large phylogenetic distance among reaction center proteins and their rates of evolution, then the appearance of reaction center proteins around 3.8 billion years ago is likely. Not only that, but if the ancestral Photosystem II before the split of D1 and D2 could have carried out some inefficient water oxidation, then primordial forms of oxygenic phototrophic bacteria 3.2 billion years ago, or before, is perfectly feasible. A detailed analysis on the evolutionary rates of reaction center proteins will be published elsewhere.Consider the following example. The standard D1 protein of bc1/b6f complexes (Nitschke et al., There are two main implications derived from the discussions in here. First, if photosynthesis is an ancient process originating 3.8–3.5 billion years ago, this should place its origin near the root or at the root of the bacterial tree of life. It means that photochemical reaction centers were crucial in the development of bacterial bioenergetics systems and were not just merely appended to it. The second ramification is the massive loss of phototrophy throughout the bacterial tree of life, regardless of how pervasive horizontal gene transfer has been. Unlike the trees for Rubisco (Tabita et al., The author confirms being the sole contributor of this work and approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


P < 0.05). Patient satisfaction using the 3D-printed prototype and the communication score were 9.3 ± 0.6 points. A 3D-printed prototype can faithfully reflect the anatomy of the fracture site; it can effectively help the doctors plan the operation and represent an effective tool for physician-patient communication. To evaluate the effect of 3D printing in treating trimalleolar fractures and its roles in physician-patient communication, thirty patients with trimalleolar fractures were randomly divided into the 3D printing assisted-design operation group (Group A) and the no-3D printing assisted-design group (Group B). In Group A, 3D printing was used by the surgeons to produce a prototype of the actual fracture to guide the surgical treatment. All patients underwent open reduction and internal fixation. A questionnaire was designed for doctors and patients to verify the verisimilitude and effectiveness of the 3D-printed prototype. Meanwhile, the operation time and the intraoperative blood loss were compared between the two groups. The fracture prototypes were accurately printed, and the average overall score of the verisimilitude and effectiveness of the 3D-printed prototypes was relatively high. Both the operation time and the intraoperative blood loss in Group A were less than those in Group B ( Three-dimensional 3D) printing , through layered processing and additive manufacturing, can output computerized data by “printing” the form of a solid object with complex geometry. In recent years, the rapid development of 3D printing technology combined with digital orthopedics technology has allowed its scope of applications to be extended from the industrial field to orthopedic research , 2. The D printinA trimalleolar fracture is a type of complex ankle fracture that mostly consists of high-energy comminuted fractures. If a complete reduction is not achieved, poor alignment of the articular surface and widening or narrowing of the ankle mortise will result in traumatic arthritis and severe dysfunction. If a complete 3D replica of the fracture type can be generated before the operation, most of the details like bone fragments, medial malleolus, lateral malleolus, trochlea of astragalus, and distal tibia can be determined, which could play an important role in producing a precise strategy for the operation and will facilitate communication between doctors and patients.In the present study, we used 3D printing technology to reconstruct the details of trimalleolar fractures in patients and evaluated its effectiveness in the surgical planning for the fracture repair and in the communication between doctors and patients.The present study consisted of 30 patients, including 16 males and 14 females between the age of 31 and 42 years, with an average age of 36.5 years. The patients were classified as follows: 18 cases of traffic accident injuries, four cases of smashing injuries by heavy objects, and eight cases of sprain injuries and 16 cases of left side injury and eight cases of right side injury. Fractures were categorized according to the Lauge-Hansen classification and included 18 patients with supination-external rotation type-IV fractures and six patients with pronation-external rotation type-IV fractures. The duration between injury and the operation was 4 to 12 days, with an average of 5.4 days. The 30 patients were average randomly divided into 3D printing assisted-design group (Group A) and no-3D printing assisted-design group (Group B). Group B operation was planned and executed according to the 3D reconstruction image results from CT scan, without 3D printing fracture prototype. All patients underwent a CT scan.Each patient was scanned by 64-slice spiral CT in the supine position. The scan settings were as follows: a voltage of 120 kV, a current of 180–220 mA, and a slice thickness of 1 mm. The scan ranged from the distal tibia and fibula area to the metatarsals, using the ankle joint space as the center to select an appropriate scan range, and included the proximal tibia and fibula. The CT scan data of the ankle joint fractures were saved as DICOM files, which were input into Mimics l0.01 software to perform region growing for threshold-based 3D image segmentation. The masked original layer was carefully patched, and each contour line was accurately selected to compute a 3D digital model of the ankle joint. The optimal quality for performing model computation was selected, and 3D imaging data of the ankle joint was obtained. The computed imaging data were saved in STL format using Mimics software and were output to a 3D printer . Polylactic acid (PLA) was used as the printing material ; several parameters were used to section the prototype of the ankle joint fracture see , and a 1Questionnaire 1 for doctors was designed according to the literature to evaluate the 3D-printed prototype , 6. The General information about 3D printing and the basic process of printing the fracture prototype were presented to the four joint surgeons and they completed questionnaire 1 to evaluate the usefulness of the prototype of the actual fracture generated by 3D printing for preoperative planning and its verisimilitude to the actual fracture. Two surgeons designed an individualized surgical strategy according to a preoperative 3D-printed prototype of patient's fracture and a surgery simulation. Then, they performed the surgery and compared the surgical approach, the position of the internal fixation plate, with the preoperatively designed strategy. Meanwhile, the operation time and intraoperative blood loss (a tourniquet was used) of Group A and Group B were compared. The operation time was recorded from incision skin to suture, and the amount of intraoperative blood loss included the amount of blood in the gauze and the suction device. After the operation, the surgeons completed questionnaire 1 to evaluate the verisimilitude of the prototype to the actual fracture and its effectiveness for surgical planning.Open questionnaire 2 for patients and nonmedical professionals was designed to evaluate the preoperative conversation. The questionnaire included four questions: (1) How would you rank your overall satisfaction of the conversation? (2) Is the 3D prototype useful for helping you to understand the surgical plan? (3) Is the 3D prototype useful to you to obtain a clear understanding of your condition? (4) Would you like the doctor to use a 3D prototype to communicate with you about your condition? The scores ranged from 1 to 10 points; one point indicated that the model was useless/very poor/very unrealistic, and 10 points suggested that the model was very useful/very good/very realistic.The surgeons used the 3D-printed fracture prototype to inform the patient or the family members about the patient's conditions so that they could understand the fracture characteristics, the fracture segment location, the surgical treatment strategy, and the possible postoperative complications. After the conversation, the patients and their family members completed open questionnaire 2 to evaluate the effectiveness of the conversation and indicate their preference for communicating with the doctor using a 3D-printed fracture prototype.X ± S), and the data comparison between groups was conducted using Student's t-test. By correlation analysis, Pearson correlation test was used to assess the correlations between the operation time and intraoperative blood loss, P < 0.05 was considered as significant.SPSS 17.0 was used for statistical analysis. Data are expressed as mean ± standard deviation  and Group B , which revealed a positive correlation between the two indexes. Furthermore, the operation time and the intraoperative blood loss in Group A were less than those in Group B (P < 0.05). Postoperative X-ray films showed that the reduction and fixation of the ankle joint fracture were satisfactory, and the plate and the screws were in good positions. Images of a typical case are shown in Ankle joint fracture prototypes in Group A were successfully printed, and all 15 fracture prototypes clearly showed the anatomical structure of the ankle joint, which can help the surgeon to determine the specific type of fracture, the displacement direction of the fractured fragment, the location of bone fragments, and the injury degree of the articular surface. Through the analysis and study of the 3D-printed fracture prototypes (1 : 1 ratio), intraoperatively designed surgical plans were successfully achieved. The surgical entry point, position of the internal fixation plate, and direction and length of the implanted screws were all the same as those in the preoperative plan. The correlation between operation time and intraoperative blood loss was carried out in both Group A  A 3D-printed fracture prototype allows imaging data output to be transformed from a two-dimensional image to a three-dimensional form  and from a static to a dynamic model, which gives a realistic impression to doctors. Using the 3D-prototype, the fracture can be viewed in any direction (0–360°) to allow an accurate description of fracture properties; therefore, it allows the doctor to more effectively and comprehensively understand the specific details of the patient's fracture , 9, veri3D printing technology can be a powerful tool for orthopedists, and it has received much attention in orthopedic applications. It has advantages for the application of surgical planning and surgery training; however, some obstacles need to be overcome. Although CT scans are made in very thin slices, the imaging modality can only provide the accumulation of the multiple slices; error can often occur between the slices. Other restrictions include system error rate and relative long learning curve. At the same time, its current applications mainly include stomatology and orthopedics. We hope that future 3D printing technology can be developed for soft tissue modeling to produce prototypes that can accurately mimic the shape and texture of human organs , 12, whiOur study revealed that 3D printing can reflect the anatomy of the fracture accurately; it effectively helps the doctors plan the operation and provide more effective communication between doctors and patients.


The claustrum is a surprisingly large, sheet-like neuronal structure hidden beneath the inner surface of the neocortex. We found that the portions of the claustrum connected with V4 appear to overlap considerably with those portions connected with other cortical visual areas, including V1, V2, MT, MST and FST, TEO and TE. We found extensive reciprocal connections between V4 and the ventral portion of the claustrum (vCl), which extended through at least half of the rostrocaudal extent of the structure. Additionally, in approximately 75% of the cases, we found reciprocal connections between V4 and a more restricted region located farther dorsal, near the middle of the structure (mCl). Both vCl and mCl appear to have at least a crude topographic organization. Based on the projection of these claustrum subdivisions to the amygdala, we propose that vCl and mCl are gateways for the transmission of visual information to the memory system. In addition to these crude visuotopically organized regions, there are other parts of the claustrum that obey the topographical proximity principle, with considerable overlap of their connections. There is only an overall segregation of claustrum regions reciprocally connected to the occipital, parietal, temporal and frontal lobes. The portion of the claustrum connected to the visual cortex is located ventral and posterior; the one connected to the auditory cortex is located dorsal and posterior; the one connected to the somatosensory cortex is located dorsal and medial; the one connected to the frontal premotor and motor cortices is located dorsal and anterior; while the one connected to the temporal cortex is located ventral and anterior. The extensive reciprocal connections of the claustrum with almost the entire neocortex and its projections to the hippocampus, amygdala and basal ganglia prompt us to propose its role as a gateway for perceptual information to the memory system. The claustrum is a thin, irregular, sheet-like neuronal structure hidden beneath the inner surface of the neocortex. It is a very narrow nucleus (1–6 mm), except for its ventral portion (3–16 mm). Laterally, it wraps around the basal nuclei, mainly putamen . It resembles a leaf with two segments, one extending anteriorly into the frontal lobe and the other extending anteriorly into the temporal lobe. A lateral reconstruction of the claustrum reveals that this nucleus is surprisingly large in its anterior-to-posterior extent (30 × 20 mm). There was a debate concerning the ontogenetic origin of the claustrum, with three different opinions being argued: that the structure is derived from the adjacent insular cortex Meynert, , that itCrick and Koch  summariz3H), wheat germ agglutinin conjugated to horseradish peroxidase (HRP) and retrograde fluorescent tracer injections, placed under physiological control, into 19 different retinotopic locations of V4. We compare the organization of these visual regions of the claustrum with other portions of the claustrum connected with other sensory, motor, and association cortices.In this study, we review the total extent of the connections of V4 with the claustrum, as well as their topographic organization in the context of the connections of the claustrum with other cortical areas. We describe the overall extent of the claustrum in one macaque and its connections in nine macaque monkeys with combined tritiated amino acid , diamidino yellow (DY), and bisbenzimide (Bis) were injected in 10 hemispheres of 9 adult Macaca mulatta, weighing between 3.2 and 4.4 kg. In all animals, the injections of the tracers were placed into retinotopically specified sites (n = 21) in V4, which were determined by electrophysiological recordings. The injection sites, two or more in each animal, spanned eccentricities from central to peripheral vision  and lower (n = 18) visual fields . The horizontal meridian was considered a line through both these points, and the vertical meridian was an orthogonal line that passes through the fovea.All experimental procedures were approved by the NIMH Animal Care and Use Committee and described in detail previously , and tritiated leucine ], specific activity 100–140 Ci/mmol). The labeled amino acids, which had been evaporated and then reconstituted in 0.9% saline to give a final concentration of 50 μCi/μl, were injected at the rate of 0.02 μl/2 min. To minimize leakage of the tracer up the electrode track, the syringe was left in place for 30 min after the injection and then withdrawn into the guide tube, which was then removed from the brain. In 7 cases, one to three injections (0.15–0.3 μl each at each site) of aqueous solutions of 2% FB, 4% DY, or 10% Bis were placed in V4. In 5 cases, two to four injections (0.2 μl each) of 5% of HRP were placed in V4. In the animals with injections involving both HRP and other tracers, the other tracer(s) were injected into the designated V4 sites during one procedure; then, 4–6 days later, HRP was injected into another V4 site.The amount, concentration, and liquid vehicle of the tracer injections as well as the survival times were selected to produce anterograde and retrograde labeling of equivalent size. However, the nature of the tracers caused small differences in sensitivity. Among the tracers used, HRP was the most effective as both an anterograde and a retrograde tracer. Among the fluorescent dyes, the most effective retrograde tracer was FB, which was closely followed by DY.After survival times of 2 days for HRP and 6–8 days for the other tracers, the animals received a lethal dose of sodium pentobarbital and were then transcardially perfused with 0.9% saline followed by 10% formaldehyde-saline. The brains were blocked with the aid of a stereotaxic apparatus, removed from the skull, photographed, and stored in 30% sucrose in 10% formaldehyde-saline until they sank. Frozen sections, 33 μm in thickness, were cut in the frontal plane. One case (Case 6) was cut in the parasagittal plane. Every fifth section was mounted onto gelatinized slides, dehydrated, defatted and processed for autoradiography according to the procedures of Cowan et al. . These sIn this section, we show the size, shape and location of the claustrum in the macaque and we revisit the data on the connections of the claustrum with V4 to reveal the location and organization of two visuotopic organized regions in the claustrum that are connected to virtually all visual areas of the occipital, temporal, and parietal lobes.The claustrum is a narrow nucleus  that laterally wraps around the basal nuclei, mainly putamen and a very small portion of the anterior thalamus  did not reveal projections to the claustrum  and peripheral (blue) injections reveal feedback projections from the claustrum to V4, while the intermediate injection (yellow) reveals feedforward projections to the claustrum. Six representative coronal sections show the feedback and feedforward projections in the claustrum. The lateral reconstruction of the claustrum shows that these projections are contiguous in the ventral portion of the nucleus. The projections are spatially segregated and occupy both the ventral claustrum (vCl) and the mid-claustrum (mCl). An expansion of the central field representation was observed in vCl.Figure 3H, Bis, and HRP in central (red), intermediate (yellow) and peripheral (blue) representations of the lower field of V4 in Case 3 revealed feedback and feedforward projections into two regions of the claustrum in three representative coronal sections. The receptive fields corresponding to the injections significantly overlap and the projections are poorly segregated  and peripheral (blue) representations of the lower field in V4 revealed segregated patches in the ventral portion of the claustrum and a central patch in the mid-claustrum. These bidirectional connections are shown in 5 representative coronal sections and illustrated on the lateral reconstruction of the claustrum in Case 2  that are critical for object recognition  that extended through at least half of the rostrocaudal extent of the structure  but also label the visual related areas vCl and mCl . It is worth noticing that neurophysiological recording in the claustrum revealed mostly unisensory responses (Remedios et al., Based on our data and other connectional studies of the claustrum (Ungerleider et al., Using constrained spherical deconvolution tractography, Milardi et al.  describeThe Review Editor David A. Leopold declares that, despite being affiliated to the same institution as authors Robert Desimone and Leslie G. Ungerleider, the review process was handled objectively and no conflict of interest exists.


Fig. 1 was published in error. The error was corrected in the HTML and PDF versions of this article on March 23, 2016, and the corrected figure is available in the supporting information of this article.S1 File(TIF)Click here for additional data file.


Gastroenteritis may result in loss of zinc by feces, decreased tissue levels of zinc and negative zinc balance in children.The first aim of the study was to determine the plasma zinc level on admission in the hospital and 10 days after the recovery from acute gastroenteritis, in children aged 0-3 years, from the region of Bihor, Romania. Second aim of the study was to analysis the plasma zinc level according to the etiology of gastroenteritis. Zinc sulfate  was given to the patients in the study group, for 10 days. The colorimetric method with Br-PAPS final point (CV% 0.98%-4.64%) was used for the determination of the zinc level. The program IBM SPSS statistics version 22 was used for analysis of the data.th day versus day 0, increased in the study group (n=53)  and decreased in the control group (n=50) . In the day 0, there were no significant differences between plasma zinc level in children with bacterial gastroenteritis compared with viral gastroenteritis .During three years (2009-2011), 103 children with acute gastroenteritis were enrolled in the study. The mean plasma zinc level in the 10Plasma zinc level decreased after 10 days of acute gastroenteritis. The etiologic agent of diarrhea did not influence the plasma level of zinc.


In contrast to a previous report, we propose that the 9p21 locus is a single genetic contribution to CAD in Taiwan because: 1) the penalized logistic regression and the follow-up conditional analysis suggested that rs1537372 accounts for all of the CAD association in 9p21, and 2) the high linkage disequilibrium observed for all associated SNPs in 9p21. We also observed evidence for the following loci at a false discovery rate >5%: SH2B3, ADAMTS7, PHACTR1, GGCX, HTRA1, COL4A1, and LARP6-LRRC49. We also took advantage of the fact that penalized methods are an efficient approach to search for gene-by-gene interactions, and observed that two-way interactions between the PHACTR1 and ADAMTS7 loci and between the SH2B3 and COL4A1 loci contribute to CAD risk. Both the similarities and differences between the significance of these loci when compared with significance of loci in studies of populations of European descent underscore the fact that further genetic association of studies in additional populations will provide clues to identify the genetic architecture of CAD across all populations worldwide.By means of a combination of genome-wide and follow-up studies, recent large-scale association studies of populations of European descent have now identified over 46 loci associated with coronary artery disease (CAD). As part of the TAICHI Consortium, we have collected and genotyped 8556 subjects from Taiwan, comprising 5423 controls and 3133 cases with coronary artery disease, for 9087 CAD SNPs using the CardioMetaboChip. We applied penalized logistic regression to ascertain the top SNPs that contribute together to CAD susceptibility in Taiwan. We observed that the 9p21 locus contributes to CAD at the level of genome-wide significance (rs1537372, with the presence of C, the major allele, the effect estimate is -0.216, standard error 0.033, p value 5.8x10 Recent large-scale association studies of populations of European descent have now identified over 46 loci associated with coronary artery disease (CAD)–3. In orWith respect to populations of Chinese descent, a meta-analysis of two genome-wide association studies of Han subjects from Beijing by the CARDIoGRAM consortium (over 1500 cases and over 5000 controls), followed by a replication study , replicated four of the European loci, 9p21, PHACTR1, TCF21, and C12orf51 (now known as HECTD4), and identified four additional four loci, TTC32-WDR35, GUCY1A3, the MHC, and ATP2B1 As amply demonstrated by these and other studies of non-Mendelian or complex genetic traits, the genome-wide association study represents one of the most powerful tools for the identification of the genetic contribution of individual SNPs to specific phenotypes. Most of these studies have been limited to analyses of the association of phenotype with individual genetic loci or single-nucleotide polymorphisms (SNPs), taken one at a time. This is because applying multivariate regression to such a large dataset, with the goals of investigating the contribution of all SNPs to phenotype and of constructing models of the genetic architecture of the phenotype, becomes computationally impossible with the large number of SNPs to be considered; the requisite matrix inversion procedures or linear equations scale with the number of SNPs to the power of three . One meaFor the study of the genetics of CAD in Taiwan, we took advantage of the fact that the CAD SNPs identified in populations of European descent were incorporated into the design of the Illumina CardioMetaboChip . This deThe TAICHI Consortium is a collaborative study in Taiwan, with the aims to genotype a large number of Taiwanese Chinese subjects with the Illumina CardioMetaboChip and to identify genetic determinants of atherosclerosis- and metabolic-related traits –20. AcadInstitutional Review Boards at all participating centers have approved this study: Taichung Veteran’s General Hospital, Tri-Service General Hospital, the National Taiwan University Hospital, and the National Health Research Institute of Taiwan. Written informed consent was obtained from each participant and is on file in Taiwan. All data and samples that are transferred to the Los Angeles Biomedical Research Institute in the US are identified by codes so that all personal identifying information is kept in Taiwan. The Institutional Review Board (IRB) of the Los Angeles Biomedical Research Institute has approved collection and handling of data and samples from Taiwan as well as molecular studies of the samples including genotyping, all data analyses, and publication of results. The IRB’s of the Hudson-Alpha Research Institute and Stanford University have given approval to this project as exempt from the regulations at 45 CFR 46 or 21 CFR56.Patients admitted to the several Hospital Cardiovascular Departments were enrolled. Subjects were defined as CAD positive with one of the following criteria: 1) history of myocardial infarction; 2) history of coronary artery bypass graft (CABG) or percutaneous coronary angioplasty (PTCA) and or stenting; or 3) at least 1 major vessel with stenosis ≥ 50% demonstrated by angiography. Age was defined as age of event, graft or angioplasty, or angiography.Subjects were enrolled from an epidemiology study and from the several Hospital Endocrinology and Metabolism Departments either as outpatients or as their family members. Subjects with history of CAD were excluded. Age was defined as age of recruitment.Genotyping was performed using the Illumina CardioMetaboChip v1.0  following the manufacturer’s protocol , 22 in aBecause the set of subjects includes family members, subjects were removed for relatedness and cryptic relatedness with PI-Hat values >0.125. Population outliers were defined as those subjects with eigenvalues from principal components 1, 2, and 3 greater than 3 standard deviations from the mean and were removed after calculating principal components using standard methods . Only suhttp://csg.sph.umich/kang/MetaboChip. The results from 9087 SNPs are reported here.“CAD SNPs” were selected from the 23 fine-mapping regions and GWAS hits contributed by the Cardiogram Consortium to the original design of the CardioMetaboChip. This lihttp://www.genetics.ucla.edu/software/mendel with age, gender, and eigenvalues from principal components 1 and 2 added as covariates. As applied to this study, lasso penalized regression is a method for continuous model selection on all of the CAD SNPs and covariates, together and analyzed at the same time, and is of particular value when the number of SNPs exceeds the number of subjects, as in the standard GWAS design.[Penalized logistic regression was employed using the software MENDEL v14  availablS design. The ordeTwo-way interactions were also explored by penalized lasso logistic regression within the top 100 associated SNPs in order to reduce the number of interactions to be computed using the Mendel software.http://biostats.usc.edu/Quanto.html).As implemented in R software, the Fisher exact test, calculation of Odds Ratios, logistic regression, and the Cochran-Mantel-Haenszel test were used to explore the effects of gender in the association of CAD, both within and across gender, and across genotypes , 26. ConHerein we report observations from 8556 Taiwan subjects (3133 cases and 5423 controls) and 9087 coronary artery disease (CAD) SNPs genotyped using the Illumina CardioMetaboChip.Characteristics of the subjects by gender and by case/control status are given in -8 .The 9087 coronary artery disease (CAD) SNPs identified by the CARDIoGRAM Consortium for populations of European descent were tested for association with CAD in Taiwan using a penalized logistic regression method. This method allows consideration of the contribution of all SNPs together and is appropriate for a dataset with more SNPs than subjects. With age, gender, and the eigenvalues from principal components 1 and 2 as covariates, the p-values and effect estimates are given in As a check of the result of the penalized logistic regression, we investigated the fact that only a single SNP in the 9p21 region was retained. A “conventional,” or single-SNP logistic regression of the 9p21 region was conducted with age, gender, and the eigenvalues from principal components 1 and 2 as covariates; the results without and with conditioning on the 9p21 SNP that was retained by the penalized logistic regression, rs1537372, are compared in In order to place our work within the context of the previous study of CAD in a Chinese population, we show Since penalized methods are also efficient for exploring two-way interactions, we explored possible interactions among the 100 SNPs with the highest association with CAD . Please These interactions were studied further using frequency tables Tables  and 5. F-8 in our dataset  SNPs dataset , and 3)  dataset , and 4) 2 value > 0.87 in data from Asian subjects in the Thousand Genomes Project Click here for additional data file.S1 TableAssociation assessed by logistic regression with age, gender, and the eigenvalues from Principal Components 1 and 2 as covariates; this table provides data from (XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.


Noise levels in ICUs are often very high, potentially affecting patient health outcome, which are also considered to be among the risk factors contributing to ICU delirium . In the Acoustic measurements were taken in eight single-bed patient rooms in a level 3 ICU, while patient information was also collected, including APACHE IV score and mortality rate together with other potentially relevant variables; for example, the usage of mechanical ventilators, and so on. The hourly and daily trends of the acoustic data were obtained, and an analysis of variance was carried out to investigate the effect of relevant factors . Furthermore, multiple linear regression models were established to relate the different types of patient-related data (as independent variables) to each type of the acoustic data (as dependent variable), where independent variables were selected based on an exhaustive search method.P = 0.023) and on the time of the day (day/night) (P < 0.001). The average noise level decreased from day 1 to day 2 with a significant reduction at night (P = 0.008), but it was found to rebound from day 2 where the increase of the daytime noise level from day 2 to day 4 was significant (P = 0.005). The results of the multiple linear regression showed that various patient conditions influenced different types of noise-level parameters. For example, the location of the patient room, the usage of mechanical ventilators and the mortality rate were found to correlate to the 10th-percentile SPL (L90) in the first 48 hours . Also, the room location, gender and the usage of mechanical ventilators were found to be related to the 50th-percentile SPL (L50) in the same period .Data were collected for 3 months for 106 patients. The 24-hour trends of acoustic parameters corresponded well to the daily routine events in the ICU. The analyses for the first 4 days of the patients' ICU stay showed that the average SPL varied depending on the day (Noise-level parameters were found to vary depending on the day of ICU stay, the time of day, and other indicators of the patient's status. For a rigorous analysis of the influence of noise on patients' outcome, the effects of these factors must first be controlled or corrected.