Stochastic fluctuations in the protein turnover underlie the random emergence of neural precursor cells from initially homogenous cell population. If stochastic alteration of the levels in signal transduction networks is sufficient to spontaneously alter a phenotype, can it cause a sporadic chronic disease as well – including cancer?Expression in >80 disease-free tissue environments was measured using Affymetrix microarray platform comprising 54675 probe-sets. Steps were taken to suppress the technical noise inherent to microarray experiment. Next, the integrated expression and expression variability data were aligned with the mechanistic data covering major human chronic diseases.Measured as class average, variability of expression of disease associated genes measured in health was higher than variability of random genes for all chronic pathologies. Anti-cancer FDA approved targets were displaying much higher variability as a class compared to random genes. Same held for magnitude of gene expression. The genes known to participate in multiple chronic disorders demonstrated the highest variability. Disease-related gene categories displayed on average more intricate regulation of biological function vs random reference, were enriched in adaptive and transient functions as well as positive feedback relationships.A possible causative link can be suggested between normal  state gene expression variation and inception of major human pathologies, including cancer. Study of variability profiles may lead to novel diagnostic methods, therapies and better drug target prioritization. The results of the study suggest the need to advance personalized therapy development. The studies of gene expression variability started relatively lately, with the advent of high-throughput technologies of analysis A study was published comparing non-disease and disease state, detecting de-regulation, as a signature of disease mechanism While providing a link between expression variability and disease, the prior publications appear to be confined to particular diseases  and certain genes, thus they do not provide a global view of the possible connection between normal gene expression variance and mechanism of subsequent sporadic disease emergence. By contrast, this work presents a genomic scale study into all major chronic diseases, including aging and such a scope may be of interest.Since variability was measured in normal state, its link to the propensity of a gene to become a successful target is significant. Typically, participation in the essential mechanism of pathogenesis establishes a gene as a target. On the other hand, the elevated expression variation was measured prior to development of a disease – hence it may be causative to the subsequent pathogenesis. A concern exists that the extent of clinical development may not objectively reflect the extent of mechanistic participation of a gene but may be distorted by other factors, such as market niche, the historical duration of study, dominant opinions in the field etc. To ensure that the extent of variability indeed parallels the objective extent of mechanistic participation, the variability data were aligned with differential expression consistency and metric of tissue-specific expression. Prior works show that differential expression consistency is an objective metric providing significant enrichment in the FDA-approved and proposed anti-cancer targets N, MAXc/VULNERABLES the level of variability in the norm was by far the highest. At the same time, this group of genes was strongly enriched in FDA-approved targets and proposed target candidates, such as MAGE , MS4A1, REG4, MSLN, IL1A, ENPEP, TYR, RARA, FCLRA. The data by To ensure that these observations are not specific for cancer alone, similar analysis was conducted for other classes of disease-related genes, see A possibility exists that the differences between random control and disease-associated genes are not objective, but arise accidentally due to a particular composition of the integrated panel. To rule this possibility out, multiple (8) sub-panel compositions were generated by random bootstrapping and in each composition random genes were compared with therapeutic target genes (P3.3). The difference between the groups under comparison exceeds the relevant confidence intervals.A hypothesis was advanced that the elevated variability in disease-related categories may be a function of higher expression also observed in these categories. To test this possibility, variability as a function of copy-number was plotted  using muStill another possibility of an artifact resides in the fact that a very sensitive measure was employed as a variation criterion. A propagated error, associated with a ratio of outliers can be very significant and an additional test is needed to evaluate its neutralization by aggregation of multiple datasets in a panel. Coefficient of Variation (CV) was chosen as a less sensitive, but more reliable alternative metric, taking into account the scattering behavior of the entire population of N values in a project. The Per processing as discussed in The FENR values were assembled in the panel, with two sub-profiles in every populated functional category, one for random negative control and another for the diseases being grouped together. Such grouping allowed exploration of the features generic to all chronic disorders using the rationale presented in According to the plotting, top-ranking disease-related functional categories respond to the keyword “regulation” twice as frequently as the total population of categories and four fold more frequently if compared with lowest-ranking categories (representing random gene population). More surprising, however, was the finding that positive regulation is much more prominent among top-ranking disease-related categories as compared to the lowest ranking categories or total list of categories. The total population of categories appears to display approximate balance between positive and negative regulation, according to our analysis. This balance between positive and negative regulation appears to shift in favor of positive regulation in the categories most associated with disease and this observation suggests some fundamental biological role.Practical applications of the current project were explored below. The trend observed for non-cancer targets appears to be the opposite: the candidate genes  appear tE.coli gene expression  and found to be absent. Assuming validity of these findings, the above trend was observed in healthy state and a causal link to inception of pathology may be hypothesized (hypothesis 1). An alternative (hypothesis 2) calls for elevated variability to be a hallmark of a gene's disease-relatedness, but no direct role in pathological mechanism can be attributed.The references 5, 6, 8, 10–12, 21 support the hypothesis 1, pointing to a possibility of disease inception due to dramatic positive or negative variation in expression of a single gene. Indeed, decrease in a single gene expression level due to haplo-insufficiency of tumor suppressors is carcinogenic. Conversely, engineered over-expression of a signal protein triggers cancer in normal skin Indeed, increased cell-to-cell variation of a given gene expression would indicate loose regulatory control and the possibility that the given gene expression may reach extreme values, high or low. Such extreme values are argued in this report to be the “flashpoints” of sporadic disease. In some types of pathology  only a single “extreme” cell may be sufficient to cause systemic effect. In other pathologies  a critical weight of deregulated population has to be reached. In all cases, increased variability in expression levels would facilitate reaching of the critical “triggering” parameters, thus its direct measurement may have diagnostic and prognostic value. A combination of instabilities in regulation of expression of several mechanistically important genes may exert effects comparable to mutations. Currently, the classifiers of disease etiology and prognosis utilize SNP, microarray, proteomic and metabolomic data aligning particular pattern signatures with clinical correlates Variability studies performed over single monocytes of a peripheral blood sample from the given patient and covering the most informative high-variability gene subset may bring an additional dimension to genetic marker methodology. Thus, variametric component should enhance genetic polymorphism analysis regarding predisposition to chronic disease, diagnostics of congenial disorders, longevity, personalized diet and therapy. On the technical side, multiple expression levels in single cells comprising a sample can be measured by the novel methods of single cell arrays, phosphocytometry and cellomics Our data facilitate understanding of limitations existing in cancer therapy and also suggest novel therapeutic possibilities. For example, we demonstrate , 3, 8 thThe effect is especially striking for tissue-specific anti-cancer targets that are in the current focus of research relying on differential expression .We have conducted a genomic-scale differential expression study. The latter comprised 40 pairs of normal versus cancer Affymetrix array datasets, covering most of tissue environments in a single computational space. The goal was to observe cancer-specific hyper-expression absent in the entire panel of norm. The initial hypothesis stated that such hyper-expression would be a reliable basis for high quality target candidates. Preferably, such hyper-expression should have been tissue-specific. Surprisingly, we observed that such target candidates display the highest variability in all categories studied in this report . Based opersonalized target visualization approaches taking into account increased target variability described in this report. Some genes – such as metalloprotease MMP12 – display very favorable variation profile, being almost uniformly over-expressed in cancer and almost absent in norm. Conjugating visualizing and therapeutic moieties to MMP12 ligands may be promising. Similar use of other MMP ligands can be considered. Attachment of colloid gold nanoparticles to such ligands would enable selective gold build-up in tumor sites with subsequent enhancement of therapeutic X-ray absorption. The long-term cancer survival rate in the presence of such gold nano-particles during a systemic radiotherapy comprises 86% against only 20% with X-ray alone in mice We show (based on reliability theory) that the survival probability would be impacted by these fluctuation factors in the most dramatic manner. Consequently, we emphasize The proposed therapy was described as an example of approaches suggested by the results of our work. Currently, significant investment in time and funds is consumed by the study of molecular signaling associated with cancer targets. At the same time, especially high expression variation associated with such targets questions uniformity of their presence in malignant clonal population and the significance of using blockers against such targets. However, using over-expressing clones as attachment sites for selective delivery of radioactivity appears to be bypassing these difficulties. Absorption and scattering of radiation by such attachment sites would create “killing zones”, encompassing the malignant clones that insufficiently express a particular target and do not depend on it for survival.The high cost of new therapeutics against chronic disease drains the resources of society by forcing higher health care spending and by detracting from the vital task of anti-infective development. In the context of SARS outbreak and our new knowledge of pandemic flu genesis it becomes imperative to produce computational signatures: of the anti-infective targets, of the successful targets against chronic disease, and of the ligands capable of being viable leads. Apparently, lagging in these technologies opens vulnerabilities at global scale, considering the issues of microbial drug resistance, bioengineering and bio-terror.a-priori in large integrated datasets, based on the gene's expression behavior. In the current report we attempted to rationalize this link.Based on the findings of this report, we rationalized and advanced the criteria of target prioritization, previously published in are statistically more error-prone and thus the link between variability and disease can be rationalized at mechanistic level. Namely, the most of variations occur in the expression levels of genes carrying out sophisticated regulatory functions.Ontological analysis reveals that the genes of high variability class may require more sophisticated orchestration of their functions (P4.13-P4.14). At the same time low variability genes  appear to be regulated in a more static (conservative?) manner. Such a result is in agreement with external variability model published in As an example, genes expressed only in a particular tissue lineage display more variable expression than the genes expressed systemically compare  and 3. TAt the same time, broad variations in the levels of the most upstream regulators are unlikely to be compatible with life - therefore distribution of variation across the tiers of significance should be optimized, by maximized population survival. One can observe these principles following the trends discovered in target success analysis . Indeed,The goal of anti-cancer therapy is to eliminate the cell population of interest and such therapies – in final reckoning – are cytotoxic. Using the uppermost significance tier of signal transducers for such purposes would endanger normal cells. The signal transducers of lower significance tiers may be more important for tumor than norm and a therapeutic window opens. However, at this level of significance the expression stability controls are less strict and the inherent increased expression variability of signal transducers prevails in this tier. As a result, the expression variability of successful anti-cancer targets exceeds the one for candidate genes  and 4. TE. coli, but only dacB  displays low expression variation. The target candidates in development appear to behave similar to random population in this regard and appear to be less evolutionally conserved  divergence, there is a “luxury” of inhibiting the most significant tier of functional elements in the prokaryotic cell with minimal risk to normal human tissues. Correspondingly, the prokaryotic targets display increased evolutional conservation and decreased expression variability as compared to random genes . Instruconserved  . Our reThe limited volume of a journal publication does not allow answering all numerous questions raised by our findings. First, there is a paradox: according to The answers to this and other questions are provided in Supporting material, www.mayburd.comThe Supporting data are available online at the link: The primary data and processing files in Excel format are designated by letter P. They comprise initial downloads (the folders P1), datasets covering tissue environments in norm  and in cancer . The file P2.4 comprises integrated panel of differential expression. The supporting materials further comprise assembled panel of variability , alignment of expression parameters (including variability) with target mechanistic data , the files P4 supports ontological analysis. Each Excel file is also described and annotated in its top left part.Supporting material in Word format is referred to by E. coli Antisense Genome Array platform .Large-scale microarray profiling of disease and norm as well as smaller scale datasets were downloaded from Global Expression Omnibus (GEO) platform at NCBI Aggregating of multiple microarray experiments by diverse authors poses unique challenges due to a significant component of technical noise, overlaid with biological variability. Several steps were taken to maximize the benefits of dataset aggregation in terms of signal-to-noise ratio.Low quality datasets were excluded from the analysis if they presented low levels of signal (that may indicate insufficient hybridization to the probes), evidence of missing genes, imputed data, datasets that are too small (<4 samples).The results pertaining to N samples identically processed were defined as “project”. The averages for each sample were computed among 54670 probe-set readings comprising all genes included in U133 Plus 2.0 microarray by Affymetrix . Each individual gene expression value in the column of 54670 probe-sets was normalized by that average. Variability was measured as a ratio of maximal and minimal outliers in the profile of N normalized samples obtained under identical conditions and representing the same tissue lineage. The ratios (MAX/MIN) were combined in a large-scale panel of 80 values per each gene, each value representing a dataset (project) component of a panel . Xi, Xm and σM all refer to ranked values of MAX/MIN. This procedure allows integrating experiments where levels of variability were very different and thus prevents skewing of the resulting panel data in favor of accidentally higher variability values . In each sub-panel variability was computed. The procedure produced two sets of 8 values for FDA-approved anti-cancer target variability and random gene variability. The reproducibility in the sets of bootstrap-generated values was assessed by plotting confidence intervals at α  = 0.05.The metrics comprised: a) coefficient of variation (CV) defined as the ratio of variance in the profile to the average of the profile b) kurtosis To compute gene expression levels, each dataset component of the integrating panel was normalized as described above (each sample divided by array average intensity). The paired panels of 31 matching cancer and normal datasets produced a profile of differential expression values for each probe-set. Those values that exceeded 3-fold up-regulation were preserved and the rest were replaced by zeros, to maximize signal-to-noise ratio. The resulting indexes of consistent up-regulation were computed for the panel of data .C is the maximal expression level in the panel of M2 normalized cancer environments; MAXN is the maximal expression level among M1 normalized disease-free tissue environments, AV is the average level in the norm (average of M1 experiments) and VULNERABLES is the average level measured in the sub-panel of normal tissues most often suffering from side effects of therapy. Cancer expression was characterized by ratios of MAXC/MAXN; MAXC/AV; MAXC/VULNERABLES. Simultaneously high ratios indicate a potentially cancer-specific expression level, only minimally expressed in norm. Such profiles were assumed to indicate potential target candidates, specific for a particular cancer lineage and minimally expressed in normal tissues .Microarray data were organized in gene expression panels, each composed of M experiments, each experiment comprising N samples. The expression data were normalized as described above and averaged for each experiment. Thus Normal Expression panel and Cancer Expression panel contained M1 and M2 averaged values each. Several criteria of tissue-specific expression were defined. The MAXThe disease-association status follows key-word querying of the database “Genes” at NCBI FENR is functional enrichment coefficient; Ci is population in the category of interest generated by a studied sub-set of genes; Pi is population in the studied sub-set of genes; Ct is population in the same category of interest generated by a total sub-set of genes; Pt is population in the total subset of genes. The FENR for high and low variability groups were compared. The FENR were also computed for individual diseases and FDA-approved target datasets. The values of FENR were organized in profiles, each functional category corresponding to N values for major human diseases.The genes comprising the datasets of study (∼54675 probe-sets) were ranked based on variability and the highest and lowest groups by rank were selected, ∼500 probe-sets in each. The classes were compared by GO-MINER methodology FENR arises accidentally and does not have a biological meaning, 12 randomly selected sets of genes of the size 500–1000 were processed by GO-MINER, establishing a negative control. These values of FENR were also organized in profiles per each functional GO-MINER category. The sub-profiles for random genes and diseases were compared using T-test and the resulting p-values were ranked. The most disease-associated functional categories were defined by difference between negative control FENR profiles vs. disease-related FENR profiles (p<10−11). With the T-test p-values being sorted in ascending order, this category forms the top 10% of a rank.To rule out the possibility that any given To produce the minimal p-value (the strongest T-test), the disease-related FENR profile has to display minimal scattering, thus the highest ranking belonged to the functional categories corresponding to the most generic features of chronic disease, equally displayed by all pathologies and absent in the negative random control. For illustration see P4, file “Analysis” in SM. The top-ranking and lowest-ranking functional categories, as well as the total list were text-mined with the keywords of interest and the data were plotted.Text S1Supporing text and in-depth presentation(0.61 MB DOC)Click here for additional data file.


We note the comments of Dr. Aaby Dr. Aaby and co-workers are concerned that the apparent reduction in severe adverse events (SAEs) in the RTS,S groups may represent an adverse consequence of the control vaccines (rabies and hepatitis B vaccines). To evaluate this hypothesis Dr. Aaaby and co-workers request: “we hope the authors will report the malaria and non-malaria serious adverse events and vaccine efficacy in the two trials separately for each sex” Many parents, the FDA, local regulatory authorities and IRBs, want children to benefit from a vaccine rather than a placebo where an appropriate choice for a control exists. Young children are at risk of rabies in East Africa, and the disease is fatal if acquired. Pre-exposure prophylaxis is not widespread in Kenya and Tanzania, but is of proven benefit In EPI (extended programme of immunisation) schedules children are routinely vaccinated against hepatitis B, and it would not have been acceptable to withhold this vaccination in the control group Dr. Aaby and co-workers have described non-specific deleterious effects of inactivated vaccines in Sub-Saharan Africa Efficacy against clinical malaria was 53% (95%CI 28–69) in all 5–17-month-old children Our phase II studies were not designed to examine inpatient morbidity in detail. Phase III studies to clarify these findings are underway. There seems little evidence of a non-specific effect by Dr. Aaby's proposed analysis, and control vaccinations will be used in accordance with regulatory authority and IRB advice.


Fifty-one patients with advanced non-small cell lung carcinoma were treated with a combination of mitomycin C, vinblastine and cis-platin (MVP). Most cycles were given on an out-patient basis. Major side effects were leukopenia and peripheral neurotoxicity; one patient died of sepsis while leukopenic. In 44 evaluable patients the response rate was 50%, with one complete response. Overall median survival time was 280 days and median duration of responses was 232 days. A better performance status, disease limited to one hemithorax and no prior exposure to chemotherapy positively influenced the survival. MVP is an effective chemotherapy for non-small cell lung cancer and further experience with this combination is warranted.


The differentiation of naive T and B cells into memory lymphocytes is essential for immunity to pathogens. Therapeutic manipulation of this cellular differentiation program could improve vaccine efficacy and the in vitro expansion of memory cells. However, chemical screens to identify compounds that induce memory differentiation have been limited by 1) the lack of reporter-gene or functional assays that can distinguish naive and memory-phenotype T cells at high throughput and 2) a suitable cell-line representative of naive T cells.Here, we describe a method for gene-expression based screening that allows primary naive and memory-phenotype lymphocytes to be discriminated based on complex genes signatures corresponding to these differentiation states. We used ligation-mediated amplification and a fluorescent, bead-based detection system to quantify simultaneously 55 transcripts representing naive and memory-phenotype signatures in purified populations of human T cells. The use of a multi-gene panel allowed better resolution than any constituent single gene. The method was precise, correlated well with Affymetrix microarray data, and could be easily scaled up for high-throughput.This method provides a generic solution for high-throughput differentiation screens in primary human T cells where no single-gene or functional assay is available. This screening platform will allow the identification of small molecules, genes or soluble factors that direct memory differentiation in naive human lymphocytes. After antigen encounter populations of naive lymphocytes cells differentiate through an effector state into memory cells that confer protective immunity to the host ,2. MemorHistorically, vaccines have been used to induce antigen-specific T and B cell memory and are highly effective against many pathogens . Howeverex-vivo expansion of antigen-specific T cells or as molecularly targeted vaccine adjuvants. Applying this approach to memory differentiation, however, is limited by significant obstacles. Differentiation screens rely on high-throughput assays that can detect the emergence of a more differentiated state. This is often achieved by the use of a reporter gene assay that monitors the expression level of a single differentiation marker gene [High-throughput small-molecule screens are being increasingly used as a tool to identify compounds that can direct cellular differentiation from a precursor cell into a more differentiated cell-type . This raker gene ,8. Reporker gene . Other sker gene , but theGenome-wide transcriptional profiling of naive, effector and memory T cells has demonstrated that extensive reprogramming of gene expression occurs during memory differentiation ,11. We hLigation-mediated amplification with bead-based detection has been described in detail previously -16. In bAs proof of principle, we tested whether we could use this approach to distinguish between naive human CD4 cells and memory-phenotype cells on the basis of a well-defined differentiation signature. To identify a differentiation signature that was characteristic of the memory state, we analyzed our previously-published gene expression profiles of human and mouse memory lymphocytes . In a twmemory-phenotype cells, rather than those with known functional properties of memory lymphocytes. Unequivocally identifying human T cells with the functional properties of memory – such as antigen-independent survival and the ability to self-renew – is not feasible; instead we chose a to study cells with a well recognized memory-phenotype as this population is presumably enriched for those cells that have memory function. The method of purifying populations of naive and memory-phenotype cells did not influence the expression levels in the signature, and comparison of results from cells flow-sorted or purified with MACS were highly correlated (data not shown). However, MACS purification allowed more rapid isolation of the number of cells needed for screening applications.CD4 T cells were chosen for these experiments because of the availability of reagents for magnetic isolation of large numbers of naive or memory-phenotype cells. It is important to note that for these proof of principle experiments, our test populations of lymphocytes were ACTB, TUBB, TUBG1, and HNRPAB) that showed low signal-to-noise ratios of expression level in Affymetrix data between naive and memory-phenotype CD4 T cells, and which spanned the range of expression levels seen in the transcripts of the differentiation signature. In subsequent analyses each gene in the differentiation signature was normalized to the mean of these four genes.Gene-specific primers were designed for each of the 55 genes in the differentiation signatures and for four control genes with which to normalize expression level data between replicate wells. Control genes were chosen  gave Z' factors above 0. Thus the use of a signature-based assay was superior to any single marker gene in distinguishing naive and memory-phenotype CD4 T cells.To quantify this difference, Z' factors were calculated for individual genes and for the summary score of expression values of all genes. The Z' factor is used to evaluate the ability of assays to detect true positive results in high-throughput screens . The theWe next considered how well the method recapitulated the changes in gene expression measured by Affymetrix microarrays. We compared the difference in gene expression levels between memory-phenotype and naive CD4 T cells measured by LMA/bead-based detection with the original Affymetrix microarray data shown in Figure We next evaluated the variability of the gene expression level detection across multiple replicates of naive and memory-phenotype CD4 T cells from two donors. Being able to perform screens involving large numbers of compounds would require pooling data from multiple donors. To identify the extent of donor-to-donor variability, we compared the expression levels of all genes in naive and memory-phenotype CD4 T cells in two donors Fig . The expTo function as a screening tool that can be scaled up to high-throughput, the assay should ideally be robust and precise, even when used at high-throughput with robotic fluid handling. We therefore performed the assay in large numbers of replications (~100 per cell type) in CD4 T cells obtained from a leukapheresis product from a healthy donor.After MACS purification of naive and memory-phenotype CD4 T cells, cells were plated into a 384 well plate. The cells were incubated for 18 hours (to simulate exposure to test compounds), followed by harvest, LMA, and bead detection using robotic fluid handling Fig . ReplicaThe ability to identify compounds that direct the cellular differentiation of naive T cells into memory cells could have significant therapeutic impact. However, the lack of reporter assays suitable for high-throughput that can accurately distinguish between discrete differentiation states in T cell development has limited this approach. Here we describe the application of a gene expression-based assay that detects complex, multigene signatures and distinguishes samples of naive and memory-phenotype CD4 T cells from human peripheral blood at high-throughput.Traditionally, reporter gene assays measure the transcriptional activity of a single gene that is characteristic of the differentiated state. However, in the case of memory lymphocyte differentiation, no single gene can accurately distinguish between naive and memory-phenotype cells. For instance, the naive markers CCR7 and CD62L are both re-expressed by central memory cells preventing these genes in isolation from being used to separate the two cellular states . Similarex vivo more closely recapitulates the cellular state in vivo than does the analysis of cell lines. Moreover, primary cells may be more sensitive to toxicities of compounds identified within a screen allowing earlier detection of potentially harmful drugs. Three factors suggest that using primary human T cells for memory differentiation screens will prove feasible. First, T cells are relatively abundant and can be accessed in normal volunteers with minimal morbidity. Second, selection of subsets within the peripheral T cell compartment is technically straightforward with a number of available cell separation techniques. Third, we found a remarkable degree of consistency in the relative expression level of genes in the differentiation signature between two different donors. This fact is of great practical significance as several different donors would be needed to contribute naive T cells to a screen for a high-complexity compound library. Further evaluation across a larger number of normal donors will be necessary to define rigorously how significant an obstacle donor-to-donor variability in gene-expression will be in the conduct of large screens spanning more than one donor. However, the lack of donor-to-donor variability in these initial studies suggests that analysis of pooled screening data from multiple donors may be possible.We developed this assay to allow screening to be done using primary human lymphocytes. The use of primary human cells as starting material is highly unusual for differentiation-based screens but offers significant advantages over cell lines. The analysis of primary cells in toto is highly correlated with the differentation state of functional memory cells. Never-the-less, future studies will be required to determine whether it is possible to elicit the complex gene expression profile representative of a given differentiation state without inducing its attendant functional properties. Second, the use of primary human cells is technically challenging. Like all primary cells we found that increasing time in culture altered the gene expression profiles, presumably due to the lack of input signals from the normal milieu found in vivo (data not shown). However, data from mouse models suggests that the lineage commitment step for naive T cells differentiating after antigen-encounter occurs within hours or days of initial TCR signaling [ex vivo effects are apparent in the differentiation signature.There are clearly caveats associated with a signature-based approach to HTS in primary cells. The signature of genes was defined empirically, and so the biological relevance of any of the genes in the signature to the differentiation states of interest is unknown. However, the differentiation signature used in this study is highly evolutionarily conserved, shared by memory differentiation in both T and B cell lineages, and disrupted in functionally exhausted T cells . This suignaling -22. Thisex-vivo expansion of antigen-specific T cells or improve the efficiency of current vaccination approaches. Although widescale differentiation of naive T cells into memory cells would be undesirable in a clinical situation, antigen-specificity on promoting memory differentiation could be achieved by co-administration with vaccines, by optimization of dose and/or timing, or by the use of ex-vivo treatment of antigen-specific T cells.Our data suggests that it is now possible to screen for compounds that induce memory differentiation in naive human T cells. Compounds identified in such a screen would have significant biological and therapeutic uses. First, as tool compounds, hits in a memory differentiation screen could afford new insights into the mechanisms underlying memory development. Second, as therapeutic agents, memory-differentiating drugs could be used to accelerate the The use of LMA with bead-based detection offers a generic solution for T cell differentiation-based screens where no single-gene or functional assay is available. The same experimental platform can be easily adapted to other screening applications by using a panel of genes specific to the biology of interest. For instance, screens to detect the differentiation of effector memory T cells into those with a central memory gene expression signature could be designed based on their different expression profiles. Screens to convert conventional CD4 T cells into regulatory T cells could be developed by using a transcriptional profile of genes differentially expressed between conventional CD4 T cells and Tregs ,24. SimiPeripheral blood samples were obtained from healthy volunteers who gave informed consent for research and were enrolled on a protocol approved by the DFCI IRB. Written informed consent was obtained from the subjects for publication of data in this report. A copy of the written consent is available for review by Editor-in-Chief of this journal. Peripheral blood mononuclear cells were purified by density centrifugation. Naive or effector/memory phenotype CD4 T cells were obtained either by negative selection over a magnetic column  or by flow sorting. For sorting, cells were stained with a cocktail of antibodies designed to exclude irrelevant lineages, Annexin V to exclude dead/dying cells, and CD4, CD45RA and CD62L to identify memory-phenotype and naive CD4 T cells as shown in Figure 2 for ~18 hours. After incubation, 40 uL of medium was removed from the wells with a Multimek robot (Beckman Coulter). Cells were lysed with the addition of 10 ul of TCL lysis buffer (Qiagen) for 20 minutes at room temperature. The lysate was transferred to a 384-well oligo-dT coated plated  and incubated for 1 hour at room temperature. Excess lysate was removed by briefly centrifuging inverted uncovered plates onto absorbent paper towel. Reverse transcription was carried out in a 5 uL reaction volume using MMLV reverse transcriptase at 37°C (Promega). After a 90 minute incubation, liquid was removed from the wells by inverted centrifugation of the plates. Gene-specific oligonucleotide probes  and antibiotics, and incubated at 37°C with 5% CO6) coupled to FlexMap barcode sequences that were complementary to barcode sequences contained in the gene-specific probe pairs. Coupling reactions were performed as previously described [Amplicon detection was carried out using xMAP Multi-Analyze COOH microspheres  in the corresponding well to minimize well-to-well variability. Filtering was performed to eliminate wells with aberrant control gene expression from further analysis, using the mean plus or minus two times the standard deviation of the control genes as a filtering threshold. Aggregate scores of all genes in the panel created a summed score of the indexed expression values of the memory and naive genes with a sign determined by the expected direction of expression in the two differentiation states. For some visualizations, normalization of the gene expression values were performed using a Z-score derived by subtracting the mean expression level of the gene in that class from a the raw gene expression value in a given well and then dividing the difference by the standard deviation of the gene's expression in that class.The median fluorescence intensity of PE corresponding to a gene-specific bead was used as a measure of the gene's expression level. Expression levels of each signature gene in the well were indexed to a mean of 4 control genes (WNH conceived and designed the experiments, analyzed the data and wrote the manuscript. JA, KB, KR, CH, and SN performed the experiments and acquired the data. KR analyzed the data. KS helped design the experiments, analyze the data and write the manuscript.Supplementary Table 1. Sequence information for gene-specific probes.Click here for file


The depth of photodynamic treatment-induced tissue necrosis was measured in a subcutaneous prostate cancer model in Copenhagen rats. A comparison between continuous wave irradiation and pulsed irradiation with the same average incident irradiance showed no statistically significant difference in the depth of necrosis at 48 h after irradiation. These results indicate that photosensitizer population-state changes are measurable in tissues and may provide a method for measuring triplet-state properties of photosensitizer in vivo, but for BPD-MA at clinically used concentrations these changes do not significantly affect the depth of photodynamically-induced tissue damage. © 1999 Cancer Research CampaignHigh intensity pulsed-laser light can be used to excite absorbing molecules to transient states in large proportions. The laser-induced spectral changes can be characterized by transient changes in light propagation; through the tissue provided the excited states of these molecules have altered absorption spectra. Characterization of these transient changes may then be used to exploit new mechanisms in photosensitization and/or to optimize photobiological effects. In this study, transmittance and reflectance were measured as a function of laser pulse energy, from tissue-simulating media as well as in rat muscle and liver slices, both with and without the photosensitizer benzoporphyrin derivative monoacid (BPD-MA) present. There was a transient decrease in absorption from the photosensitizer at peak pulse irradiance in the range of 100–1000 W cm


Maelstrom (MAEL) plays a crucial role in a recently-discovered piRNA pathway; however its specific function remains unknown. Here a novel MAEL-specific domain characterized by a set of conserved residues  was identified in a broad range of species including vertebrates, sea squirts, insects, nematodes, and protists. It exhibits ancient lineage-specific expansions in several species, however, appears to be lost in all examined teleost fish species. Functional involvement of MAEL domains in DNA- and RNA-related processes was further revealed by its association with HMG, SR-25-like and HDAC_interact domains. A distant similarity to the DnaQ-H 3'–5' exonuclease family with the RNase H fold was discovered based on the evidence that all MAEL domains adopt the canonical RNase H fold; and several protist MAEL domains contain the conserved 3'–5' exonuclease active site residues . This evolutionary link together with structural examinations leads to a hypothesis that MAEL domains may have a potential nuclease activity or RNA-binding ability that may be implicated in piRNA biogenesis. The observed transition of two sets of characteristic residues between the ancestral DnaQ-H and the descendent MAEL domains may suggest a new mode for protein function evolution called "active site switch", in which the protist MAEL homologues are the likely evolutionary intermediates due to harboring the specific characteristics of both 3'–5' exonuclease and MAEL domains.This article was reviewed by L Aravind, Wing-Cheong Wong and Frank Eisenhaber. For the full reviews, please go to the Reviewers' Comments section. Germline cells among different species are characterized by the presence of a morphologically unique organelle called the germ plasm  ,2. This Drosophila mutant, whose germline cells exhibit incorrect posterior localizations of several transcripts  [The molecular functions of most germ plasm proteins in the piRNA pathway have been assigned based on domain examination, biochemical and genetic characterizations. For instance, PIWI proteins contain the PAZ and PIWI domains, which contribute to recognition of single-stranded RNA  and sequ Bicoid) . It is a Bicoid) ,13,21,28 Bicoid) ,29. The  Bicoid) , which i Bicoid) . Therefo Bicoid) . In the Entamoeba histolytica, Entamoeba dispar, and Trypanosoma brucei). We also examined NCBI nucleotide and Ensembl genome databases and identified eight other homologues in insects and urochordates . Three more protist homologues were obtained through searching GeneDB database. A multiple sequence alignment was built for all the retrieved sequences  and mosquito (A. aegypti), three copies in Culex pipiens, and five copies each in amoeba E. dispar and E. histolytica. Phylogenetic tree construction suggests that multiple MAEL copies are generated from a series of ancient lineage-specific duplication events . It can be inferred that it is the ancestor of the fish lineage after the divergence of teleost and tetrapod lineages that underwent the loss of MAEL domain. The timing of the loss is probably related to the ancient fish-specific genome duplication [For the majority of species, only one copy of MAEL domain exists. However, there are multiple MAEL homologues in several other species; for instance, two copies are found in sea squirts . The HDAC_interact domain is known to bind to histone deacetylases (HDACs), core enzymes for removing acetyl group from lysine residue of histones during chromatin remodeling process [Three other domains are associated with MAEL domains, including HMG (SMART: SM00398), HDAC_interact (SMART: SM00761), and SR-25-like domain  Figure . HMG is  process . It has  process . Mosquit process . The ass process  and PRP3 process , It is a process . Therefometa server was utilized, which assembles various state-of-the-art fold recognition methods and further evaluates modeled structures based on a consensus score computed by a 3D-JURY system [X. tropicalis, Ciona and Drosophila were first used as queries and several structural hits were identified by MetaBasic, ORFeus and BasicDist with consensus scores from 21 to 46. Although these 3D-Jury scores are below the cutoff 50, which corresponds to correct assignment with statistical significance [E. histolytica MAEL domain  as a query. Eleven structural hits were identified with high scores around 58–69, and they all belong to the DnaQ-H 3'–5' exonuclease family. Structural fold similarities between DnaQ-H and MAEL domains encouraged us to re-examine this relationship using PSI-BLAST. We noticed that several DnaQ-H exonucleases can be retrieved as insignificant candidates in our initial PSI-BLAST searching with a profile inclusion expectation (E) value of 0.005. However, when we set inclusion E value at 0.05, significant similarity between the first 100aa segment of MAEL domains and several prokaryotic DnaQ-H exonucleases was achieved in the fourth iteration.We applied a fold recognition strategy to identify remotely related homologues of MAEL domains. The rationale is that in the case of remote homology, conserved protein structural folds can be kept despite limited sequence identity . A meta Y system . MAEL doificance , domain ificance ,42. We eWe next examined this homologous relationship by building structure-based multiple sequence alignments for MAEL domains and DnaQ-H 3'–5' exonucleases. Since sequence identity among different DnaQ-H domains is very low, their alignment was first generated based on structural information as assessed by a CE-MC server  followedThe tertiary structures of protist and chicken MAEL domains were further constructed by comparative modeling. Like DnaQ-H domains Figure , these M2+, Mg2+, or Mn2+) are involved [The proposed evolutionary link of MAEL domains to DnaQ-H 3'–5' exonuclease with RNase H fold may provide functional clues for MAEL domains. The DnaQ-H 3'–5' exonuclease family, also known as DEDDh exonuclease family or Exonuc_X-T domain (Pfam ID: PF00929), is one member of RNase H fold superfamily SCOP: 53098) which also includes RNase H, mu transposase, crossover junction resolvase RuvC, and PIWI domain families ,49-52. T098 whichinvolved -47, theiinvolved . Therefo2+ or Mn2+) and is responsible for the hydrolysis of the RNA in a DNA/RNA duplex [2+ [Escherichia coli proteins including ColE7 endonuclease [2+ [In contrast to the protist MAEL domains, most recent MAEL domains do not contain the DnaQ-H specific residues but are characterized by the EHHCHC residues. What is the functional contribution of these residues to MAEL domains? Structural observations showed that a structural core can be potentially formed by the MAEL-specific residues EHHCHC or EHHH. This may provide a structural basis for an active site. On the one hand, this active site may confer RNA-binding ability for MAEL domains because of the lack of DnaQ-H specific residues. In this way, MAEL may contribute to stabilizing or positioning the RNA substrate in piRNA pathway. On the other hand, MAEL-specific residues and its potential active site may define another nuclease activity. We noticed that although all related families with the RNase H fold have low sequence identities and contain different active site residues, they all have DNA/RNA 3' or 5' end-directed nuclease activities with metal ion coordination in their own active sites ,51. For A duplex ,54. In cA duplex ,24,55-57plex [2+ . Moreovenuclease , Zinc trnuclease , and Aldease [2+ ,Experimental evidence have suggested that MAEL may be involved in piRNA biogenesis since its loss-of-function mutant impairs the production of piRNAs or rasiRNAs and increases the transcript level of transposable elements . DifferePhylogenetic analysis has revealed several unique characteristics of MAEL domains including single-copy status in most species, ancient lineage-specific expansion and the loss in the teleost fish lineage. It has been long recognized that during evolution eukaryotic species have high duplication rates  and vertWe also observed the loss of MAEL domain in all examined teleost fish species. Gene loss in protein family evolution is well-recognized. The lost member may be functionally replaced by another member of the same family. However MAEL does not belong to this case because of its single-copy nature especially in the vertebrates. What happens in teleost fish germline cells without the MAEL protein? One possibility is that fish have a distinct but functionally similar counterpart, which remains to be characterized. Another possibility is that MAEL loss results in a unique piRNA pathway or a unique developmental morphology in fish germline cells compared to mammals and flies. Indeed, a distinct cellular distribution of Vasa protein, a marker for germline cells, has been observed in fish . MoreoveProtista that represents the earliest eukaryotic branches [Eukaryota or during the divergence of the prokaryotes and eukaryotes, corresponding to the time when small RNA pathways emerged. Thereafter, the duplicated one (MAEL ancestor) obtained a protein motif comprising CHC residues, forming an evolutionary intermediate which has both DEDHD and EHHCHC residues. The original DnaQ-H activity was attained by some ancestral protist MAEL domains. However, driven by relaxed evolutionary constraint associated with functional specification, other MAEL domains generated by further lineage-specific duplications or species speciation (duplication 2) may have lost the original active site with DEDHD residues, but at the same time developed a new active site with EHHCHC residues while keeping RNase H structural scaffold  of MAEL with EHHCHC residues, we now only present structural evidence and give discussion based on other evolutionary and functional information. We agree with the reviewer on the question of whether both active sites (DnaQ-H and MAEL-specific activities) can be realized in the same protist MAEL domain. We believe they could since their conservation likely reflects functional contribution; otherwise the conservation of these residues should have been lost during evolution. It seems that because of the structural conformation constraints, the same protist MAEL domain cannot form two active sites at the same time; instead, it may adopt different conformations for each of the different activities.Further, there are several points in the paper that need to be addressed for it to be suitable for publication.-Nomenclatural: Currently the authors use the term DNAQ-H for the superfamily. This is confusing as one could imagine that these indeed arose from DNAQ itself. Instead they should use the terminology "3'–>5' exonuclease superfamily in the RNAse H fold".-Phylogenetic analysis: The resolution is probably insufficient and the topology of the tree appears suspect as a result. Further some proteins are evolving very rapidly and at different rates and distort topology (e.g. Entamoeba). The tree is not critical for the argument and it is best that it is presented in the additional file.-Introduction is too long. The authors can very briefly state the importance of Mael and its biology rather than attempting the current detailed description.-The key functional prediction and sequence/structure analysis can be also briefly presented.-Rename DUF1777 to something more meaningful.In conclusion a modified version of the paper, which is suitably condensed and presents the major findings succinctly would be suitable for publication as a discovery note in Biology Direct.We thank the reviewer for these suggestions. We have revised the introduction, results and discussion sections and transferred the methods section to additional file We considered the proposal to put Fig 2A in the supplement but feel strongly that it should be in the main paper. We discuss MAEL evolution extensively in the main text, so the tree will be important for readers to understand MAEL evolution, especially the transition between DnaQ-H and MAEL residues. We agree that some supporting values are weak, so we only present supporting value greater than 75% for the nodes. We renamed DUF1777 as SR-25-like domain because of its similarity to SR-25 domain family (pfam: PF10500) as revealed by the SCOOP program. For terminology, we used DnaQ-H 3'–5' exonuclease family with the RNase H fold. The reason why we emphasized DnaQ-H  is to differentiate another DEDDy family of 3'–5' exonuclease with the RNase H fold, which is characterized by conserved residues DEDYD.In this paper, Dapeng Zhang et. al. attempt to decipher the molecular function of the germ plasm-specific protein, Maelstrom (MAEL) which has been implicated in the piRNA pathway and also in chromatin remodeling from previous experimental studies. The authors conjectured that Maelstrom has nuclease-like activity from three main findings: Firstly, the novel MAEL-specific domain is defined by a set similar sequence segments (related via a few PSI-BLAST searches) with a conserved motif involving residues (Glu-His-His-Cys-His-Cys) from mostly metazoans (except of fish species) and some protists. Some of these protist MAEL sequences also contain the DnaQ-H specific site (Asp-Glu-Asp-His-Asp) that exhibits a 3–5' exonuclease catalytic activity. Therefore, it seems likely that the metazoan MAEL proteins had inherited nuclease-like activity from their protist MAEL ancestors. Secondly, domain architecture analysis of MAEL-related proteins showed the association of the MAEL domain with the HMG (SMART: SM00398), DUF1777 (PFAM: PF08648) and HDAC_interact (SMART: SM00761) for DNA binding, RNA binding and chromatin remodeling respectively. Finally, structural modeling showed that the MAEL-specific domain (Glu-His-His-Cys-His-Cys) in metazoan is able to form a structural core despite the lack of the DnaQ-H active residues. The authors also argued that the residues His, Cys and Glu are the most frequently residues capable of interacting with Zn2+ and also utilized by ColE7 endonuclease, Zinc transport protein ZnuA and Aldolase; analogous to metal ion-binding DnaQ-H.There are several critical points with this manuscript:Danio Rerio , oxidoreductases, DNA polymerases III and 3–5' exonucleases  from numerous species can be found. Thus, there are homologs among fish species.(1) The sequence segment family collection of homologous Maelstrom protein sequences is incomplete. Using the fan-like search methodology as described in Schneider et al. BMC Bioinformatics 2006 v.7, 164), more MAEL-like sequences including sequences from We thank you for your insights and suggestions. We also appreciate your attempts at retrieving additional sequences using your novel methodology. The sequences you identified all are DnaQ-H domains and some sequences you mentioned like 1W0H:A have been included in our study as representatives of the DnaQ-H domain. As we mentioned in the main text, PSI-BLAST searching with a profile inclusion E value of 0.05 can retrieve several DnaQ-H exonucleases as significant hits. However, they are not included in our initial sequence analysis for MAEL domains since they do not have MAEL specific residues (EHHCHC), and introducing these sequences may dilute conserved characteristics of MAEL domains. We used an E value of 0.005 for PSI-BLAST searches with different MAEL domains as queries. They all retrieved the same set of MAEL domain sequences. We also tried the HHsenser server, another sensitive sequence searching program, which retrieved similar results. We could not detect any fish MAEL domain from protein, nucleotide/EST or even Ensembl genome databases of five fish species. So, we are proposing that the MAEL domain is lost in fish species according to these observations. This discovery should be very interesting to experimental biologists who are working on the piRNA pathway. We agree that other distant homologs exist in fish, such as DnaQ-H members and other RNase H members (like PIWI).The emphasis on a set of conserved positions  does not make the definition of a domain. Most importantly, the notion of globular domains unifies protein sequence segments having similarity of their fold . Besides understanding the types of protein families that are in the vicinity of the starting sequence, the purpose of performing fan-like search is also to determine if the search space of the starting sequence for its orthologous sequences is well sampled. When sequences are been collected, the relationship of orthology or paralogy is not obvious. But eventually, with sufficient sequence collection, sequences from different taxonomic groups will be able to form distinct group of protein families. Finally, with reference to these neighboring protein families, one can then use clustering or phylogenetic methods to determine the orthology coverage of the starting sequence. This has not been done in the work of the authors. We have carried out a full sequence family collection with a fan-like PSI-BLAST search , aligned the family and created a phylogenetic tree from hits . It looks as if the so-called maelstrom group is surrounded by the bloom syndrome proteins (DNA helicases), DEAD-domain containing RNA helicases followed by bacterial nucleases as next hits. The fish sequences mentioned by us are in the neighboring helicase groups and, apparently, are not nucleases.We have conducted profile-profile alignments between MAEL, DnaQ , and DEAD helicase  domains using the logomat-p program  and that no structural fold considerations were made. Therefore, the evolutionary tree inferred from unrelated sequences is not reliable. We do not agree with the assessment of the reviewers.(2) An exhaustive search for homologous sequences across all species is the foremost important task in function annotation transfer via homology. This exhaustive list of the homologous sequences enables one to construct clusters of orthologous and paralogous genes and to group them in a phylogenetic tree. Among orthologous sequences, function annotation transfer is able to hold well especially for one-to-one orthologs, with decreased confidence at greater evolutionary distances. On the opposite end, paralogous sequences are generalized to be functional diversified and specialized. This makes the task of function annotation transfer more complex . In this paper, the exact homology relationships among the collected sequences were not well established and, thus, function annotation transfer in this context is problematic. It appears to us that the exonucleases are in another branch of the tree compared with maelstrom sequences; thus, the predicted function might not be correct.This paper presents an evolutionary relationship between MAEL domains and DnaQ-H domains with the RNase H fold based on structural fold similarity as well as the evidence that protist MAEL domains have DnaQ-H specific residues. We do agree that a direct function annotation transfer may not be guaranteed based on this evolutionary link because of functional divergences during protein evolution. But considering the general functions in nuclease activities of DnaQ-H family as well as its distantly related RNase-H superfamily members, we predicted that MAEL may have a similar function with either nuclease or RNA-binding activity. We provide a preliminary evolutionary tree between DnaQ-H and MAEL domains in the additional file 4 and combined this with Figure 2A for extensive discussion in the last section. We hope this discovery will facilitate the further investigation on MAEL function.We think that the conclusion about the functional relationship to the DnaQ-H domain is premature in this form. A hit with 3D-jury is, at best indicative. Our family search and the resulting phylogenetic tree (see attachment) bring the maelstrom group equally close to various helicases and nucleases. This more stringent homology search results  revealed that the Maelstrom sequences are in close vicinity to a group of Bloom syndrome proteins (belonging to the DNA helicase family), bacteria nucleases and helicases while the exonucleases were not significant enough to be found . A preliminary phylogenetic study (with exonuclease as the out-group) showed that the Maelstrom sequences are most homologous to the Bloom syndrome protein sequences in comparison to the other sequences. DnaQ-H is by far not the closest functionally characterized neighbors. In the absence of further structural and catalytic information of the MAEL motif (Glu-His-His-Cys-His-Cys), the functional evolution relationship between Maelstrom and exonuclease is still unclear except for a potential similarity of fold. to collect more homologous sequences to clarify the relationship of Maelstrom to its adjacent protein families.If you do not have an own resource for correct family collection, we strongly suggest the authors to use protein family searcher like HHsenser As we indicated previously, no sequence similarity can be detected between MAEL and DEAD helicase domains. We tried HHsenser to retrieve MAEL homologues sequences, and it generated similar results as PSI-BLAST. We thank reviewers for this suggestion.(3) Furthermore, the suggestion of nuclease-like activity in metazoan MAEL proteins is weak given that the DnaQ-H active residues were not conserved even if the predicted tertiary structure is correct and, probably, conserved in the family. A structural is only a plausibility argument; it does not prove the conclusion. Doubts are the more appropriate since the homology model involves a translocation/shifting of the active site.At the end, a set of sequentially similar sequence segments without any trustworthy molecular function prediction remains. This result is not necessarily demanding another publication.We present general discoveries about MAEL, its evolutionary link to DnaQ-H domains and structural predictions on active sites. We agree that functional prediction is not the definitive conclusion. However, we believe that our rigorous analysis may give us a strong basis to hypothesize on function. Firstly, the evolutionary link and possible DnaQ-H active site in protist MAEL domains may suggest that protist MAEL domains have a 3'–5' exonuclease activity. Secondly, for the MAEL domains with EHHCHC residues, the high conservation of these residues likely reflects their functional contributions. Structural examinations direct our attention to an active site since these conserved EHHCHC residues are located closely together. We then found other evidence including the property of E, H and C residues to interact with metal ions and general functions of evolutionarily related RNase-H fold families. Although we do not have experimental support, these lines of evidence provide structural, chemical and evolutionary basis for an active site, and thus lead to our hypothesis that it may have nuclease activity or RNA-binding ability. Thirdly, translocation/shifting of the active site is common in evolution of protein families as reviewed by Todd et al. (2002) and Anantharaman et al. (2003). It is also true for the RNase H fold superfamily in which the DnaQ-H family and other families use their own specific residues to form different active sites. Therefore, we believe that the DnaQ-H active site is lost during MAEL evolution and the MAEL domain developed its own active residues. More importantly, we identified some protist domains which have both sets of active residues of DnaQ-H and MAEL domains. They can serve as an evolutionary intermediate during this translocation/shifting, thus suggesting a new mode for protein function evolution.Firstly, the authors utilized the 3D-jury results to indicate that the maelstrom protein segment might confer a similar fold to that of DnaQ-H domain exemplified by pdb 1W0H:A. It appears to us that the evolutionary distance to these exonucleases is considerable and that other groups are much more closely related. We found the Maelstrom sequences to be most homologous to the Bloom syndrome protein sequences. Therefore, the structural fold prediction might not be reliable and, at such evolutionary distances, it would be not surprising if the relative positions of important residues are scrambled. Furthermore, the metazoan Maelstrom proteins have lost these residues that appear indispensable for the nuclease activity. Unless it can proven experimentally, the suggestion that metazoan Maelstroms have nuclease activity seems less plausible, especially given the presence of a more closely related group of Bloom syndrome proteins.Secondly, Anantharaman et al. state that the presence of a characteristic set of conserved active residues is important for the identification of enzymes in sequence analysis. The set of conserved active residues are typically derived from known set of sequences and structures of related enzymes. For those proteins with preserved structure but varying catalytic residues, the detection of evolutionary relationship is far more difficult. In the case of the Maelstrom, the structure is purely hypothetical and the MAEL motif (Glu-His-His-Cys-His-Cys) has yet to show nuclease-like activity. Therefore, to say that a translocation or shifting of the active nucleatic site has occurred in the Maelstrom in the course of its evolution simply cannot be proven at this point without further experimentation or other type of compelling information. Thus, the molecular function of the maelstrom domain remains unclear and the current stage of research does not justify a report; otherwise, any additional branch of the phylogenetic tree would deserve another article.Firstly, DEAD Helicase domains belong to the P-loop containing nucleoside triphosphate hydrolases fold, whereas DnaQ-H domains belong to the RNase H fold. It is not possible that a reliable searching with MAEL sequences can retrieve both DnaQ-H domains and DEAD helicase domains. Secondly, since no similarity exists between the MAEL/DnaQ and DEAD domains, it is not reasonable to align them together and infer their evolutionary history. Thirdly, we agree with the reviewer that similar structural fold alone does not provide sufficient evidence of common ancestry [However, significant sequence conservation, structural resemblance and catalytic residue conservation may strongly indicate evolutionary relationship [In our study, the proposed evolutionary relationship is established on the basis of three lines of evidence: 1, sequence similarity via PSI-BLAST, which provides the most straightforward evidence of homology [2, similar structural fold; 3, ancestral protist MAEL domains have DnaQ-H characteristic residues. We thank the reviewers for their efforts.ancestry . Howevertionship ,75. In oA complete multiple sequence alignment of MAEL domains. The domain sequences are represented by an abbreviation of species name followed by database ID and domain regions. The consensus in 75% of the sequences is shown below the alignment based on default amino acid classes in Chroma. The numbers in bracket are indicative of the excluded residues from sequences. Species name abbreviations refer to Figure Click here for fileStructural alignments between five different DnaQ-H domains showing the plasticity of structural conformations of α5 and α6. (A) The cartoon structures of four DnaQ-H domains are shown with different colors, in which 2P1J is colored with red. For the structure of 1J53, the NewCartoon diagram is shown with α helices in pink and β sheets in yellow. (B) The structural alignment of 1J53 and 2P1J. The structure of 2P1J is colored in red whereas for 1J53, its α helices are colored in pink and β sheets in yellow. (C) The structural locations of active site residues in both 1J53 and 2P1J domains.Click here for fileE. coli DnaQ-H domain (1J53:A)The conservation of MAEL-specific residues in . The cartoon drawing of protein structure is shown. The α helices are shown in pink, β sheets in yellow and loops in cyan. The DnaQ-H specific residues (DEDHD) are highlighted with acidic residues (D and E) in light red and basic His in light blue, whereas three MAEL-specific residues (EHH) are highlighted with acidic Glu in red and basic His in blue.Click here for fileEvolutionary relationship between DnaQ-H and MAEL domains.Click here for fileMaterials and methods. Detailed description on materials and methods used in the present study.Click here for fileProfile-profile alignment among MAEL, DnaQ , and DEAD (Pfam: PF00270) domains by the logomat-p program.Click here for file


Reduction of gross diarrhea rate in excess of that seen over time with intravenous therapy and appropriate antibiotics is not usually achieved by oral glucose-electrolyte rehydration therapy for cholera and cholera-like diarrheas.adj<0.001). Survival analysis (Kaplan-Meier) showed faster recovery from diarrhea in the HAMS-ORS group . Total diarrhea fecal weight in grams  was not significantly lower in the HAMS-ORS group  compared to HO-ORS  (Padj = 0.08). However, stool weight at 13-24 hours  and 25–48 hours  were significantly lower in HAMS-ORS compared to HO-ORS group . ORS intake after first 24 hours was lower in the HAMS-ORS group. Subgroup analysis of patients with culture isolates of Vibrio cholerae indicated similar significant differences between the treatment groups.This prospective randomized clinical trial at a tertiary referral hospital in southern India was undertaken to determine whether amylase resistant starch, substituting for glucose in hypo-osmolar oral rehydration solution, would reduce diarrhea duration and weight in adults with acute severe dehydrating diarrhea. 50 adult males with severe watery diarrhea of less than three days' duration and moderate to severe dehydration were randomized to receive hypo-osmolar ORS (HO-ORS) or HO-ORS in which amylase resistant high amylose maize starch 50g/L substituted for glucose (HAMS-ORS). All remaining therapy followed standard protocol. Duration of diarrhea (ORS commencement to first formed stool) in hours was significantly shorter with HAMS-ORS  compared to HO-ORS  (Bonferroni adjusted P, PCompared to HO-ORS, HAMS-ORS reduced diarrhea duration by 55% and significantly reduced fecal weight after the first 12 hours of ORS therapy in adults with cholera-like diarrhea.ISRCTN72841333Current Controlled Trials  Cholera and acute dehydrating diarrhea continue to be a major cause of morbidity and mortality in resource poor settings. Oral rehydration therapy using glucose salt oral rehydration solution (ORS) is the cornerstone of treatment iso-osmolar glucose ORS The colon is capable of absorbing a considerable amount of salt and water under conditions of stress as occurs in secretory diarrhea per se does not achieve in cholera. This present study in adults was designed to determine whether ORS efficacy could be further increased by substituting HAMS for glucose in hypo-osmolar ORS, an intervention that would have the consequences of both further lowering osmolarity and providing carbohydrate substrate to the colon for SCFA generation. A similar trial in chidren under the age of five, with non-cholera diarrhea, was registered along with this trial in adults but will be completed by a different team and reported separately.We hypothesized that HAMS would reduce stool output and diarrhea duration in cholera when substituted for glucose in a low sodium hypo-osmolar ORS, thereby achieving what the hypo-osmolar ORS The protocol for this trial and supporting CONSORT checklist are available as supporting information; see Consecutive patients presenting to the Emergency Services of the Christian Medical College between May 2003 and June 2005, who met the entry criteria and consented to participate, were referred to the study medical officers. Patients were considered for eligibility if they were males aged between 12–65 years with watery diarrhea of less than 3 days duration, and moderate to severe dehydration Individual participants gave written informed consent. The study protocol and consent forms were approved by the Institutional Review Board of the Christian Medical College, Vellore.After admission into the study, patients were randomly allocated to receive one of two ORSs, either hypo-osmolar ORS (HO-ORS) or HO-ORS in which glucose was completely replaced by 50 g/L high amylose maize starch  (HAMS-ORS). As can be seen from the composition of the two ORSs , there wThe hypothesis was that substitution of amylase-resistant starch for glucose in hypo-osmolar ORS would significantly reduce fecal weight (volume) and diarrhea duration in adults with cholera or cholera-like illness, with at least therapeutic equivalence in hydration. The specific objectives were to conduct a randomized controlled clinical trial in adults with cholera or cholera-like illness using either hypo-osmolar glucose ORS or hypo-osmolar HAMS ORS (i.e. with glucose replaced by high amylose maize starch) and to determine the effect on fecal weight, diarrhea duration, unscheduled intravenous fluid administration, and total ORS intake.Primary outcomes were: (1) Duration of diarrhea defined as time from commencing ORS to first formed stool and (2) Total diarrheal fecal weight. Secondary outcome measures included: (1) Fecal weight in the time periods 0–12 hours, 13–24 hours, and 25–48 hours after commencing ORS; (2) Total ORS intake; (3) Need for unscheduled intravenous fluids; and (4) Serum sodium of 134 mE/L or less. The occurrence of elevated serum creatinine (>1.5 mg/dL) at 48 hours and of hypokalemia (serum K <3.5 mmol/L) was also recorded.The study was designed as a randomized controlled clinical trial. Sample size was calculated using PS Calc A table of random numbers was computer-generated by randomization such that there was equal distribution among the two groups in fifty participants. Sachets of the appropriate ORS were packaged, as per the random table, in sealed opaque covers bearing serial study numbers.When a participant entered the study, the next available serial number was allocated and the cover bearing this serial number, containing the packaged ORS, was handed by the study physician to the nursing team. The sequence was concealed until the interventions were assigned.The allocation sequence was generated by SV prior to the start of the study. Participants were enrolled by the study medical officers (including VM and BKS) who assigned participants to their groups by handing over the sealed cover bearing the next serial study number (containing the appropriate packaged ORS) to the study nurses.The participants could not be blinded to the intervention. Those assessing the outcomes were blinded to group assignment. The physicians assessing hydration of the patient were unaware of the intervention received by the patient. The fecal weight was assessed by a para-medical worker who was unaware of the exact nature of the study and did not come into contact with the participants.adj) values are presented. Two tailed P values less than 0.05 were considered statistically significant. Statistical analysis was performed using InStat for Windows version 3.06 and Prism4 for Windows version 4.03 .Survival curves were estimated by the Kaplan-Meier method and compared using the log-rank test. Categorical variables were reported as numbers and the Fisher exact test was used to assess significance of differences between groups. Continuous variables that were not distributed normally were reported as median with interquartile range (IQR) and significance of differences between groups tested using the Mann-Whitney U test. Continuous variables that were normally distributed were reported as mean with standard deviation (SD) and significance of differences between groups was tested using the two-tailed unpaired Student t-test. A Bonferroni correction was used to adjust for the multiple end points as well as multiple time points for fecal weight . Both unadjusted and adjusted P . One patient in the HO-ORS group had prolonged diarrhea lasting longer than four days. The difference between the two groups remained highly significant even when this outlier value was excluded.The time  to first formed stool (duration of diarrhea) was significantly reduced in patients receiving HAMS-ORS  compared to those receiving HO-ORS   .adj = 0.08) (adj = 0.048). Fecal weight in the 24–48 hour study period was also significantly lower in the HAMS-ORS group  compared to that of the HO-ORS group  .Total fecal weight  from entry into the study until the first formed stool was substantially less but not statistically significantly different in the HAMS-ORS group  compared to the HO-ORS group   . When an = 0.08) , whereasadj<0.001) (adj = 0.12).ORS intake  in the first and second 12 hours after admission was similar in the HAMS-ORS group  and the group receiving HO-ORS  (P = 0.79 and 0.46 respectively). However, in the second 24 hour period after entry, ORS intake was significantly lower in the HAMS-ORS group  compared to the HO-ORS group  (Pj<0.001) . For theAdministration of intravenous fluids after commencement of ORS was considered necessary in 9 patients randomized to HAMS-ORS compared to 12 patients randomized to HO-ORS (P = 0.56) . All theVibrio cholerae compared to those whose stool culture was negative. There was no difference in recovery from diarrhea between Vibrio positive and Vibrio negative patients. Treatment with ORS type, not the presence of cultivable Vibrio, was the determinant of recovery time .Subgroup analysis was performed to compare stool weight and diarrhea duration in patients with positive stool culture for ery time . In the Hyponatremia (serum sodium less than 134 mEq/L) was noted in 4 patients in the HAMS-ORS group and 3 patients in the HO-ORS group prior to ORS treatment, and in 3 and 2 patients in the respective groups 24 hours after commencement of ORS. None of these patients had any clinical deficit. Four of the 5 patients who had serum sodium of 134 mE/L or less at 24 hours were the same as the ones who had low levels at entry. One patient who received HO-ORS had serum sodium of 136 mE/L at entry and sodium of 128 mE/L at 24 h. There was no significant difference in serum potassium or serum creatinine between the two groups.The present study demonstrates that diarrhea duration and fecal weights 13–48 hours after start of oral therapy were reduced in cholera and cholera-like diarrhea by treatment with a hypo-osmolar HAMS-containing ORS compared to hypo-osmolar glucose ORS. The findings are particularly important because reduction in diarrhea of the magnitude noted in these studies has not been achieved by hypo-osmolar glucose ORS which at best reduced diarrhea by 20% in children with diarrhea Previous studies suggest that when diarrhea duration is reduced by an intervention, total fecal weight is also reduced. In this study, although there was a substantial decrease in stool output for the entire 48 hours (5210 vs 2190 g), the difference between the two groups did not reach statistical significance primarily because the initial 0–12 hour stool collection did not reveal any decrease (1970 vs 2160 g). Significant differences in fecal weight (which can be perceived as severity of diarrhea) became apparent only after the first 12 hours suggesting that there was a latent period before the action of HAMS-ORS. This supports the hypothesis that the effect of HAMS is secondary to metabolism and absorption in the colon. Two major end points were studied, i.e. diarrheal stool loss and duration of diarrhea. Since there were multiple analyses, a Bonferroni correction was applied, and significant differences were noted between the two groups in diarrhea duration as well as in fecal weight after the first 12 hours. In the prior study of HAMS-ORS using an iso-osmolar ORS solution in patients with cholera Vibrio cholerae isolates from southern India are presently sensitive to tetracycline although isolates from several parts of the world exhibit tetracycline-resistance. It has been shown that the anaerobic bacterial flora of the colon, which is responsible for starch fermentation to SCFA, is not affected by oral doxycycline ORS use is based on the concept that absorptive and secretory processes in the intestine and colon are separate and distinct; that stimulation of secretion does not affect absorption; and that enhancement of absorption does not alter secretion. Cyclic AMP induces fluid and electrolyte secretion, but enhances glucose-stimulated sodium absorption Vibrio cholerae only in 22 of these 50 patients, in southern India, severely dehydrating acute infective diarrhea in adults is almost always due to cholera. Such diarrhea may also be caused by enterotoxigenic E. coli that elaborate heat labile enterotoxin and produce a syndrome similar to cholera E. coli. When sub-group analysis was done on those patients with positive stool cultures for Vibrio cholerae, the difference between the two treatment groups remained statistically highly significant. In an earlier study, our group demonstrated that addition of HAMS to conventional (iso-osmolar) glucose ORS reduced diarrhea duration and fecal weight in patients with cholera Participants in this study had severe acute watery diarrhea with significant dehydration at presentation, necessitating initial intravenous rehydration. Although fecal culture was positive for The large volume of ORS given in both trial arms resulted from following the recommendation to administer 200 ml of ORS every hour in addition to 200 ml after each loose stool, until such time as the stool became formed. This large volume of ORS led to increased urine volume in both groups. Although not to be recommended as physiological, the apparently excessive ORS administration and urine volume served to protect against acute renal failure which remains a significant complication in patients with acute severely dehydrating watery diarrhea. Despite the excessive ORS administration, ORS intake in the period 25–48 hours after commencement of treatment was significantly less in the HAMS-ORS group (median 0 ml) compared to the HO-ORS group (median 2200 ml). It is possible that total ORS intake would have been much less in both groups had intake been exactly matched to stool output. Thus, as earlier mentioned in connection with fecal volumes, the methodology used could possibly have confounded the outcomes by affecting the parameters measured.Concerns have been raised about the possibility of symptomatic hyponatremia in patients with cholera treated with HO-ORS In designing a new ORS that would reduce stool weight and diarrhea duration, we felt that it would be important for the difference to be not only statistically significant but also both quantitatively appreciable and clinically substantial. In the present study, substitution of glucose in hypo-osmolar ORS by high amylose maize starch reduced the duration of diarrhea by 55% and very significantly reduced fecal weight after the first 12 hours of illness. Hypo-osmolar ORS has become the recommended standard of therapy for the oral therapy of cholera. However in patients with cholera, hypo-osmolar ORS does not reduce fecal weight or shorten diarrhea Protocol S1Trial Protocol(0.04 MB DOC)Click here for additional data file.Checklist S1CONSORT Checklist(0.05 MB DOC)Click here for additional data file.


The heart carries out its pumping function by converting the chemical energy stored in fatty acids and glucose into the mechanical energy of actin-myosin interaction of myofibrils. Development of congestive heart failure is usually preceded by a period of compensated left ventricular hypertrophy (LVH) and alterations in myocardial bioenergetics have been considered to play an important role in this transition. Myocardial energetic state that is reflected by the ratio of Phosphocreatine to Adenosine Triphosphate (PCr/ATP) is significantly decreased in hearts with LVH. The severity of this abnormality is linearly related to the severity of cardiac hypertrophy as well as left ventricular (LV) dysfunction, and is independent of a persistent myocardial ischemia. The decrease in PCr/ATP is accompanied by a decrease in creatine kinase flux and alterations in substrate utilization in LVH hearts. Moreover, there is a profound heterogeneity in alterations in myocardial energy metabolism in hearts with post-infarction hypertrophy with the most severe abnormality present in the inner layers of the periscar border zone (BZ). This review will discuss various aspects of myocardial energetics in animal models of three different types of LVH  with a brief description of myocardial energetics in humans with LVH. The second component is oxidative phosphorylation which involves transfer of electrons from NADH to oxygen through the mitochondrial respiratory chain that creates a proton electrochemical gradient across the inner mitochondrial membrane subsequently driving the F1-F0 ATP synthase to produce Adenosine Triphosphate (ATP) by phosphorylating Adenine Diphosphate (ADP). The third component is ATP transfer and utilization that involves the creatine kinase shuttle. Cardiac energy metabolism has three components. The first is substrate utilization which involves the cellular uptake of substrates (mainly fatty acids and glucose) and their breakdown by beta-oxidation and glycolysis resulting in the formation of acetyl coenzyme A (CoA), which is fed into the Krebs cycle and produces Nicotinamide Adenine Dinucleotide (NADH) and carbon dioxide (CODerangements in cardiac energy metabolism occur with the development of left ventricular hypertrophy (LVH). LVH is a manifestation of cardiac remodeling and has been classified into pressure overload hypertrophy, volume overload hypertrophy and post-infarction hypertrophy. LVH is initially adaptive but ultimately leads to maladaptive hypertrophy and transition to congestive heart failure (CHF). The exact mechanisms underlying this transition from compensated LVH to CHF are likely multifactorial but alterations in myocardial bioenergetics have been considered to play an important role.in vivo is through Nuclear Magnetic Resonance (NMR) spectroscopy. Our laboratory has specialized in performing in vivo NMR spectroscopy in large animals to study abnormalities in high energy phosphate metabolism, oxidative phosphorylation, creatine kinase kinetics and flux and substrate utilization. In this review, we will focus on the myocardial energetics in different animal models of ventricular hypertrophy (summarized in Table 1).The principal method for studying myocardial energy metabolism Pressure overload hypertrophy leads to concentric hypertrophy due to an increase in wall thickness that can be seen clinically in patients with hypertension or aortic stenosis. Different animal models have been used to create pressure overload leading to concentric hypertrophy. The degree of ventricular hypertrophy varies in these models and thus the results of the studies vary slightly. The most commonly used method is placement of a constrictor band around the ascending aorta to cause an increase of left ventricular systolic pressure.  vs. 4.3±0.14 g/kg, p< 0.01) which represented a doubling of the relative LV mass in the banded dogs . FAO enzyme mRNA levels were coordinately downregulated (>70%) during both the LVH and HF stages in the SHHF rats compared with controls. In contrast, the activity and steady-state levels of medium-chain acyl-CoA dehydrogenase, which catalyzes a rate-limiting step in FAO, were not significantly reduced until the HF stage, indicating additional control at the translational or posttranslational levels in the hypertrophied but nonfailing ventricle [i), and fructose-2,6-bisphosphate , all activators of the rate-limiting enzyme phosphofructokinase (PFK), were increased (up to 10-fold) [2 and increased sarcolemma localization of glucose transporters, leading to coordinated increases in glucose transport and activation of PFK.Recent advances in molecular biology have enabled us to study the molecular mechanisms underlying the observed substrate switch in pressure overload hypertrophy. These studies have revealed the importance of peroxisome proliferator-activated receptor gamma co-activator (PGC-1α) and peroxisome proliferator-activated receptor (PPAR) in mitochondrial dysfunction. The expression of the PGC-1 gene, as well as nuclear respiratory factor 2 and mitochondrial transcription factor A, which act downstream from PGC-1 were shown to be downregulated in rats with pressure overload due to aortic banding . In a moentricle . In a ra10-fold) . This acVolume overload leads to eccentric hypertrophy as a result of increase in cardiomyocyte length. Left ventricular volume overload can be of two types: low-pressure or high pressure . Low preOur group has studied the myocardial energetics in a canine model of volume overload LV hypertrophy by tearing the chordae tendineae and causing severe mitral regurgitation (MR) . This re125I-iodophenyl 9-methylpentadecanoic acid was increased in the volume-loaded animals, suggestive of a greater fatty acid uptake and oxidation [Substrate metabolism has also been studied in volume overload LV hypertrophy. In a rabbit model of volume overload created by carotid-jugular shunt formation, eccentric hypertrophy developed as seen by an increase in LV end-diastolic dimension without an increase in anterior or posterior wall thickness . The myoxidation . In cont vs. 3.65±0.25 vs. 4.99±0.42). It caused a decrease in ejection fraction to about 35% and 27% in the LVR and LVR+CHF group respectively. LV end-systolic volume increased significantly in both groups of animals with remodeling and was significantly higher in LVR+CHF hearts than in LVR hearts. Both LV diastolic wall stresses and mean LV systolic wall stresses were significantly increased in hearts with LVR and were further increased in LVR+CHF hearts.Post-myocardial infarction hypertrophy is a complex phenomenon in which the stretched and dilated infarcted tissue increases left-ventricular volume with a combined volume and pressure load on non-infarcted areas . Our labThe bioenergetic abnormalities in our post-infarction LV hypertrophy model were characterized by a lower ATP, CP, creatine and PCr/ATP ratio in the LVR hearts and the LVR+CHF hearts had an even lower PCr/ATP ratio. The myocardial calculated free ADP levels were significantly increased only in CHF hearts. The increase in myocardial free ADP is associated with reductions in mitochondrial F0F1-ATPase protein expression . These HIt is known that the decrease of PCr/ATP in LVR animal during dobutamine infusion is not the result of insufficient myocardial oxygen availability . FurtherIt has been shown that the CK-MM and CK-mitochondrial protein levels are decreased in animals with pos-infarction hypertrophy while the CK-MB levels are increased . HoweverSubstrate utilization in post-infarction LV hypertrophy also exhibits shifts in fatty acid and glucose metabolism. In a post-infarction rat model, glucose oxidation was increased by 84% while there was no change in palmitate oxidation 8 weeks after MI . The patPost-infarction hypertrophy differs significantly from other forms of hypertrophy because of the presence of a discrete scar region. The alterations in myocardial HEP and mtATPase have been shown to be substantially more severe in the peri-infarct region than in the remote zone of hearts with compensated postinfarction LV remodeling . These a2) have shown similar results to previously obtained from animal studies. Clinical studies of cardiac energy metabolism (summarized in Table 2) than healthy control subjects . LV filling was impaired in patients, reflected by a decreased peak rate of wall thinning (PRWThn), E/A ratio, early peak filling rate, and early deceleration peak , whereas systolic function was still normal. The myocardial PCr/ATP ratio determined in patients at rest (1.20±0.18) and during stress (0.95±0.25) was lower than corresponding values obtained from healthy control subjects at rest  and during stress . The PCr/ATP ratio correlated significantly with PRWThn , early deceleration peak , and with the rate-pressure product . Creatine kinase kinetics is also affected in patients with pressure induced LVH. In hypertrophic human hearts in the absence of CHF, ATP flux through CK is reduced by 30% in LVH patients, and this reduction can be accounted for by a proportionate loss of substrate (PCr) [The PCr/ATP ratio is decreased in patients with pressure induced LVH , 37. In te (PCr) . Patientte (PCr) . These dPCr/ATP is also lower in humans with volume overload hypertrophy such as chronic MR and the severity of abnormal myocardial energetics is linked to disease severity as measured by dimensional indexes of left ventricular dilatation and hypertrophy . In pati201Tl scintigraphy, but ATP content was decreased significantly only in subjects with fixed thallium defects [There have been some myocardial energetic studies in ischemic heart disease patients. In patients with ischemic cardiomyopathy, PCr/ATP ratio was significantly lower as compared to controls . Cardiac defects . The data on substrate metabolism in patients with LV hypertrophy is scant, while in patients with congestive heart failure it is conflicting. In a study of NYHA class II and III patients, fatty acid uptake was increased while glucose uptake was decreased . This waLeft ventricular hypertrophy is characterized by derangements in cardiac energy metabolism including lower PCr/ATP, abnormal creatine kinase kinetics and deranged substrate utilization. The PCr/ATP is significantly decreased in hearts with LVH and the severity of this abnormality is proportional to the degree of cardiac hypertrophy and accompanying LV dysfunction. These myocardial energetic abnormalities are also not due to a persistent myocardial ischemic state. There is a profound heterogeneity in the myocardial energy metabolism in hearts with postinfarction LV remodeling with the most severe abnormality occurring in the inner layers of the periscar border zone (BZ). Increased wall stress and overstretching of myocytes could contribute to the observed decrease in energetic efficiency in the BZ. CHF probably develops as this energetically inefficient BZ expands over time and eventually involves the entire heart. Preserving myocardial energetic efficiency through metabolic therapies is a promising therapy for heart failure in the future. To achieve this goal, further molecular studies are required to understand the mechanisms underlying the myocardial energetic abnormalities. Furthermore, development of effective and time feasible methods of non-invasive NMR spectroscopy along with magnetic resonance imaging (MRI) will allow us to study temporal changes in myocardial energetics in patients with LVH and CHF and correlate these with structural changes in the hearts. Finally, new drugs need to be developed and tested that target energy metabolism pathways with the ultimate goal of improving survival in patient with LVH and CHF.