The Proteome section contains extensive data on each of 19 HIV-1 proteins, including their functional properties, a sample analysis of HIV-1HXB2, structural models and links to other online resources. The HIV-1 Protease Cleavage Sites section provides information on the position, subtype variation and genetic evolution of Gag, Gag-Pol and Nef cleavage sites. The HIV-1 Protein Data-mining Tool includes a set of 27 group M (subtypes A through K) reference sequences that can be used to assess the influence of genetic variation on immunological and functional domains of the protein. The BLAST Structure Tool identifies proteins with similar, experimentally determined topologies, and the Tools Directory provides a categorized list of websites and relevant software programs. This combined database and software repository is designed to facilitate the capture, retrieval and analysis of HIV-1 protein data, and to convert it into clinically useful information relating to the pathogenesis, transmission and therapeutic response of different HIV-1 variants. The HIV-1 Proteomics Resource is readily accessible through the BioAfrica website at: Most Internet online resources for investigating HIV biology contain either bioinformatics tools, protein information or sequence data. The objective of this study was to develop a comprehensive online proteomics resource that integrates bioinformatics with the latest information on HIV-1 protein structure, gene expression, post-transcriptional/post-translational modification, functional activity, and protein-macromolecule interactions. The BioAfrica HIV-1 Proteomics Resource  Although the HIV-1 genome contains only 9 genes, it is capable of generating more than 19 gene products. These products can be divided into three major categories: structural and enzymatic ; immediate-early regulatory , and late regulatory  proteins. Tat, Rev and Nef are synthesized from small multiply-spliced mRNAs; Env, Vif, Vpu and Vpr are generated from singly-spliced mRNAs, the Gag and Gag-Pol precursor polyproteins are synthesized from full-length mRNA. The matrix (p17), capsid (p24) and nucleocapsid (p7) proteins are produced by protease cleavage of Gag and Gag-Pol, a fusion protein derived by ribosomal frame-shifting. Cleavage of Nef generates two different protein isoforms; one myristylated, the other non-myristylated. The viral enzymes  are formed by protease cleavage of Gag-Pol. Alternative splicing, together with co-translational and post-translational modification, leads to additional protein variability [Phylogenetic analysis, on its own, provides little information about the conformational, immunological and functional properties of HIV-1 proteins, but instead, focuses on the evolution and historical significance of sequence variants. To understand the clinical significance of genetic variation, sequence analysis needs to be combined with methods that assess change in the structural and biological properties of HIV-1 proteins. At present, information and tools for the systematic analysis of HIV-1 proteins are limited, and are scattered across a wide-range of online resources ,3. To faWe have categorized the Proteomics Resource into the following main subject headings Figure  &3:HIV Proteome – Information about structure and sequence, as well as references and tutorials, for each of the HIV-1 proteins  proteins using information available in public databases and tools ; the other from singly-spliced mRNA (p16) [MEPVDPRLEPWKHPGSQPKTA-21; the hydrophobic residues are highlighted in bold, and polar residues are italicized) at the N-terminus of the protein; the cysteine-rich disulphide bond region (22-CTNCYCKKCCFHCQVC-37); the core, basic and glutamine-rich region (49-RKKRRQRRRAHQNSQTHQASLSKQ-72) that is important for nuclear localization and TAR-binding activity, and the RGD cell-attachment site that binds to cellular integrins. In addition to being expressed in HIV-1-infected cells, Tat is also released into the extracellular fluid where it acts as a growth factor for the development of Kaposi's Sarcoma. Additional information about Tat and its protein-protein interactions can be found on the proteome page of the BioAfrica website located at .In the HIV-1 Proteome section, each of the 19 HIV-1 proteins has a webpage that is divided into six parts: "general overview", "genomic location", "domains/folds/motifs", "protein-macromolecule interactions", "primary and secondary database entries", and "references and recommended readings" Figure . The oveHXB2 , sequencB2 [HXB2 , and proB2 [HXB2 ,6  . ImportaPost-translational cleavage of the Gag, Gag-Pol and Nef precursor proteins occurs at the cell membrane during virion packaging, and is essential to the production of infectious viral particles. Drugs that inhibit this process, the protease inhibitors (PIs), are the most potent antiretroviral agents currently available. Thus it is important to collect information, not only on the sequence of protease enzymes from different HIV-1 subtypes, but also on the natural polymorphisms and resistance mutations that may effect their catalytic activities, drug responsiveness, substrate specificities, and cleavage site characteristics. Studies have shown that resistance mutations in the protease of subtype B are associated with impaired proteolytic processing and decreased enzymatic activity, and that compensatory mutations at Gag and Gag-Pol cleavage sites can partially overcome these defects . These fThe cleavage site section of the BioAfrica webpage is the direct extension of a recent publication in the Journal of Virology describing the location and variability of protease cleavage sites  Figure . TogetheHXB2 isolate, which, in addition to a premature stop codon, contained no cAMP/cGMP, PKC or CKII phosphorylation sites.The HIV-1 Protein Data-Mining Tool contains twelve sequence analysis techniques for assessing protein variability among different strains of HIV-1 Figure . These tThe HIV-1 BLAST Structure Tool facilitates the analysis of HIV-1 protein structure by allowing for rapid retrieval of archived structural data stored in the public databases Figure . Users mThe HIV-1 Proteomics Tools Directory is divided into two web pages. The initial webpage is a concise compilation of some of the most commonly used protein-specific Internet resources Figure . This "bThe impending rollout of antiretroviral therapy to millions of HIV-1-infected people in sub-Saharan Africa provides a unique opportunity to monitor the efficacy of non-B treatment programs from their very inception, and to obtain critical new information for the optimization of treatment strategies that are safe, affordable and appropriate for the developing world. An integral part of this massive humanitarian effort will be the collection of large amounts of clinical and laboratory data, including genetic information on viral subtype and resistance mutations, as well as routine CD4+ T-cell counts and viral load measurements. The mere collection of this data, however, does not ensure that it will be used to its maximum potential. To achieve full benefit from this explosive source of new information, the data will need to be appropriately collated, stored, analyzed and interpreted.[The rapidly emerging field of Bioinformatics has the capacity to greatly enhance treatment (and vaccine) efforts by serving as a bridge between Medical Informatics and Experimental Science. By correlating genetic variation and potential changes in protein structure with clinical risk factors, disease presentation, and differential response to treatment and vaccine candidates, it may be possible to obtain valuable new insights that can be used to support and guide rationale decision-making, both at the clinical and public health levels. The HIV-1 Proteomics Resource, described in this report, is an initial first step in the development of improved methods for extracting and analyzing genomics data, converting it into biologically useful information related to the structure, function and physiology of HIV-1 proteins, and for assessing the role these proteins play in disease progression and response to therapy. The Resource, developed at the Molecular Virology and Bioinformatics Unit of the Africa Centre of Health and Population Studies, is a centralized user-friendly database that is easily accessed through the BioAfrica website at .AA – Amino AcidBLAST – Basic Local Alignment Search ToolCKII – casein kinase IICTLs – cytotoxic T-lymphocytesDIP – Database of Interacting ProteinsDNA – deoxyribonucleic acidEnv – envelope glycoproteinGag – group-specific antigen polyproteinGIF – Graphics Interchange FormatHIV – Human Immunodeficiency VirusHIV-1 – Human Immunodeficiency Virus Type-1HTTP – Hypertext Transfer ProtocolLTR – long-terminal repeatmRNA – messenger RNANCBI – National Center for Biotechnology InformationNef – negative factorPDB – Protein Data BankpI – isoelectric pointPIs – protease inhibitorsPKC – protein kinase CPol – polymerase polyproteinRev – ART/TRS anti-repression transactivator proteinRNA – ribonucleic acidRNase H – ribonuclease HTat – transactivating regulatory proteinVif – virion infectivity factorVpr – viral protein RVpu – viral protein UThe author(s) declare that they have no competing interests.RSD created and maintains BioAfrica's HIV proteomics resource, HIV proteome section, proteomics tools directory, HIV-1 protein data-mining tool and HIV structure BLAST tool; performed protein sequence and structural model analyses; and wrote the manuscript.TDO conceived and maintains the BioAfrica website, and continues to oversee its rapid expansion; created the cleavage sites section; and participated in the design and implementation of the HIV proteomics resource.CS participated in the design of the HIV proteomics resource, with an emphasis on the proteomics tools directory.SD participated in the design and creation of the HIV proteome section, with an emphasis on the HIV-1 Tat protein.MG participated in the design of the HIV proteomics resource, with an emphasis on the HIV proteome section.SC supervised the project, and participated in the design and implementation of the HIV proteomics resource.All authors read and approved the final manuscript.A table containing a comparative summary of potential functional motifs  in the HIV-1 Tat proteins of subtypes B and C, as identified using PROSITE.Click here for file


This paper analyzes the effect of the mean-square error principle on the optimization process using a Special Case of Hopfield Neural Network (SCHNN).The segmentation of multidimensional medical and colour images can be formulated as an energy function composed of two terms: the sum of squared errors, and a noise term used to avoid the network to be stacked in early local minimum points of the energy landscape.Here, we show that the sum of weighted error, higher than simple squared error, leads the SCHNN classifier to reach faster a local minimum closer to the global minimum with the assurance of acceptable segmentation results.The proposed segmentation method is used to segment 20 pathological liver colour images, and is shown to be efficient and very effective to be implemented for use in clinics. Segmentation is an important step in most applications that use medical image data. For example, segmentation is a prerequisite for quantification of morphological disease manifestations and for radiation treatment planning ,2, for cA Number of algorithms based on approaches such as histogram analysis, regional growth, edge detection and pixel classification have been proposed in other articles of medical image segmentation. In recent years, Artificial Neural Networks (ANNs) have been proposed as an attractive alternative solution to a number of pattern recognition problems. In our previous works , we haveHopfield network for the optimization applications consists of many interconnected neuron elements. The network minimizes an energy function of the form:Vk is the output of the kth neuron, Ik is the bias term, and Tkl is the interconnection weight between the kth and lth neurons. The energy function used in the segmentation problem is slightly different from the one defined by Hopfield and the arguments are given in [where N is the number of neurons, given in .The results that have been obtained in  were preThe segmentation problem of an image of N pixels is formulated in  as a parRkl is the Mahalanobis distance measure between the kth pixel and the centroid of class l, Rkl is also equivalent to the error committed when a pixel k is assigned to a class l. The index n in  is the power or weight of the considered error in the energy function of the segmentation problem, and Vkl is the output of the klth neuron. Nkl is a N × M vector of independent high frequency white noise source used to avoid the network being trapped in early local minimums. The term c(t) is a parameter controlling the magnitude of noise which is selected in a way to provide zero as the network reaches convergence. The minimization is achieved by using SCHNN and by solving the motion equations satisfying:where Ukl is the input of the kth neuron, and μ(t) is a scalar positive function of time, used as heuristically motivated stopping criterion of SCHNN, and is defined as in [where ed as in  by:β(t) = t(Ts - t)     (4)t is the iteration step, and Ts is the pre-specified convergence time of the network which has been found to be 120 iterations [kth pixel and the centroid of class l given by:where erations . The netXk is the P-dimensional feature vector of the kth pixel (here P = 3 with respect to the RGB color space components),  is the P-dimensional centroid vector of class l, and Σl is the covariance matrix of class l. The segmentation algorithm is described as follows [where  follows .Step 1 Initialize the input of the neurons to random values.Step 2 Apply the following input-output relation, establishing the assignment of each pixel to only and only one class.Step 3 Compute the centroid  and the covariance matrix Σl of each class l as follows:nl is the number of pixels in class l, and the covariance matrix is then normalized by dividing each of its elements by .where Step 4 Update the inputs of each neuron by solving the set of differential equations in (2) using Eulers approximation:Step 5 if t <Ts, repeats from Step 2, else terminated.For this study, a total of 20 liver tissue sections were provided by the pathological division of National Cancer Center in Tokyo. These sections were taken using needle biopsy, stained with hematoxylin and then magnified with an optical microscope. Figure Ts values between 30 and 120 iterations. Similar curves were obtained for the rest of the images of the dataset. As it is illustrated in Figure Ts = 120 iterations gives the optimal solution, the same as it is with MRI data [Figure MRI data .Rkl in the cost function (2), we have provided a simple modification to the above algorithm as follows:In order to study the effect of the weight of the Mahalanobis distance Step 1 Use the same random initialization N × M matrix, as input of the neurons, when minimizing the energy function (1) with different error's weight n.This condition is added to the algorithm in order to make sure that the random field does not have any effect on the generated results.Step 2 trough Step 5 remain the same.n in equation (2). As aforementioned, the pre-specified convergence time of SCHNN is fixed to Ts = 120 iterations. However, we can clearly see from Figure n in Equation (2), the same convergence point or a close position is reached in half the time of the one reached with n = 2 and Ts = 120 iterations. So, this raises the following question: what is the type of relation between the variable n in (2) and the pre-specified convergence time Ts?Figure n that corresponds to the optimum solution with Ts = 120 iterations. From Figure n = 6 gives the optimum solution with Ts = 120. Similar figures to Figure Before answering this question, it is essential to know at this level what is the best value of n, in Equation (2). When both (Ts and n) used together they give a local optima in the energy landscape of SCHNN. Figure n in Equation (2) that are obtained with Ts values 120, 60, and 30. We realized that the curves corresponding to Ts = 120 and Ts = 60 intersect in their optimum solutions obtained with n = 6, and the two curves are similar when n is in the range 5–10. However, the curve corresponding to Ts = 30, shows higher error at convergence of all values of n.In order to study the effect of the pre-specified time, we repeated the above experiments with different Ts values. We realized that each value of Ts corresponds to a value of n in (2) when it takes the value of six where SCHNN gives an optimum and acceptable results that agree with the pathological experts point of views. However, with other values of n, the random initialization may affect the solution of the problem, or in other words, may affect the error of the SCHNN at convergence as shown in Figure In order to see the effect of the random initialization on the results of the algorithm described in section 3, we have executed the same algorithm with different initialization matrices and the curves of the convergence values of SCHNN corresponding to these initializations are shown in Figure We analyzed the effect of considering the mean-square error in formulating the segmentation problem of multidimensional medical images. We have shown, empirically, that considering an integer power equal to six, of the error in the energy function of the problem, helped SCHNN to converge twice as fast as the same optimal solution obtained with the mean-square error algorithm. This result is promising to make our segmentation method useful for a Computer Aided Diagnosis (CAD) system for liver cancer and the like. In our future work, we will study deeply the effect of the random initialization and its effect on the segmentation result and on the SCHNN classifier.The author(s) declare that they have no competing interests.Rachid carried out the theoretical study, the sequence alignment and drafted the manuscript. Mohammed participated in the design of the study and performed the analysis and helped to draft the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


ABCG5 or ABCG8, comprising the STSL locus, are now known to cause this disease and their protein products are proposed to function as heterodimers. Under normal circumstances cholesterol, but not non-cholesterol sterols, is preferentially absorbed from the diet. Additionally, any small amounts of non-cholesterol sterols that are absorbed are rapidly taken up by the liver and preferentially excreted into bile. Based upon the defects in sitosterolemia, ABCG5 and ABCG8 serve specifically to exclude non-cholesterol sterol entry at the intestinal level and are involved in sterol excretion at the hepatobiliary level.The molecular mechanisms that regulate the entry of dietary sterols into the body and their removal via hepatobiliary secretion are now beginning to be defined. These processes are specifically disrupted in the rare autosomal recessive disease, Sitosterolemia (MIM 210250). Mutations in either, but not both, of two genes Here we report the biochemical and immuno-localization of ABCG5 and ABCG8 in human liver, gallbladder and intestine using cell fractionation and immunohistochemical analyses.We raised peptide antibodies against ABCG5 and ABCG8 proteins. Using human liver samples, cell fractionation studies showed both proteins are found in membrane fractions, but they did not co-localize with caveolin-rafts, ER, Golgi or mitochondrial markers. Although their distribution in the sub-fractions was similar, they were not completely contiguous. Immunohistochemical analyses showed that while both proteins were readily detectable in the liver, ABCG5 was found predominately lining canalicular membranes, whereas ABCG8 was found in association with bile duct epithelia. At the cellular level, ABCG5 appeared to be apically expressed, whereas ABCG8 had a more diffuse expression pattern. Both ABCG5 and ABCG8 appeared to localize apically as shown by co-localization with MRP2. The distribution patterns of ABCG5 and ABCG8 in the gallbladder were very similar to each other. In the small intestine both ABCG5 and ABCG8 appear to line the brush border. However, at the level of the enterocyte, the cellular distribution patterns of ABCG5 and ABCG8 differed, such that ABCG5 was more diffuse, but ABCG8 was principally apical. Using standard deglycosylation methods, ABCG5 and ABCG8 do not appear to be glycosylated, suggesting a difference between human and mouse proteins.We report the distribution patterns of ABCG5 and ABCG8 in human tissues. Cell fractionation studies showed that both proteins co-fractionated in general, but could also be found independent of each other. As predicted, they are expressed apically in both intestine and liver, although their intracellular expression patterns are not completely congruent. These studies support the concept of heterodimerization of ABCG5 and ABCG8, but also support the notion that these proteins may have an independent function. The gastrointestinal tract is the initial barrier to dietary constituents and is important in regulating nutrient entry, as well as keeping non-nutrients out. Additionally, the hepatobiliary system acts as an additional filter to rapidly excrete such non-nutrients into bile, thus keeping the net retention of these potential toxins low. Mammals have evolved many mechanisms in the gastrointestinal tract to select out usable dietary constituents from those that maybe potentially toxic to the body. It is apparent that the ATP-binding cassette proteins (ABC proteins/transporters) are the machinery that mediate the ATP-dependent transport of a wide variety of substrates that range from xenobiotics to peptide fragments . A subsede novo synthesis and hepatobiliary secretion. Sitosterolemia, a rare autosomal recessive disorder of sterol metabolism results in the disruption of dietary sterol entry and hepatobiliary sterol secretion [STSL, are known to cause this disease [in vitro and in vivo data, these 'half-transporters' are proposed to function as obligate heterodimers. In vitro experiments have shown that both proteins are needed to be co-expressed for apical expression and that these may function as mutual chaperones in the ER for maturation [In vivo experiments in mice have not been consistent. Using the Abcg5/Abcg8 double knockout mice, Graf et al has shown that by inoculating them with adenoviral constructs for Abcg5 and Abcg8 that both are required for expression of both proteins [et al and our group have constructed mouse models deficient in either Abcg5 [One of these processes involves the regulation of sterol entry and excretion. Whole body cholesterol homeostasis is a tightly regulated process, involving dietary absorption, ecretion -6. Under disease -9. Basedturation ,11. In ver Abcg5  or Abcg8er Abcg5  that shoIn this report, we examined the location of these two proteins using cellular fraction and immunohistochemical analyses of human liver, gallbladder and small intestine. We found a general concordance of co-expression of both proteins, but we also noted that ABCG5 and ABCG8 could be found in plasma membranes, as well as in intracellular membrane locations independent of each other. Additionally, deglycosylation of human liver membranes with peptidyl N-glycosidases did not alter the mobility of the proteins after SDS-PAGE, suggesting that these proteins may not be glycosylated in human liver. This differential localization suggests that perhaps ABCG5 and ABCG8 may have functions independent of each other, as well as functioning as heterodimers.Tissue aquistion. Human liver donated for transplantation, but deemed unsuitable for transplantation on inspection by the transplant service  was obtained in accordance with IRB approval. As soon as the liver was deemed unsuitable  pieces were either snap-frozen in liquid nitrogen and stored at -80°C or liquid nitrogen until use, or placed in ice-cold 2-methylbutane and stored in liquid nitrogen. Samples from more than nine different donors were used in these studies. Additionally, human gallbladders and segments of proximal small intestine were obtained from patients under going either laproscopic cholecystectomy or pancreatoduodenectomy (Whipple procedure). These tissues were directly taken from the operating room in normal saline on ice to be processed directly for frozen sectioning.Anti-membrin, anti-transferrin, and anti-calnexin antibodies were obtained from Stressgen , anti-caveolin antibody from BD Bioscience , anti-MDR1 that also detects MDR2/3 (C219) from Centocor Inc. , anti-MRP2 (cMOAT) from Chemicon International  and secondary antibodies were purchased from Jackson Immuno research . Polyclonal rabbit anti-sera to human ABCG5 and ABCG8 peptides were generated in-house, using a 20-peptide immunogen from human ABCG5  and a 22-peptide sequence from human ABCG8 . The anti-sera were further purified using peptide affinity columns and stored at a concentration of 0.8 mg/ml in Immuno Pure Binding Buffer . For peptide blocking experiments the peptides were dissolved in DMSO , incubated with the corresponding peptide for 1 1/2 hours at 37°C then used for immunoblotting as described below.g for 10 minutes, the pellet containing any undisrupted cells and nuclear debris were re-homogenized with one-half the initial volume of homogenization buffer, centrifuged at 1000 g for 10 minutes and this process was repeated once more. Supernatants were pooled and subjected to centrifugation at 100,000 g for 40 minutes. The resulting pellet, deemed the crude membrane fraction, was used as the starting material for Western blotting and fractionation experiments.Crude total membrane isolation was carried out with minor modifications as previously described . All theHuman liver crude total membrane proteins were re-suspended in 30% Nycodenz solution (Nycodenz in 5 mM Tris-HCl pH 7.5 and 1 mM EDTA). This suspension was loaded on top of a 40% Nycodenz solution cushion in an ultracentrifuge tube, overlaid by consecutive 23%, 20%, 15% and 10% Nycodenz solutions and subjected to centrifugation at 39,000 rpm for 16 hours at 4°C in a SW41 rotor . After centrifugation, 800–1000 μl fractions were sequentially removed from the top, combined with two volumes of homogenization buffer (see above) and centrifuged at 39,000 rpm at 4°C for 40 minutes to remove the Nycodenz. The resulting pellets were re-suspended in buffer , the protein content determined by the method of Lowry and fractions analysed by SDS-PAGE. Equal amounts of protein (25 μg) per lane were loaded.The procedure for membrane fractionation was essentially as described for the Nycodenz fractionation, except for the homogenization buffer used . The sucrose density gradient fractionation was modified as previously described . Human l® Chemiluminescence Reagent Plus .Proteins resolved by SDS-PAGE were transferred onto nitrocellulose membranes. Membranes were then blocked for 1 hour in 5% dry milk in PBS-T  and incubated with primary antibody against either ABCG5 or ABCG8 in 5% milk in PBS-T overnight at 4°C. Blots were washed three times for 5 minutes in TBS-T  with 150 mM NaCl, incubated with goat-anti-rabbit conjugated HRP antibodies (1:10000 dilution), washed for three times 5 minutes and developed with Western LightningSnap-frozen liver, gallbladder and intestine tissues were used to cut 8 μm thick frozen sections, air-dried for 30 minutes onto glass slides and kept at -80°C until used. The slides were stained with hematoxylin, rinsed with PBS three times, fixed for 10 minutes with cooled methanol at -20°C and rinsed with PBS three times. The slides were treated with blocking solution (10% donkey serum in 0.1 M glycine/PBS) for 30 minutes at room temperature and incubated with primary antibody overnight at 4°C. The slides were washed with PBS and incubated with secondary antibody (goat-anti-rabbit conjugated with Cy3™ or rhodamine or FITC) for 20–30 minutes at room temperature, rinsed with PBS three times and examined under an Olympus BX-5 confocal microscope with Fluoview.Peptide antibodies were raised against human ABCG5 and human ABCG8 and affinity purified prior to use (see Methods). The immunogen peptides used for the antibodies were selected since they were sequences that lay outside of the predicted transmembrane domains and based upon antigenicity.Western blotting experiments Figure  showed tCrude total membrane proteins from human liver were fractionated by Nycodenz gradient centrifugation and examined for localization markers by western blot analyses Figure . After NTo examine whether ABCG5 and ABCG8 were associated with membrane rafts, total membrane proteins from human liver were solubilized with ice-cold 1% Triton X-100 detergent and fractionated by sucrose density gradient centrifugation Figure . FractioThus, ABCG5 and ABCG8 have significant overlap with each other suggesting co-localization. However, these proteins did not seem to co-localize with any specific membrane marker except transferrin, when two different methods of fractionation were utilized.It has been shown previously that ABCG5 and ABCG8 are expressed only in the liver and intestine . To furtSerial sections of human gall bladder were incubated with each antibody and pre-immune serum was used as a negative control. Both ABCG5 and ABCG8 were detected in the epithelium of gall bladder mucosa Figure  and 5B. Both ABCG5 and ABCG8 were detected in the apical surfaces of the enterocytes in biopsy samples of the small intestine , the apical patterns of expression of these proteins in both the liver and intestine would seem to suggest that these proteins seem to traffic to these specialized membranes normally. In the absence of an independent method, and the lack of a direct assay for function, whether these proteins form an active heterodimer can not be resolved at present.Finally, although rodent sterolins are glycosylated and In summary, we report the first immunolocalization of ABCG5 and ABCG8 in human liver, gall bladder and intestine. Our data show that these proteins are located in membranes and can have an apical expression in all of these tissues. Biochemical, as well as immunolocalization studies show that while both proteins co-localize in general, they can also seem to have expression patterns that may be independent of each other.None declared.ELK and MHL performed the experiments, KDC and DBA provided the liver and surgical samples respectively SBP was responsible for supervision, data analyses and obtaining funding for these experiments. ELK and SBP wrote the paper.The pre-publication history for this paper can be accessed here:


The present paper expresses the author's views about the practical utility of Health Behavior Theory for health behavior intervention research. The views are skeptical and perhaps even a bit exaggerated. They are, however, also based on 20-plus years of in-the-trenches research focused on improving health behavior practice through research.The author's research has been theoretically driven and has involved measurement of varying variables considered to be important theoretical mediators and moderators of health behavior. Regretfully, much of this work has found these variables wanting in basic scientific merit. Health Behavior Theory as we have known it over the last 25 years or so has been dominated by conceptualizations of behavior change processes that highlight cognitive decision-making. Although much of health behavior practice targets what people do rather than what they think, the logic of focusing on thoughts is that what people think about is the key to what they will do in the future, and that interventions that can measure and harness those processes will succeed to a greater extent than those that do not. Unfortunately, in the author's experience, the premise of cognitive theories has fallen short empirically in a number of ways. The cognitive schemata favored by most health behavior theories are difficult to measure, they do not predict behavioral outcomes very well, there is little evidence that they cause behavior, and they are hard to change directly.It is suggested that health behavior researchers reconsider their use of these theories in favor of models whose variables are more accessible to observation and experimental manipulation and that most importantly have strong empirical support. The author has been conducting research on behavioral treatment of obesity for about 25 years. During that time, the dominant conceptual models guiding intervention development have been cognitive behavior models that have their origin in psychological theory. Those most often cited include the Health Belief Model , ProtectWeinstein's summary is illustrative of the fact that Health Behavior Theory has tended to be particularly interested in understanding people's motivation to change behavior rather than ability to change. Moreover, motivation is thought to be the result of a relatively complex, but logical, interpretation of large quantities of information about self and environment. The theories that Weinstein reviewed deal almost exclusively with behavioral decision processes in people's minds. They have few if any terms relating to how information gets into peoples minds or how subsets of it receive more or less attention. Broader health behavior theories such as Social Cognitive Theory or the Transtheoretical model have addressed issues and variables outside the person to a greater extent, but the fundamental interest in and belief in psychological variables as the key force in determining health behavior remains.The implications of the focus of health behavior theory on psychological determinants of behavioral decision-making for my own research area of interest, obesity treatment, are several. One is the inclusion of measures of psychological characteristics in most research protocols . A second is the inclusion of treatment elements that specifically target psychological perceptions and processes independent of the diet and physical activity behaviors that actually produce weight change . A third is the belief that psychological reactions to treatment experiences themselves are very important and deserve independent attention. Common behavioral prescriptions for weight-loss goals and frequency of self-weighing are exemplary .The problem with the emphasis on cognitive variables in weight-control research is that they have so far failed to meet fundamental scientific criteria for empirical verification. Thus, they also have not led to a better understanding of the weight-loss process, have not improved our ability to predict weight-loss outcomes, and have not led to improvement in treatment methods. In some cases it is even arguable that they have made treatment worse. I will illustrate these problems with results from my own research.Like most behavioral researchers in the obesity area, I have attempted to measure elements of health behavior theory in every obesity intervention project I have ever conducted. I have assessed weight-loss goals, behavioral and weight-loss self-efficacy, psychological well-being, perceived barriers to diet and physical activity change, stages-of-change, and perceived social support. How well have empirical examinations of these factors fared as predictors of success in weight control?We have examined the predictive value of self-efficacy assessments in several of our studies and describe the results from three of these here in more detail -10. In tThe second study examined mood and situational self-efficacy in 55 men and 58 women before and after a 16-week weight-loss treatment with a 1-year follow-up . Women hThe third study examined predictors of weight change over a 2-year period in 460 men and 1172 women who received a low-intensity weight-loss intervention delivered through their HMO . The selOur overall conclusion from the analyses described above, as well as others not pursued in as great detail, is that self-efficacy is a weak predictor of weight loss and is inconsistent across study populations and gender. It tends to increase with weight loss. However, treatment-induced increases in efficacy are not predictive of longer-term weight-loss success.We have also attempted to measure barriers to adherence to weight-control behaviors in many of our studies -14. The Goal-setting has long been of interest to health behavior theory and in recent years has attracted attention in weight-loss research when it was realized that most people who enter weight-loss treatments want to lose a lot more weight than is realistic given the potency of current weight-loss methodologies . When asPerceived social support is another psychological factor thought to influence health behavior decision-making. We have measured social support in a variety of ways in our studies, ranging from single-item questions to multipaged assessments attempting to differentiate among informational, instrumental, and emotional support. The results, unfortunately, have closely paralleled those we have seen with other assessments of barriers to adherence. Assessments of social support prior to treatment do not predict weight loss. Average reports of social support tend to parallel weight loss itself. When people lose weight they report more social support. When they regain, they report less. In other words, perceptions of social support are not predictive of success in weight-loss treatments.Self-monitoring of health behavior is incorporated into many health behavior theories, usually as part of a person's assessment of achieved outcomes. Although self-monitoring is usually considered a positive element in the adoption of health behavior, in obesity treatment frequent self-monitoring of weight has tended to be down-played or even discouraged on the grounds that disappointing results  may undermine motivation. This is another example in which health behavior theory may have indirectly led to incorrect treatment recommendations. In weight-loss treatments, active discouragement of frequent self-observation of weight has become popular based on the premise that more frequent weighting will cause psychological stress and lower self-efficacy. Recently, we have examined the relationship between frequency of self-weighing and body weight in both clinical and population samples and have found, somewhat to our surprise, that frequency of self-weighing is one of the strongest single predictors of body weight cross-sectionally, and change in the frequency of self-weighing is one of the strongest predictors of weight change . The direction of predictions, however, is opposite that derived from theory. People who weigh themselves more weigh less and are more successful in losing weight.A final failure of current health behavior theory to prove useful in weight-control research is a recent examination of the relationship between a stage-of-change measure adopted from Prochaska and short- and long-term weight loss . CategorOur most recent effort to utilize health behavior theory in obesity intervention research is a study that attempted to examine the effectiveness of experimentally-induced outcome expectancies on weight loss . Obese men and women participated in an 8-week weight-loss program with 18-month follow-up in which they were assigned to one of two expectancy groups. The optimistic group was told that focusing exclusively on the positive benefits of weight loss would be valuable in ensuring that they remained motivated in their weight-loss efforts and was given assignments during weekly group sessions and homework between sessions to reinforce this optimistic mindset. A "balanced" expectancy group received the instructions that focusing on both the positive and negative aspects of weight loss, a balanced approach, would be most conducive to maintaining weight-loss motivation. This group also received assignments to reinforce their message. Results of this study indicated that the expectation induction was successful initially but difficult to maintain in the face of real weight-loss experience. We were also unable to show that experimentally-induced expectations influenced weight-loss success.To summarize the findings described above, I have had considerable difficulty over the last 25 years in confirming that the psychosocial variables favored by health behavior theory are of much value for obesity intervention research. They do not predict weight loss well, either as mediators or moderators. There is little evidence to support the idea that targeting them for intervention improves weight-loss outcomes. It is, of course, arguable that the weak findings relating to health behavior theory variables are due in large part to methodological weaknesses, either in measurement tools and/or their frequency of measurement. I would argue, however, that 25 years is long enough to wait for improved methods and that it is time to look elsewhere for variables that better predict weight-change outcomes and that, therefore, may form a better basis for improving future treatments.Given the lack of success finding support for cognitive mediators of behavior change in weight loss, one might surmise that progress in improving weight-loss interventions over the last 20 years must have been dreary indeed. Somewhat surprisingly, however, that is not the case. In fact, the short-term (6 to 12 months) success of weight-loss treatments has approximately doubled over that time and several variables have been identified that reliably enhance treatment outcomes. It has been clearly shown experimentally that increasing treatment length , prescriThe argument above about the practical limitations of many popular theories of health behavior is not meant to be a call to abandon theory. Behavior scientists have amassed much useful information about the principles underlying human behavior that should be valuable for health behavior interventions. Much is known about human perception, learning, motivation, and responsiveness to environmental opportunities and contingencies. Health behavior intervention lies at the interface between people and their environment. Interventionists change aspects of the environment  with the intention of producing changes in how people behave. What is needed to advance health behavior intervention is theory that addresses relationships between modifiable aspects of the environment and behavior. There is no doubt that cognitive processes are involved in these relationships. However, the extent to which current theories capture this is questionable. Data now available suggest that easily obtainable information about people's cognitive processes adds little to our ability to predict the results of interventions. Thus, it may be wise to pay more attention to applied theories like classical behavior theory , communiNone declared.


Proteins having similar functions from different sources can be identified by the occurrence in their sequences, a conserved cluster of amino acids referred to as pattern, motif, signature or fingerprint. The wide usage of protein sequence analysis in par with the growth of databases signifies the importance of using patterns or signatures to retrieve out related sequences. Blue copper proteins are found in the electron transport chain of prokaryotes and eukaryotes. The signatures already existing in the databases like the type 1 copper blue, multiple copper oxidase, cyt b/b6, photosystem 1 psaA&B, psaG&K, and reiske iron sulphur protein are not specified signatures for blue copper proteins as the name itself suggests. Most profile and motif databases strive to classify protein sequences into a broad spectrum of protein families. This work describes the signatures designed based on the copper metal binding motifs in blue copper proteins. The common feature in all blue copper proteins is a trigonal planar arrangement of two nitrogen ligands [each from histidine] and one sulphur containing thiolate ligand [from cysteine], with strong interactions between the copper center and these ligands.Sequences that share such conserved motifs are crucial to the structure or function of the protein and this could provide a signature of family membership. The blue copper proteins chosen for the study were plantacyanin, plastocyanin, cucumber basic protein, stellacyanin, dicyanin, umecyanin, uclacyanin, cusacyanin, rusticyanin, sulfocyanin, halocyanin, azurin, pseudoazurin, amicyanin and nitrite reductase which were identified in both eukaryotes and prokaryotes. ClustalW analysis of the protein sequences of each of the blue copper proteins was the basis for designing protein signatures or peptides. The protein signatures and peptides identified in this study were designed involving the active site region involving the amino acids bound to the copper atom. It was highly specific for each kind of blue copper protein and the false picks were minimized. The set of signatures designed specifically for the BCP's was entirely different from the existing broad spectrum signatures as mentioned in the background section.These signatures can be very useful for the annotation of uncharacterized proteins and highly specific to retrieve blue copper protein sequences of interest from the non redundant databases containing a large deposition of protein sequences. Most proteins can be grouped, on the basis of similarities in their amino acid sequences, into a limited number of protein families. Proteins or protein domains that belong to a particular family usually share functional attributes and are derived from a common ancestor. Highly conserved sequences in protein families are generally important for the function of a protein and/or for the maintenance of its 3-dimensional structure. Within the last decade, the sensitivity of sequence searching techniques has been improved by profile or motif-based analysis, which uses information derived from multiple sequence alignments to construct and search for sequence patterns -4. By stBy doing a keyword search, the protein sequences mined out from different databases is highly varied owing to different levels of redundancy. This could be due to the different strengths and weaknesses underlying the analysis algorithms used in different databases. The usage of the broad range signatures existing in databases, for the retrieval of blue copper proteins like the type 1 copper blue, multiple copper oxidase, cyt b/b6, photosystem 1 psaA&B, psaG&K, and reiske iron sulphur protein brings out different kinds of copper proteins and a lot more of unrelated proteins. A search once again becomes necessary for sorting out the required blue copper proteins. The usage of pattern database would be more selective as it can identify family members based on the conserved functional region patterns. Keeping these broad spectrum signatures in mind, more specific and targeted protein signatures for each of the blue copper proteins was designed. The diagnostic success of these specified signatures over the wide range signatures mentioned lies in the number of true positives picked over the minimal or nil false positives picked from the non redundant databases.Blue copper proteins, which are also known as cupredoxins, are small, soluble proteins 10 – 14 kDa) whose active site contains a type 1-copper . All the – 14 kDaThe use of active site patterns or signatures is very rapidly becoming one of the essential tools of sequence analysis ,15. Althth position, cysteine at the 74th position, histidine at 79th position and methionine at the 74th position are bound to the copper atom involved in electron transport chain.The eukaryotic blue copper proteins chosen for the study were plantacyanin, plastocyanin, cucumber basic protein, stellacyanin, umecyanin, uclacyanin, and cusacyanin. The prokaryotic blue copper proteins were rusticyanin, sulfocyanin, halocyanin, azurin, pseudoazurin, auracyanin, amicyanin and blue nitrite reductase. Plastocyanins are found both in eukaryotes and prokaryotes. Table The number of sequences retrieved for a protein from different databases by keyword search are tabulated in Table The signatures already available for each of the blue copper proteins retrieved from the Prosite motif database are listed in Table As shown in 'Appendix 1 see ' the newThe members of a protein family can be identified by collecting the matching sequences to profile or motif databases. Protein signatures are sequence motifs diagnostic to a protein family indicating function. Signatures are matched to protein sequences in the non redundant databases and is scored using a dynamic programming algorithm which permits permeability in gap distance and residue type . GeneratBy doing a keyword search, we get varied results from the different databases as indicated in Table The protein signatures in a way can be compared to primers used for amplification. The more specific and concise a primer, the more specific is the amplification, similarly more specific the protein signature more significant are the picks from the non redundant databases. Specific signatures in a way reduce the time taken to pool related sequences from the abundantly available sequences from the non redundant databases. For example as shown in Table Signatures designed around the functionally important regions of a protein are valuable for annotation. In this study, specific signatures were designed around the active site regions of each of the blue copper proteins plantacyanin, plastocyanin, uclacyanin, stellacyanin, rusticyanin, sulfocyanin, amicyanin, halocyanin, pseudoazurin, azurin and nitrite reductase. These will be very useful for annotating uncharacterized proteins as blue copper proteins. Further, because of their high specificity to each subclass, they can be used in classifying the various subtypes of blue copper proteins.The blue copper proteins were distinguished based on the source of origin as prokaryotic and eukaryotic. The active site residues of the eukaryotic and prokaryotic blue copper proteins, which bind the copper metal atom, were identified from the Protein Data Bank and tabulated.The name of each of the blue copper protein was given as a query in the keyword searches at NCBI, SwissProt, TrEMBL, PIR, EMBL and PDB databases to check for the number of sequences retrieved from each database and the results were tabulated.The signatures already existing for each of these blue copper proteins were identified from the Prosite motif database and tabulated. These already existing signatures were used as query patterns in the PIR Motif/Peptide match and a search was made against the PIR-nREF database. The PIR-nREF database consists of sequences from the PIR , SwissProt , TrEMBL , GenPept , Ref Seq  and PDB . The number of protein sequences matching the query  was retrieved and the query results were tabulated.Each of the blue copper proteins from different sources were chosen from the different sequence databases and aligned using the multiple sequence alignment tool ClustalW. Conserved regions, which include the amino acids bound to the copper, reflecting the active site imparting a vital biological role of electron transport were chosen to design signatures. In regions showing 100% conserved sequences they were identified as conserved peptides. An example of how the functional protein signatures were designed is shown for plastocyanin in Appendix 1 see . The sigPG, AVG and SA participated in the design and coordination of the study. AVG carried out the bioinformatics search, the designing of the signatures and drafted the manuscript. PG and SA read and approved the final manuscript.Designing protein signatures: Illustrated example Plastocyanin. Plastocyanin sequences of eukaryotic and prokaryotic origin were retrieved from the PDB and SwissProt databases. The eukaryotic sequences were subjected to a ClustalW multiple sequence alignment. Signatures were designed based on the conserved pattern around the active site region [copper binding to four amino acids in plastocyanin]. The same procedure was adopted for plastocyanin sequences of prokaryotic origin. The newly designed signatures were used as queries in the Pattern/peptide match search at the PIR database [Protein Information Resource]. The numbers of plastocyanin sequences retrieved are tabulated in Table 6. The results were compared with the already existing signatures for plastocyanins and the number of sequences that these signatures picked up from the PIR database [data shown in Table 4 & 5].Click here for file


E. sakazakii is considered to be an opportunistic pathogen, implicated in food borne diseases causing meningitis or enteritis especially in neonates and infants. Cultural standard identification procedures for E. sakazakii include the observation of yellow pigmentation of colonies and a positive glucosidase activity. Up to now, only one PCR system based on a single available 16S rRNA gene sequence has been published for E. sakazakii identification. However, in our hands a preliminary evaluation of this system to a number of target and non-target strains showed significant specificity problems of this system. In this study full-length 16S rRNA genes of thirteen E. sakazakii strains from food, environment and human origin as well as the type strain ATCC 51329 were sequenced. Based on this sequence data a new specific PCR system for E. sakazakii was developed and evaluated.E. sakazakii species. The newly developed 16S rRNA gene targeting PCR system allows identification of E. sakazakii strains from both lineages. The assay's ability to correctly identify different E. sakazakii isolates as well as to differentiate E. sakazakii from other closely related Enterobacteriaceae species and other microorganisms was shown on 75 target and non-target strains.By phylogenetic analysis of the new full-length 16S rRNA gene sequence data obtained we could show the presence of a second phylogenetic distinct lineage within the E. sakazakii isolates from both phylogenetic distinct lines within the E. sakazakii species. The impact of this second newly described phylogenetic line within the E. sakazakii species in view of clinical and food safety aspects need further investigation.By this study we are presenting a specific and reliable PCR identification system, which is able to correctly identify  E. sakazakii is considered to be an opportunistic pathogen, implicated in food borne diseases causing meningitis or enteritis especially in neonates and infants , was published up to now for akazakii . HoweverE. sakazakii strains from various sources  and of E. sakazakii type strain ATCC 51329 were determined. This sequence data has been deposited in the GenBank . Thereafter, a sequence comparison was performed between the 16S rRNA genes of the fourteen E. sakazakii strains and the E. sakazakii type strain ATCC 29544 by calculation of a distance matrix using the ARB programme. Thirteen E. sakazakii strains used in the study exhibited 99.4 to 100% sequence similarity to the E. sakazakii type strain ATCC 29544 [GenBank: AB004746]. Meanwhile similarity between strain ATCC 51329 and the strain ATCC 29544 was significantly lower (97.9%).As a starting point, the full-length 16S rRNA sequences of thirteen E. sakazakii species. In figure E. sakazakii type strain ATCC 29544, whereas the E. sakazakii strain ATCC 51329 forms a second branch within this group. The subtree was calculated using the TREEPUZZLE tool within the ARB package exhibiting a "consensus tree" from different calculation methods. The value on each branch is the percent occurrence of the branching order in 500 bootstrapped trees. The 92% bootstrap value for the E. sakazakii ATCC 51329 branch strongly supports the theory about the second lineage.Phylogenetic affiliation of the newly obtained sequences to tree_jul04_1450 comprising >28.000 almost full-length (>1449 nucleotides) 16S rRNA gene sequences revealed the presence of two phylogenetically distinct lineages within the E. sakazakii 16S rRNA gene . Within these regions substitutions ranging from two to seven bases are present in all strains investigated and are most pronounced in E. sakazakkii strain ATCC 51329, thus providing the bases for the formation of the second phylogenetic branch. In table E. sakazakii strains investigated in the study in relation to the E. sakazakii typestrain ATCC 29544, E. cloacae typestrain ATCC13047 and E. coli type strain ATCC 11775. Recently, a number of E. sakazakii partial sequences were deposited into the public database , E. sakazakii 1084 fruit powder isolate [GenBank: AY803192], E. sakazakii 954 fruit powder isolate [GenBank: AY752938], E. sakazakii 858 fruit powder isolate [GenBank: AY752936], E. sakazakii 759 fruit powder isolate [GenBank: AY752939], E. sakazakii 236 fruit powder isolate [GenBank: AY752943], E. sakazakii FSM393 baby food isolate [GenBank: AY752941], E. sakazakii FSM33 milk isolate [GenBank: AY752940], E. sakazakii 265 milk powder isolate [GenBank: AY803191], E. sakazakii FSM468 production environment isolate [GenBank: AY752942], E. sakazakii 266 production environment isolate [GenBank: AY803190], E. sakazakii ES4 human isolate [GenBank: AY803186], E. sakazakii ES11 human isolate [GenBank: AY803187], E. sakazakii ES Vo7/24922 human isolate [GenBank: AY803189].AL and TT carried out the experimental part of the study. RS carried out the conception of the study. All authors participated in production and approval of the manuscript.


Patients hospitalized with community acquired pneumonia (CAP) have a substantial risk of death, but there is evidence that adherence to certain processes of care, including antibiotic administration within 8 hours, can decrease this risk. Although national mortality data shows blacks have a substantially increased odds of death due to pneumonia as compared to whites previous studies of short-term mortality have found decreased mortality for blacks. Therefore we examined pneumonia-related processes of care and short-term mortality in a population of patients hospitalized with CAP.We reviewed the records of all identified Medicare beneficiaries hospitalized for pneumonia between 10/1/1998 and 9/30/1999 at one of 101 Pennsylvania hospitals, and randomly selected 60 patients at each hospital for inclusion. We reviewed the medical records to gather process measures of quality, pneumonia severity and demographics. We used Medicare administrative data to identify 30-day mortality. Because only a small proportion of the study population was black, we included all 240 black patients and randomly selected 720 white patients matched on age and gender. We performed a resampling of the white patients 10 times.Males were 43% of the cohort, and the median age was 76 years. After controlling for potential confounders, blacks were less likely to receive antibiotics within 8 hours , but were as likely as whites to have blood cultures obtained prior to receiving antibiotics , to have oxygenation assessed within 24 hours of presentation , and to receive guideline concordant antibiotics . Black patients had a trend towards decreased 30-day mortality .Although blacks were less likely to receive optimal care, our findings are consistent with other studies that suggest better risk-adjusted survival among blacks than among whites. Further study is needed to determine why this is the case. Pneumonia (with influenza) is the leading infectious disease cause of death in the United States and the sixth leading cause of death overall  AccordinAs part of the Pneumonia Medical Quality Improvement Study (MQIS) a national expert panel established a set of process of care measures for patients hospitalized with CAP  Several The aims of this paper are to 1) examine whether there are significant racial differences in the processes of care that have been associated with mortality for patients hospitalized with CAP, and 2) to examine the relative risks of death within 30-days for blacks versus whites.KePRO, the Medicare Peer Review Organization for Pennsylvania, obtained these data as part of the Pneumonia MQIS project, whose goals is to assess and improve the quality of care for Medicare patients hospitalized with CAP.The study population was Medicare fee-for-service inpatients hospitalized at participating hospitals in Pennsylvania between 10/1/1998 and 9/30/1999. Inclusion criteria included having a primary ICD-9 diagnosis of pneumonia , or a primary diagnosis of respiratory failure (518.81) or sepsis (038. XX) with a secondary diagnosis of pneumonia. Only the first qualifying discharge was considered for each patient.Among the 204 hospitals functioning in PA during the study period, 101 agreed to participate in this study. For each hospital, a random sample of up to 60 discharges with qualifying ICD-9 codes was selected. For hospitals with fewer than 60 qualifying discharges, all charts were selected. In most cases, chart review data was collected by trained record abstractors either on site from the original record, or from photocopies sent to the offices of the Quality Improvement Organization. In two cases, data were collected by the hospital's own staff using an approved QIO data collection instrument (n = 2).Patients were excluded if they had no working diagnosis of pneumonia on admission or received care limited to comfort measures, left the hospital "against medical advice", or were transferred from another acute care hospital. Patients whose race was not white or black were also excluded.Chart review data included demographics, comorbid conditions, physical exam findings, laboratory data, and chest radiograph information. In addition, data on important processes of care for patients hospitalized with CAP were obtained by chart abstraction. These processes of care included: first antibiotics within 8 hours of admission, collection of blood cultures prior to antibiotic administration, oxygen saturation measurement within 24 hours of presentation, and concordance of antibiotic therapy with national guidelines. After initial training the abstractors performed data collection on charts were assessed using gold-standard cases that had been previously evaluated by multiple expert abstractors. If the abstractors did not achieve 95% accuracy, they underwent further training until they had an error rate of less than 5%. In addition, 10% of charts were reabstracted during the review process to monitor the accuracy of chart review. The error rate for these reabstracted charts remained less than 5%.The pneumonia severity index (PSI) was used to assess severity of illness at presentation  The PSI Due to the relatively small number of black patients in the cohort (n = 240) we performed a modified resampling procedure of the white cohort with matching to the black patients on age and gender  We incluUnivariate statistics were used to compare sociodemographic and clinical characteristics between white and blacks patients. Categorical variables were analyzed using the Chi-square test and continuous variables were analyzed using Student's t-test.Separate discrete conditional logistic regression models were estimated for each of the individual process of care measures, and for 30-day mortality  The PSI Of 4889 charts requested, the complete medical record was available for 4823. Of these, 4034 patients, 240 of whom were black, were eligible for inclusion in the study. Patients were excluded because they were neither white or black (N = 231) or because they had no working diagnosis of pneumonia on admission (n = 413), their care was restricted to comfort measures (n = 173), they were transferred from another acute care facility (n = 37), or they left "against medical advice" (n = 14).For each of the ten resamplings 720 white patients were sampled and matched to the 240 black patients based upon the age and gender as previously discussed. The clinical and demographic characteristics of the study population are presented in Table In univariate analysis mortality at 30-days was 7.8% for whites and 5.8% for blacks (p = 0.3), and 82.1% of whites received antibiotics within 8 hours as compared to 75.7% of blacks (p = 0.04). Regarding blood culture performance, 96.4% of white and 97.1% of blacks had blood cultures obtained within 24 hours, and 84.8% of whites and 77.8% of blacks had blood cultures obtained prior to antibiotics (p = 0.03). Oxygenation saturation was assessed within 4 hours of 88.9% of whites and 93.9% of blacks (p = 0.03).Figures In the regression models, after adjusting for severity of illness with the PSI, black patients were significantly less likely to receive antibiotics within 8 hours with an odds ratio (OR) of 0.63 and 95% confidence interval (CI) of 0.41 to 0.97. Black patients also had a trend towards decreased all-cause mortality at 30-day with an OR of 0.4 and 95% CI of 0.16 to 1.0. There were no significant differences between whites and blacks in regards to obtaining blood cultures prior to antibiotics , oxygenation assessment within 24 hours , or use of guideline concordant antibiotics .This study found significant racial differences in an important process of care for patients with CAP, specifically time to antibiotic administration. Our results support the previous studies of racial variation in pneumonia care which demonstrated racial variations in care for patients hospitalized with community-acquired pneumonia. -13Our study also suggests that these variations may have clinically important outcomes since the process measures used to assess quality of care in this study have been previously associated with increased 30-day mortality. -9Our study is also consistent with previous studies which found that blacks hospitalized with CAP have lower short-term mortality rates as compared to do whites ,6 It is Racial variations in CAP are important to assess since unlike coronary artery bypass surgery, hemodialysis, and many other conditions that have been studied, the inpatient treatment of CAP is largely outside of the control of the patient. Although the patient has input into being admitted to the hospital, after that point the patient has little input into the processes of care such as choice and timing of antibiotics, diagnostic testing or location of care. This has several advantages in studies where researchers seek to determine if racial differences in care reflect patient preferences, provider decisions or some negotiation between them.There are several possible explanations for our findings of racial variations in these processes of care. Besides the obvious conclusion that there may be biases that affect care there are several other possible factors that may be responsible that we are not able to examine. One possible factor is that there are geographic or other factors that results in blacks presenting for admission at hospitals with overall lower quality of care for patients with CAP. Although we were not able to control for this factor other studies have suggested that the reverse is usually true. ,13 That To attempt to adjust for imbalances between black and white patients we used a modified resampling technique to generate 10 samples of white patients, which were matched to the black population. We then pooled these results over the 10 samples. This approach allowed us to obtain a more robust estimate of the effect racial variation may have on mortality and processes of care then would be obtained from a single random matched sample  InterestThere are several limitations that should be acknowledged. First we did not have information on the physicians, hospitals, or the geographic locations of the providers so we were not able to adjust for clustering. Second our study was limited to Medicare patients hospitalized in Pennsylvania. It will also be important to examine whether patients with other types of insurance, such as Medicaid and managed care, and from other states have similar outcomes. In addition we were also unable to assess the robustness of our analysis using traditional techniques such as model cross validation on a new independent sample, or by randomly subdividing our current sample into a training and test samples, due to our small sample size. However we do have quasi-replication, at least in the white sample, by the multiple sampling that we performed. Finally we were unable to adjust for potential bias in pulse oximetry since this is a retrospective study. However recent work ,19 questDespite these limitations, we believe our results, and the results of other studies, allow us to conclude that blacks are less likely than whites to have processes of care that are considered to represent superior quality of CAP care. Despite this, blacks with CAP have similar, and perhaps lower mortality than whites. Further research should both investigate how to make sure all patients receive optimal CAP care, and identify the factors responsible for the paradoxical advantage in survival that is seen among blacks.None declared.EM and JW conceived the study. EM and JC were responsible for the analysis. EM was responsible for the initial draft of the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Amborella, as comprising the basal-most grade of all other angiosperms. A major exception is the recent study by Goremykin et al. , whose analyses of 61 genes from 13 sequenced chloroplast genomes of land plants nearly always found 100% support for monocots as the deepest angiosperms relative to Amborella, Calycanthus, and eudicots. We hypothesized that this conflict reflects a misrooting of angiosperms resulting from inadequate taxon sampling, inappropriate phylogenetic methodology, and rapid evolution in the grass lineage used to represent monocots.Numerous studies, using in aggregate some 28 genes, have achieved a consensus in recognizing three groups of plants, including Acorus and added these plus previously sequenced Acorus genes to the Goremykin et al. (2003) dataset in order to explore the effects of altered monocot sampling under the same analytical conditions used in their study. With Acorus alone representing monocots, strongly supported Amborella-sister trees were obtained in all maximum likelihood and parsimony analyses, and in some distance-based analyses. Trees with both Acorus and grasses gave either a well-supported Amborella-sister topology or else a highly unlikely topology with 100% support for grasses-sister and paraphyly of monocots . Second, we reanalyzed the Goremykin et al. (2003) dataset focusing on methods designed to account for rate heterogeneity. These analyses supported an Amborella-sister hypothesis, with bootstrap support values often conflicting strongly with cognate analyses performed without allowing for rate heterogeneity. In addition, we carried out a limited set of analyses that included the chloroplast genome of Nymphaea, whose position as a basal angiosperm was also, and very recently, challenged.We used two main approaches to test this hypothesis. First, we sequenced a large number of chloroplast genes from the monocot Amborella (or Amborella plus Nymphaea), but not monocots, is the sister group of all other angiosperms among this limited set of taxa and that the grasses-sister topology is a long-branch-attraction artifact leading to incorrect rooting of angiosperms. These results highlight the danger of having lots of characters but too few and, especially, molecularly divergent taxa, a situation long recognized as potentially producing strongly misleading molecular trees. They also emphasize the importance in phylogenetic analysis of using appropriate evolutionary models.These analyses show that  Amborella) are themselves the "earliest" angiosperms, but rather that they belong to the lineage of angiosperms that resulted from the first evolutionary split in angiosperm evolution. When the term "sister" is used to refer to a phylogenetic placement it refers to the sister group to the rest of the angiosperms unless otherwise specified.] A breakthrough in the seemingly intractable problem of identifying the earliest lineages of angiosperms occurred in 1999 and 2000, when each of many multigene studies identified the same three groups as the earliest branching angiosperms . The BSses Fig. . The SH-Amborella and Calycanthus is identical  with that for grasses-sister across the entire range of both α and PInvar values, while both of these BS values always equal 100 minus the BS value for Amborella-sister  when constant sites are removed . The 40 Acorus genes used here come from two closely related species – A. calamus (14 genes) and A. gramineus (26 genes) – and correspond to 65.6% (40/61) of the genes and 71.4%  of the nucleotide characters analyzed by Goremykin et al. . A. ML HKY85 and equal rates. B. NJ with distances calculated using an ML HKY85 model and equal rates.Click here for fileNJ analysis using ML proportion of invariant distances. Distances were calculated using the ML HKY85 model, the estimated proportion of invariant sites, and the first- and second-position matrix of Goremykin et al. [19].Click here for fileML trees using third positions only. A. HKY85 model with equal rates. B. HKY85 model with four gamma-distributed rates.Click here for fileAcorus Sister group to the rest of angiosperms found in individual gene analyses using first- and second-position data without Top, ML HKY85 with four gamma-distributed rates. Bottom, Parsimony analysis.Click here for fileAcorus addedSister group to the rest of angiosperms found in individual gene analyses using the ML HKY85 model with four gamma-distributed rates and . Top, all three positions. Bottom, first and second positions.Click here for fileAcorus added and grasses removedSister group to the rest of angiosperms found in individual gene analyses using the ML HKY85 model with four gamma-distributed rates with . Top, all three positions. Bottom, first and second positions.Click here for fileSister group to the rest of angiosperms found in individual gene analyses using parsimony on all three positions. Top, Acorus added. Bottom, Acorus added and grasses excluded.Click here for file


Schistosoma mansoni infected subjects can reduce bleeding, thereby alleviating the pathology caused by schistosomiasis.Management of patients with bleeding oesophageal varices comprises of mainly diagnostic endoscopy, sclerotherapy and band ligation. One of the major problems to do any of the above is the active bleeding which makes any intervention difficult. The neuropeptide hormone somatostatin administered exogenously has caused a reduction in portal hypertension and variceal bleeding in patients suffering from liver cirrhosis. We believe that the symptomatic use of somatostatin for variceal bleeding in Schistosoma mansoni with bleeding from oesophageal varices in the last 24 hours. One group of schistosomiasis patients will be treated with somatostatin and praziquantel, the other with propanolol and praziquantel. Survival graphs will be set up to correlate somatostatin administration with survival time. A two part questionnaire will be set up to control treatment outcomes. The pre-treatment part of the clinical questionnaire will identify inclusion criteria questions, the post-treatment part of the questionnaire will identify treatment outcomes.We herein present a study protocol for establishing this neuropeptide as a potential therapeutic agent in schistosomiasis. Adolescent subjects, age range varying from 12–17 years will be selected, based on several inclusion criteria, most important being infection with We expect that the administration of somatostatin as a bolus followed by a 24 hour long infusion, will stop bleeding immediately, delay rebleeding as compared to the control study group and delay mortality in the somatostatin treated subjects. S. mansoni infected patients. In such patients portal hypertension leads to the formation of portal-systemic collaterals, specifically gastro-oesophageal collaterals (varices). Bleeding of these oesophageal varices can occur depending on the severity of fibrosis, and can be fatal. Praziquantel, the most commonly used anti-schistosomal drug, is effective against the worm stages of the parasite, but has no activity against the pathology (fibrosis) caused by the egg stages or the variceal bleeding that can be fatal in its outcome. These observations have stressed the need to combine praziquantel treatment with symptomatic treatment like with somatostatin.Complications due to severe schistosomiasis include fibrosis, hepatomegaly, splenomegaly, haematemesis, varices, portal hypertension, ascites formation and death . ComplicSomatostatin is emerging as the ideal vasoactive drug for the control of variceal bleeding, and is as effective as sclerotherapy -4. In a Schistosoma caused morbidity. In endemic regions, at any given time, only a fraction of infected patients develop severe hepatic fibrosis. There may be a direct correlation between the development of severe fibrosis and the inability to generate required somatostatin levels. Our ongoing research at the Laboratory of Pathology give evidence to this fact, since somatostatin levels in Senegalese patients with severe morbidity  are significantly lower than that in patients with no severe morbidity [We have studied the potential role of somatostatin in modulating orbidity -10. ThisComplications linked to hepato-intestinal schistosomiasis are increasing in the area of Richard-Toll, Northern Senegal. These cases are being identified at the local health centre. However, considering the high prevalence of schistosomiasis in this region, it is likely that the number of severe cases is much higher. Clear guidelines for the management of such severe complications and criteria for referral and surgery are required in this region. The establishment of an algorithm on how to treat these patients and create the appropriate infrastructure is urgently needed. The priority is how to take care of these patients and more so what is the best way of doing this under local conditions. One of the first steps is to critically evaluate the Niamey-Belo-Horizonte ultrasound methodology in patients with severe periportal fibrosis. Efficient management of bleeding varices in the afflicted patients is imperative.Schistosoma mansoni infection. Moreover using this neuropeptide may increase time to failure of drug treatment, decrease incidences of early re-bleeding  and incidences of death during the follow up period. Decreased frequencies of late rebleeding  may occur, all indicating the safety of using somatostatin. Praziquantel cover would be given to all study patients.Given the background that somatostatin is an ideal vasoactive drug in the field of liver pathology, it is our opinion that somatostatin will be more efficacious and safe as compared to currently used beta blocker drugs like propanolol, in the control of acute oesophageal variceal bleeding due to Age and Morbidity criteria – Adolescent subjects, age range varying from 12–17 years will be selected. The inclusion criterion will be schistosomiasis patients with bleeding from oesophageal varices in the last 24 hours. A random selection will be made to form two groups, a study group and a control group. Control of active infection will be done by means of CAA-strips on urine or blood. Subjects will be asked to fill in an informed consent form and the pre-treatment part of a questionnaire.The inclusion criteria will be established fibrosis due to schistosomiasis of clinical history, physical examination and laboratory findings . Clinically active upper gastrointestinal bleeding (haematemesis of fresh or semi fresh blood and/or melena and/or haematochezia) with or without haemodynamic instability (systolic blood pressure < 80 mm Hg and heart rate > 120 bpm) will be selected. Subjects must be male or non-pregnant, non-lactating female subjects. Females of childbearing potential will have to utilize contraception for the duration of the study. Written or verbal documented informed consent will be needed from all subjects.Exclusion criteria will include participation by subjects in another investigational study within the last 14 days. Subjects may not undergo treatment with endotherapy, i.e. band ligation, sclerotherapy or other . Treatment with somatostatin, vasopressin or their analogues will also be a exclusion criteria. Subjects with end stage liver disease with hepatorenal syndrome, diffuse hepatocellular carcinoma, patent porto-systemic shunts, known diagnosis of non-fibrotic portal hypertension, severe cardiovascular diseases, i.e. acute myocardial infarction and heart failure will be excluded. Concurrent use of metoclopramide is also not advised.Conduct of trial – Active bleeding episode  from a potential variceal source should be confimed by a medical team . Patients may be outpatients or already hospitalised patients. Patients will be randomised to either arm in a sequential manner. Randomisation, and the start of study drug infusion if in adjunctive therapy arm, should be accomplished as soon as possible following identification of a patient qualifying for the study and following the conduct of pre-randomisation study procedures.Sample Size Criteria – Assuming a 80% chance of finding a significant difference <0.05 between the two study cohorts, the following statistics were established:(A) If 99% of the untreated subjects and –10% of somatostatin treated subjects bled -5 volunteers per group were sufficient;20% treated subjects bled – 7 volunteers per group were required;30% treated subjects bled-9 volunteers per group were needed.(B) If 90% of untreated subjects bled, and –10% of somatostatin treated group bled – 7 volunteers in each cohort were sufficient;20% of somatostatin treated group bled – 9 volunteers in each group were required;30% of somatostatin treated group bled – 10 volunteers in each group were needed.(C) If 70% of untreated subjects bled, and:10% of somatostatin treated group bled – 12 volunteers per group were sufficient;20% of somatostatin treated group bled – 18 volunteers per group were required;30% of somatostatin treated group bled – 28 volunteers per group were needed.In cirrhotic patients with bleeding oesophageal varices somatostatin administration controls bleeding in more than 80% of the treated patients. Based on this report, we propose to start this pilot study with 10 subjects/group.Two groups of 10 schistosomiasis patients each will be identified. Group (1) will be treated with Somatostatin  + Praziquantel (40 mg/kg). For somatostatin treatment the i.v. infusion will be started first; 3 mg somatostatin will be dissolved in the 1 ml of saline provided. This solution will be added to the saline transfusion unit and administered to the patient for the next 12 hours. Once finished the second packet of 3 mg somatostatin will be used similarly for a second saline transfusion unit for the remaining 12 hours. The bolus dose of 250 μg will be dissolved in the 1 ml of saline provided and administered over 90 seconds soon after the start of the i.v. infusion.Group (2) will be treated with Propanolol + Praziquantel.Survival graphs will be set up to correlate somatostatin administration with survival time.OR presence of a 20 mmHg drop in systolic blood pressure from the highest post resuscitation value AND achieving a heart rate of 120 bpm OR a 20 bpm increase from highest post resuscitation value OR Inability to achieve and maintain a Hct of – 27% of Hb of – 9 g/dl despite blood transfusion of 2 units or more.The primary efficacy variable is the number of patients meeting the failure of therapy definition during the infusion period. Failure criteria are defined as death during infusion, persistence of active bleeding , hematochezia, melena.A two part questionnaire will be set up to control treatment outcomes. The pre-treatment part of the clinical questionnaire will identify inclusion criteria questions:(1) When was the last haematemesis incidence?(2) When did the present incident start?The post-treatment part of the questionnaire will identify treatment outcomes. The following questions will be answered:(1) What was the reaction to somatostatin infusion?(2) When did bleeding stop after somatostatin infusion?(3) GI disturbances: Is there abdominal pain, nausea, diarrhea, after somatostatin infusion?(4) Control of early rebleeding: Is there early rebleeding in the 8 days following somatostatin administration?(5) Control of late rebleeding: When is the next rebleeding incident? Subjects will be followed up on a monthly basis, by home visits.(6) Control of mortality: Is there any difference in mortality time between the two groups.(7) Control of fibrosis: Does ultrasonography detect anti-fibrotic effect of somatostatin after treatment?It is expected that the administration of somatostatin as a bolus followed by a 24 hour long infusion, will stop bleeding immediately, delay rebleeding as compared to the control study group and delay mortality in the somatostatin treated subjects. Our protocol that is based on a pilot study will help to establish the importance of somatostatin in schistosomiasis.The author(s) declare that they have no competing interests.Both authors participated in the design of the study.The pre-publication history for this paper can be accessed here: