Little is known about treatment of multidrug-resistant tuberculosis (MDR-TB) in high HIV-prevalence settings such as sub-Saharan Africa.We did a retrospective analysis of early outcomes of the first cohort of patients registered in the Lesotho national MDR-TB program between July 21, 2007 and April 21, 2008. Seventy-six patients were included for analysis. Patient follow-up ended when an outcome was recorded, or on October 21, 2008 for those still on treatment. Fifty-six patients (74%) were infected with HIV; the median CD4 cell count was 184 cells/μl (range 5–824 cells/μl). By the end of the follow-up period, study patients had been followed for a median of 252 days (range 12–451 days). Twenty-two patients (29%) had died, and 52 patients (68%) were alive and in treatment. In patients who did not die, culture conversion was documented in 52/54 patients (96%). One patient had defaulted, and one patient had transferred out. Death occurred after a median of 66 days in treatment (range 12–374 days).In a region where clinicians and program managers are increasingly confronted by drug-resistant tuberculosis, this report provides sobering evidence of the difficulty of MDR-TB treatment in high HIV-prevalence settings. In Lesotho, an innovative community-based treatment model that involved social and nutritional support, twice-daily directly observed treatment and early empiric use of second-line TB drugs was successful in reducing mortality of MDR-TB patients. Further research is urgently needed to improve MDR-TB treatment outcomes in high HIV-prevalence settings. Multidrug-resistant tuberculosis (MDR-TB), defined as resistance to both isoniazid and rifampicin, is a growing problem in resource-poor settings where adequate diagnosis and treatment are often unavailable. Sub-Saharan Africa would seem to have all of the conditions for a “perfect storm” of HIV infection and MDR-TB Treatment of MDR- and XDR-TB is difficult even in resource-rich settings. Patients are generally treated for a minimum of 18–24 months with second-line TB drugs that have significant adverse effects Lesotho is a mountainous nation that is home to two million people and completely surrounded by the Republic of South Africa. With respect to HIV and TB, Lesotho is very similar to its neighbor, South Africa, whereas its medical infrastructure and resources reflect its significantly lower per capita GDP. It has one of the highest burdens of HIV infection in the world, with a prevalence of 23.2% The Lesotho Ministry of Health and Social Welfare recently began a national treatment program as part of a comprehensive response to MDR- and XDR-TB. International collaborators included Partners In Health, the Open Society Institute, the Foundation for Innovative New Diagnostics (FIND), and the World Health Organization. Through this program, quality-assured second-line TB drugs procured through the Green Light Committee Initiative (GLC) We conducted a retrospective analysis of early outcomes of MDR-TB treatment in the first cohort of patients treated in this program. To our knowledge, this is the first analysis of MDR-TB treatment outcomes in the midst of a generalized HIV epidemic in a resource-poor setting. It is also the first report of a community-based MDR-TB treatment model in sub-Saharan Africa.We retrospectively examined the medical records of consecutive patients registered by the Lesotho national MDR-TB program between July 21, 2007 and April 21, 2008. This included all patients starting treatment with second-line TB drugs for confirmed or presumptive MDR-TB.As this was a retrospective study of medical information previously collected in the course of routine clinical care, informed consent was not required. This study was approved by the Human Subjects Committee of the Harvard School of Public Health.At the beginning of the study period, the Lesotho National Reference TB Laboratory performed direct smear microscopy without fluorescence, but not culture or drug susceptibility testing (DST). Some patients brought DST results from various public and private sector TB laboratories in South Africa showing resistance to isoniazid and rifampicin. These patients were immediately started on treatment with second-line TB drugs, and were asked to produce sputum for culture and DST before starting treatment. Sputum was sent to the Medical Research Council (MRC) Tuberculosis Laboratory, a supranational reference laboratory in Pretoria, South Africa for first- and second-line DST.Starting in 2007, the Lesotho National Reference TB Laboratory began receiving training from FIND in culture and DST. Culture was performed initially on Löwenstein-Jensen medium, and later using a BACTEC MGIT 960 system . DST was performed to the first-line drugs of isoniazid, rifampicin, ethambutol and streptomycin. MDR isolates continued to be sent to MRC for DST to pyrazinamide and second-line drugs throughout the entire study period.In general, patients were referred to the Lesotho national MDR-TB program for evaluation of confirmed or suspected MDR-TB. Most patients were referred by clinicians working in the Ministry of Health and Social Welfare network of hospitals and health centers, but some were referred by private physicians or South African mining companies. Patients with suspected MDR-TB but without DST results were categorized according to clinical and bacteriological criteria, and high-risk patients were started empirically on second-line TB drugs. High-risk categories included: household contacts of known MDR-TB patients; probable treatment failure of a WHO Category 1 or Category 2 regimen ; and history of treatment with second-line TB drugs  were started on ART as soon as they were tolerating second-line TB drugs, irrespective of CD4 cell count. ART regimens commonly consisted of two nucleoside reverse transcriptase inhibitors (zidovudine or stavudine plus lamivudine), and one non-nucleoside reverse transcriptase inhibitor (efavirenz or nevirapine).Patients in stable clinical condition were started on MDR-TB treatment on an ambulatory basis. Patients were evaluated at least monthly by a trained clinician. All doses of second-line TB drugs and ARV's were administered under twice-daily directly observed treatment (DOT). Community health workers were trained to provide DOT, monitor for adverse effects and provide psychosocial support. Patients received daily injections from health centre nurses or by community health workers if living in remote areas. Patients who were unable to ambulate or were otherwise unstable clinically were admitted to a specialized 18-bed MDR-TB inpatient facility in the capital . Patients were also admitted if they experienced severe adverse effects or other clinical complications that could not be managed as outpatients. All treatment was free of charge. Patients received a monthly food package and were reimbursed for treatment-related travel expenses. Community health workers were reimbursed for travel expenses and were provided with a monthly cash incentive.Variables collected through a standardized chart abstraction included: demographic characteristics; previous TB treatment exposure; previous exposure to antiretroviral therapy; HIV serostatus; cavitary and bilateral disease on chest radiography; results of drug susceptibility testing; results of hemoglobin, creatinine, and hepatic function tests at baseline and during treatment, and occurrence of serious adverse effects and clinical complications (definitions shown in 2 test) were performed to determine the associations between clinical characteristics and outcome. Multiple logistic regression analysis was performed to look for potential confounders or effect modifiers. SAS software  was used for all analysis.Patient follow-up ended when an outcome was recorded, or on October 21, 2008 for those still on treatment. Possible outcomes included: death; default (greater than one month of missed doses); transfer out (transfer with records to another MDR-TB treatment program); and alive and in treatment. Autopsy was not available; cause of death was determined after a detailed review of clinical records and was a consensus decision of the first and last authors. Univariate analyses  were sputum AFB-positive before starting treatment. Of the remainder, five patients were smear- and culture-negative, including three children; the final patient was smear-negative but culture-positive. Twenty-nine patients (38%) were started on treatment with second-line TB drugs already having laboratory confirmation of resistance to isoniazid and rifampicin, mostly from South African laboratories. Forty-seven patients (62%) were started on empiric treatment with second-line TB drugs without having laboratory-confirmed MDR-TB. All patients starting empiric MDR-TB treatment had sputum samples sent for culture and DST, and 26 of these patients eventually proved to have MDR-TB; the remaining 21 patients had negative or contaminated cultures. Ten MDR isolates were tested by MRC in South Africa for resistance to second-line drugs. Of these, four isolates were found to be resistant to second-line drugs; one isolate met criteria for XDR-TB.Only two patients had never been treated for TB. Seventy-four patients (97%) had previously received TB treatment, ranging from one to five previous courses of TB treatment .Usually Fifty-six patients (74%) were infected with HIV; of the 32 patients who were tested within 90 days before or after initiation of MDR-TB treatment, the median CD4 cell count was 184 cells/μl (range 5–824 cells/μl). Of the HIV-positive patients, 24 patients were already receiving ART, a mean of 369 days (range 13 days to more than 3 years) before initiation of MDR-TB treatment. Twenty-five patients were started on ART, a mean of 35 days (range 11–117 days) after starting MDR-TB treatment. Of the remaining seven patients, six patients died without starting ART, and one was alive, in treatment and culture-negative at the end of the follow-up period.Fifty-eight patients (76%) experienced at least one hospitalization during the follow-up period. Hospitalization ranged from 2 to 188 days, and the mean length of stay was 33 days. Seventy patients (92%) experienced at least one serious adverse effect or clinical complication during the follow-up period .By October 21, 2008, study patients had been followed for a median of 252 days (range 12–451 days). Twenty-two patients (29%) had died, and 52 patients (68%) were alive and in treatment. One patient had defaulted, and one patient had moved to South Africa after culture conversion and was continuing to receive treatment at another institution. In patients who did not die, culture conversion—defined as two consecutive negative cultures greater than 30 days apart In univariate analyses, hemoglobin <10 g/dL, serum albumin <30 g/L, and an age >40 were associated with death . In multWhile there have been many previous studies of MDR-TB treatment outcomes in resource-poor settings, none have included significant numbers of HIV-positive patients The high early mortality seen in this cohort argues strongly for early initiation of second-line TB drugs in MDR-TB patients. One common theme in prior studies of HIV-positive patients with MDR-TB is the rapidity of death in the absence of effective treatment. Several studies in the 1980s and 1990s of MDR-TB in HIV co-infected patients from Europe and the United States reported mortality rates exceeding 70%, often within 4–8 weeks of diagnosis, and before laboratory confirmation of MDR-TB Clinicians are often reluctant to start second-line TB drugs empirically, without laboratory confirmation of resistance to isoniazid and rifampicin. But in situations where DST is not easily accessible, the reliance on laboratory diagnosis can become a bottleneck to life-saving treatment. In this cohort of patients, most patients were started empirically on second-line drugs, without waiting for DST results. Almost all of these patients were sputum smear-positive and would be expected to have positive cultures. However, for many patients, the cultures ended up being negative or contaminated, since in the initial phase of the program, the raw sputum had to be transported over long distances to a laboratory in South Africa. Even in settings where there is ready access to culture and DST, empiric MDR-TB treatment is a strategy that can decrease the time to effective treatment. The most commonly available DST methods utilize culture on solid or liquid media, which, at a minimum, require several weeks or months, ample time for a patient to clinically worsen or die while receiving ineffective first-line TB drugs. With the aid of clear guidelines and protocols, clinicians can be trained to identify patients at high risk for MDR-TB and initiate second-line TB drugs empirically. There is much to be gained and little to be lost by a starting a well-designed empiric MDR-TB treatment regimen in high-risk patients In this cohort, the majority of patients (62%) received empiric MDR-TB treatment, and therefore benefited from effective therapy earlier than they would have normally. Nevertheless, by many objective measures, they did not benefit early enough. In this largely HIV-positive population with advanced immunosuppression, a high proportion of smear-negative patients would normally be expected. In fact, 92% of the patients were smear-positive. Many of them may have originally had smear-negative pulmonary TB, but by the time they were referred for suspicion of MDR-TB, the sputum bacillary load was much higher. Ninety-seven percent were retreatment cases, with 65% previously receiving two or more TB treatments. This means that for most patients, there had previously been multiple missed opportunities for MDR-TB diagnosis before referral. The most common pathway to a diagnosis of MDR-TB in resource-poor settings is through multiple failed treatments with standardized regimens of first-line TB drugs. This strategy is unlikely to result in acceptable MDR-TB treatment outcomes in high HIV-prevalence settings, even in those settings with greater medical resources than Lesotho, such as South Africa. For the first cohort of MDR-TB patients treated in Lesotho, where DST and second line TB drugs had previously been unavailable, this sort of delayed diagnosis may be understandable, but probably was one of the factors responsible for the high early mortality. In the future, earlier diagnosis of MDR-TB, and in particular, increasing the diagnosis of MDR-TB among new TB patients, as has been done in other countries with more mature MDR-TB treatment programs Another major bottleneck to MDR-TB treatment in the southern African region is a reliance on hospital-based treatment. In many countries, patients with MDR-TB are routinely admitted to the hospital for several months at the start of treatment. This is partly for clinical reasons and partly to protect the community from infection In Lesotho, a community-based model of MDR-TB treatment was found to be feasible, as it has been in other countries In Lesotho, the default rate appeared to be quite low compared to typical default rates of 15–20% among MDR-TB patients in South Africa Community health workers also play an important role in ensuring that patients receive the psychological, social, and economic support required to complete treatment. Community-based MDR-TB treatment should include an assessment of the home situation and nutritional intervention when necessary. In Lesotho, all patients treated for MDR-TB were provided with a monthly food package. The high prevalence of low BMI and hypoalbuminemia in our cohort was probably caused by chronic malnutrition, not just chronic TB and HIV infection. MDR-TB patients in this region likely suffer from energy, protein and micronutrient deficiencies, all of which can contribute to early mortality, side effects and clinical complications in patients with TB or HIV There are several caveats and limitations to this analysis that should be considered before extrapolating to other patient populations and programs. This analysis included patients who did not have DST. We chose to do this because our treatment protocols strongly promoted empiric treatment of MDR-TB, and DST was not readily available during the entire study period. The inclusion of non-MDR strains, furthermore, would not have affected the results reported here, since according to national protocol, the standardized MDR-TB treatment regimen did not include isoniazid or rifampicin. No patients in this cohort benefited from the superior activity of these drugs, even in the event that some patients were infected with pan-susceptible TB. The early outcomes reported here may be different from the final outcomes; reporting of MDR-TB treatment outcomes generally requires more than two years of follow-up, in order to fully account for late death, treatment failure, default, and recurrent disease In a region where clinicians and program managers are increasingly confronted by MDR- and XDR-TB, this report provides sobering evidence of the difficulty of MDR-TB treatment in high HIV-prevalence settings. Nevertheless, in Lesotho, an innovative community-based treatment model that involved social and nutritional support, strict DOT, and early empiric use of second-line TB drugs was successful in reducing mortality of MDR-TB patients. Further research is urgently needed to improve MDR-TB treatment outcomes in high HIV-prevalence settings.


MicroRNAs have been discovered as important regulators of gene expression. To identify the target genes of microRNAs, several databases and prediction algorithms have been developed. Only few experimentally confirmed microRNA targets are available in databases. Many of the microRNA targets stored in databases were derived from large-scale experiments that are considered not very reliable. We propose to use text mining of publication abstracts for extracting microRNA-gene associations including microRNA-target relations to complement current repositories.The microRNA-gene association database miRSel combines text-mining results with existing databases and computational predictions. Text mining enables the reliable extraction of microRNA, gene and protein occurrences as well as their relationships from texts. Thereby, we increased the number of human, mouse and rat miRNA-gene associations by at least three-fold as compared to e.g. TarBase, a resource for miRNA-gene associations.http://services.bio.ifi.lmu.de/mirsel.Our database miRSel offers the currently largest collection of literature derived miRNA-gene associations. Comprehensive collections of miRNA-gene associations are important for the development of miRNA target prediction tools and the analysis of regulatory networks. miRSel is updated daily and can be queried using a web-based interface via microRNA identifiers, gene and protein names, PubMed queries as well as gene ontology (GO) terms. miRSel is freely available online at  MicroRNAs (miRNAs) are small (~22-nucleotide) non-coding RNAs (ncRNAs) that post-transcriptionally regulate the levels of a potentially large number of proteins by base-pairing to messenger RNAs (mRNAs) ,2. PerfeCaenorhabditis elegans [Since the discovery of the first miRNA, lin-4 in  elegans , thousan elegans . Tens of elegans -15. So f elegans -18. Rece elegans ,19,20 si elegans -24. Seve elegans , miRGato elegans  and TarB elegans ,27 and [ elegans . miRecor elegans . A more  elegans . They foAs a remedy to this, information extraction applied to the scientific literature can be used to significantly enhance and complement the information stored in biological databases -36. We tThe nomenclature of miRNAs and especially proteins as well as genes has evolved over time ,32 and vThe following conventions for miRNA naming are used:(i) The predicted stem-loop portion of the primary transcript is named by a 3 or 4 letter species prefix and a numerical suffix (e.g. hsa-mir-100 in Homo sapiens). Whereas, the name of the excised ~22 nucleotide sequence (mature miRNA) contains the same mir, prefix and suffix as stem-loop but with capital miR (e.g. hsa-miR-100).(ii) Orthologous miRNA sequences in different species are assigned the same names .(iii)Mature miRNA sequences can be expressed from each arm of the hairpin precursor. They are distinguished by additional suffixes (e.g. hsa-miR-1224-5p (5'arm) and hsa-miR-1224-3p (3'arm)). Previously, they also have been named for instance miR-142-s (5'arm) and miR-142-as (3'arm). In some cases, the asterisk has been used to denote the less predominant form (e.g. hsa-miR-100*).(iv) Distinct hairpin loci in a given organism that give rise to identical mature miRNA sequences are assigned names with additional numeric suffixes (e.g. hsa-mir-101-1 and hsa-mir-101-2 indicating two genomic loci of the miRNA hsa-miR-101).(v) Related hairpin loci that give rise to related mature miRNA sequences with only one or two base changes are assigned letter suffixes of the form (e.g. hsa-mir-10a and hsa-mir-10b are similar sequences).Of course, these conventions are not strictly followed in scientific publications. If complete names are used, e.g. hsa-miR-1224-5p, the author likely means the 5'arm predominant mature form of human miRNA-1224. On the other hand, an incomplete form e.g. miR-1224 could mean precursor or mature microRNAs, the 3' or the 5' variant or an unspecified variant of microRNA 1224 in some species depending on the context.In addition, there are many naming problems: For some organisms fairly different naming conventions are used ,37. For The construction of a database of miRNA-gene co-occurrences via named entity recognition (NER) requires the compilation of miRNA, gene and protein name dictionaries as well as their association to database identifiers. The extraction performance depends on the completeness and uniqueness of the entries in the dictionaries. The dictionaries for human, mouse and rat are compiled from several databases: HUGO Gene Nomenclature Committee (HGNC) , Mouse GIL = Interleukin). Secondly, inappropriate synonyms or expressions that would lead to ambiguous or wrong identifications are identified and removed. A detailed description of the curation and the involved processing steps is given in [The next steps are extension and curation of the dictionaries. For proteins, we first complement the synonym lists with spelling variants, acronyms, abbreviations and long forms . The regular expression also covers frequent spelling variants mentioned in the texts  with and without species identifiers (e.g. hsa-miR-195 and miR-195). If detected miRNA synonyms are contained in public databases we map them to their database identifiers and, if possible, distinguish matches as stem loop sequence, mature sequence and gene family matches in miRSel.The database derived synonyms are summarized in Table Proteins and genes from the dictionaries are detected in texts  by string matching using the newly developed tool syngrep . syngrepThe identification of named entities allows to identify miRNA-gene pairs both co-occurring in an abstract or in a single sentence. If not mentioned otherwise, we will focus on pairs extracted from single sentences as they are more reliable for extracting miRNA-gene associations. Information on relations or interactions between miRNA and genes is of interest for generating and analyzing network models of regulatory pathways. These pairs are extracted and stored in miRSel together with the PubMed abstracts and sentences where they have been found.We further compiled a list of 70 terms that are used to describe relations of interest between miRNA-gene pairs  (default selected).• The single-sentence filter reports only miRNA and gene pairs co-occurring in single sentences as opposed to pairs co-occurring in abstracts (default abstract).• The relation-type filter restricts matches to a particular type of miRNA-gene association (default none is selected).• The taxonomy filter aims to enforce organism specificity of the matches. Our organism specific taxonomy dictionary contains synonyms and MeSH vocabularies for all examined organisms as provided by the NCBI taxonomy database . We defi• The gene synonym filter excludes protein or gene synonyms which are assigned to multiple genes or proteins (ambiguous synonyms) in databases (default none is selected).• The database filter shows the text mining pairs only if they also contained in other databases or computational predictions of miRNA gene targets (default none is selected).The total number of associations with regard to these filters is shown in Figure miRSel provides a web interface to retrieve information on miRNA-gene pairs stored in the database Figure . The int(i) Genes can be selected based on gene names, gene symbols, protein names or database identifiers.(ii) miRNAs can be selected based on miRNA identifiers and miRNA gene families.(iii)A PubMed interface enables arbitrary PubMed keyword queries for searching miRSel, miRNA-gene pairs are reported only if found in PubMed abstracts matching the PubMed query.(iv) The gene ontology (GO) option restricts the reported miRNA-gene pairs to genes associated with the selected GO-terms .Additional filter options are described in the implementation section. Figure miRSel is based on finding occurrences of valid identifiers of genes, proteins and miRNAs in publication abstracts. Here we report on the performance of miRSel with respect to finding valid miRNA, gene and protein occurrences as well as valid miRNA-gene pairs and detailed miRNA-gene pair associations.We estimate the reliability of the detection of miRNAs in texts in the following. The performance of gene and protein name detection has already been evaluated in the BioCreAtIvE competition .For evaluation we selected PubMed abstracts that matched our regular expression for the detection of miRNAs or contained keywords such as 'microRNA', 'miRNA' 'mir', 'miR' and 'MIR'. Sentences containing a miRNA identifier or related keywords were additionally required to contain protein names from our synonym lists described in the implementation section. 50 PubMed abstracts were chosen randomly containing 89 sentences that met the above requirements. miRSel was compared against various manual analyses (see below) in terms of recall (i.e. fraction of True Positive (TP) and all True occurrences), precision  and f-measure.The evaluation of miRNA identifier occurrences is shown in Table For the detection of miRNA-gene associations we manually evaluated if a gene and a miRNA have been correctly detected my miRSel and if an association between the two is implied. As shown in Table Besides the detection of generic miRNA-gene associations, miRSel automatically annotates five different types of associations between miRNAs and genes . Out of the 1973 single-sentence human miRNA-gene pairs in miRSel 1301 (65%) were classified into one of the five types.From the test set described above, a subset of the sentences that contain association keywords have also been evaluated manually. If association keywords are present in sentences with miRNA-gene pairs, the precision of association detection increases slightly (Table The TP53 gene (Entrez Gene: 7157) encodes protein p53, which is one of the most important tumor suppressor proteins. TarBase and miRecords do not report any miRNA targeting this gene. We extracted 60 different human miRNAs that co-occur with this target gene from 79 PubMed abstracts, and some of them  are consistent with microarray-based results discussed by Shalgi et al. .hsa-miR-21 is the most frequent miRNA in miR2Disease, with 59 documented associations of this miRNA with diseases. miRSel contains 181 different genes co-occurring with this miRNA extracted from 123 PubMed abstracts. 96 pairs are retrieved if miRSel results are restricted to the more reliable single-sentence pairs.miRNA related research depends on knowledge of miRNA target genes. The available databases on literature derived miRNA target pairs demonstrate that manual curation is difficult. TarBase, for instance, contains only 1135  such pairs (TarBase version 5). Moreover, only a fraction of the content in current databases has been derived by manual curation of experimentally validated targets. Instead, the major part of the content stems from the supplemental material of few research articles describing large scale experiments. Ritchie et al  propose To keep the miRSel database up-to-date, newly available PubMed abstracts are included daily. A full refresh of the synonym list generation and, subsequently, the scan of the entire PubMed is performed monthly to ensure the validity of all identifiers.Finally, we provide a web interface for querying miRSel via miRNA names, gene or protein names and via restricting the results using gene ontology terms or PubMed queries. We provide additional filter options, for instance to ensure the taxonomy context of matches. For future development we will extend miRSel to full free text and apply miRSel to additional species.Project Name: miRSelProject home page: http://services.bio.ifi.lmu.de/mirselOperating systems: Windows, LinuxProgramming language: Java , Tomcat v6.0License: freeAny restriction to use by non-academics: NoneHN, RK, and RZ planned the project. HN acquired the data and performed validations. HN and GC implemented the database and web server. GC implemented the name entity system and HN processed and integrated the raw data. RK and HN analyzed and finalized the design and performance. HN, RK and RZ prepared the manuscript. RZ and RK supervised the project. All authors tested the database and interface. All authors approved the final manuscript.Keywords describing miRNA-target relationship types. miRSel aims to categorize detected miRNA-target associations into five different relationship types. relation_keywords.txt maps relationship types to keywords. If miRSel detects such keywords together with a miRNA-target association the corresponding relationship type is assigned.Click here for file


MicroRNAs (miRNAs) represent a class of small non-coding RNAs that control gene expression by targeting messenger RNA (mRNA). Recently, it has been demonstrated that miRNA expression is altered in many human diseases including cancers, suggesting that miRNA may play a potential role in the pathogenesis of different diseases. It has also been reported that there is a unique expression pattern of miRNAs in the disease state differing from the normal state. In this review, we focus on the miRNA signatures in different human diseases including cancers. Such signatures may be used as diagnostic and prognostic markers. Caenorhabditis elegans, miRNA are small, non-coding RNAs composed of 18–24 nucleotides. They act by negatively regulating gene expression at the post-transcriptional level through modulation of mRNA expression. Lee et al. [lin-4, an important gene that is important for post-embryonic development of C. elegans, does not code for a protein. Instead, it is transcribed into a 22-nucleotide RNA molecule. Seven years later, a second miRNA, let-7, also emerged from C. elegans genetic screening. The discovery of many new miRNAs has continued there after, with evidence emerging to suggest these small RNAs play a crucial role in gene regulation. This role has been confirmed by the identification of hundreds of conserved miRNA sequences in nematodes, flies, and mammals [Discovered in 1993 from nematode To date, more than 3% of the genes in humans have been found to encode for miRNAs. Anywhere from 40% to 90% of the human protein encoding genes are under miRNA-mediated gene regulation .MiRNAs play an important role in different biological process such as cell proliferation during development, cell growth, and organization as well as apoptosis, biogenesis, transcription, signal transduction, cell cycle, neurogenesis, and fat metabolism. The deregulation of miRNA function can lead to human diseases as the target genes of miRNA are involved in several metabolic disorders such as various forms of cancers, lymphomas, diabetes and neurological disorders such as Parkinson's and Alzheimer's diseases. Recently, there has been considerable interest in understanding the mechanism of the RNA regulatory phenomena and how miRNA functions in carcinogenesis. Specifically, determining how the regulation of miRNA may be targeted to develop cancer therapies has been a focus. In this review, we discuss recent discoveries related to miRNA in different cancer diagnoses and, in particular, treatment related to ovarian cancer (OVCA).Like proteins, miRNAs are transcribed from DNA into a primary (Pri) RNA transcript. In the first step, formation of Pri-miRNA inside the nucleus occurs from the transcription of the miRNA gene by RNA polII. The Pri-miRNA is capped, polyadenylated, and then processed by RNAase III endonuclease Drosha and the double stranded (ds) RNA-binding protein Pasha. This processing results in the formation of a 60 to 70 nucleotide stem loop precursor (Pre) called Pre-miRNA. The Pre-miRNA is then transported to the cytoplasm by the Ras-related nuclear protein-guanosine-5'-triphosphate (RAN-GTP)-dependent export receptor Exportin-5. Next, the Pre-miRNA is cleaved at the base of the loop by a second RNase III endonuclease, a cytoplasmic Dicer . ArgonuaGene regulation by miRNA can affect a wide variety of cell functions, such as regulation of cell differentiation, proliferation, and apoptosis. MiRNAs are important for normal cell functioning; dysfunction of the miRNA regulation system results in disruption of the normal cellular activity leading to induction of a disease.Neurodegenerative diseases are the result of the deterioration of neurons, which leads to disabilities and the possibility of death. Studies have shown that gene defects play an important role in the pathogenesis of neurodegenerative diseases such as Parkinson's, Alzheimer's, and Huntington's .Parkinson's disease is the second most prevalent age-associated neurodegenerative disorder. Like Alzheimer's disease, gene mutations can result in inherited forms of Parkinson's disease . Kim et Spinal muscular atrophy (SMA) is a common genetic disease characterized by the progressive degeneration of motor neurons. It has been demonstrated that deletion or loss-of-function mutations in the survival of motor neuron (SMN) protein results in SMA . Gemin 3Using RNA immunoprecipitation 3'-end labeling and miRNA cloning, Dostie and colleagues  identifiJulie Bilen and colleagues  isolatedThe heart responds to chronic and acute injury by hypertrophic growth. Cardiomyocyte hypertrophy is the dominant cellular response to virtually all forms of hemodynamic overload, endocrine disorders, myocardial injury, or inherited mutations in the various structural and contractile proteins . Multiplin vitro. Its role in myocyte hypertrophy was demonstrated by Cheng et al. in 2007 [MiR-21 is found to be consistently induced by cardiac stress and appears to function as a regulator of cardiac growth and fetal gene activation in primary cardiomyocytes  in 2007 . Their s in 2007 . This in in 2007  also con in 2007  reportedAnother miRNA that is consistently up regulated in rodent and human hypertrophic hearts is miR-195. Forced expression of miRNA-195 in transgenic mice is sufficient to induce hypertrophic growth and also results in dilated cardiomyopathy and heart failure [MiR-208 is encoded within intron 27 of the α major histocompatibility complex (MHC) gene and plays a key role in the expression of β MHC in response to cardiac stress . MiR-208MiR-1 is a muscle-specific miRNA that forms a bicistronic cluster with miR-133. Both miRNAs belong to the same transcriptional unit and have been found to play a crucial role in determining when cardiomyocyte hypertrophy occurs . While bDiabetes is a chronic disease caused by defects in insulin production, secretion, and signaling . Poy et Recent studies show that several miRNAs are differentially expressed in several human cancers, which indicate that miRNAs may have a role in the carcinogenic processes , which accounts for approximately 90% of OVCAs, continues to be the leading cause of death resulting from gynecological malignancies . HistoloNew, more effective diagnostic tests must be developed to identify OVCA in its early stage. If OVCA is diagnosed in the early stage, current treatments will be more successful, which in turn will significantly reduce the high mortality rate. The discovery that miRNAs are expressed at different levels in normal versus malignant tissues offers the possibility of identifying new methods of diagnosing early stage incidence and discovering new therapies for the treatment of OVCA.With the advent of new miRNA technology, it is possible to plan appropriate treatments and predict chemotherapy outcomes. Studies have shown that miRNAs, which are noncoding regulatory RNA products, are aberrantly expressed in many cancer types including breast , pancreaIn addition to the difference in expression levels between normal and cancerous tissues, it has been shown that miRNAs can also be histotype-specific. In endometrioid tumors, miR-21, miR-182, and miR-205 are considerably overexpressed while miR-144, miR-222, and miR-302a have reduced expression compared to normal. However, in serous and clear cell tumors, these miRNAs are normally expressed [The above tests are invasive procedures because they require a tissue sample to determine the expression levels of miRNAs for diagnostic purposes. However, a new non-invasive approach has recently been discovered that only requires a serum sample from the patient. In the serum of OVCA patients, miRNAs 21, 29a, 92, 93, and 126 were overexpressed while miRNAs 127, 155, and 99b were under expressed as compared to controls. This indicates that a patient's serum miRNA level could be used to potentially diagnose OVCA. Additionally, miRNAs 21, 92, and 93 were also overexpressed in patients with normal CA125 levels. This suggests that earlier diagnosis of OVCA may be possible through the identification of these miRNAs even though patients may present with normal CA-125 levels .Another new non-invasive method with the potential for diagnosing OVCA involves isolating tumor-derived exosomes. Exosomes are saucer shaped nanovesicles (30–100 nm) that are normally released by multiple mammalian cell types and have a significant role in cell-to-cell communication. In addition to exosomes released by normal proliferating cells, tumor cells also release exosomes. Tumor cells release exosomes at a much higher level than normal cells and the exosomes also display numerous tumor antigens that mirror the primary tumor cells. Therefore, it is proposed that these tumor-derived exosomes could be used as a diagnostic biomarker. In addition to the antigens, circulating tumor-derived exosomes have been shown to contain miRNAs that parallel those of the original tumor . This deThe current treatment utilized for patients with OVCA consists of primary cytoreductive surgery followed by platinum and taxane combination chemotherapy treatments. This method of therapy is initially successful; however, most of the patients become resistant to the platinum-based chemotherapy . Recent The difference in miRNA expression levels between chemotherapy-sensitive and resistant cells is clinically significant. New treatments could exploit these differences if the mechanism behind regulating miRNA expression is determined. Then, for instance, miR-378, which is down-regulated in malignant ovarian tissue, could be induced to over express to a normal level, possibly making the cells more sensitive to platinum-based chemotherapy. Moreover, identification of specific miRNAs between types of cancers  and even within types  could allow for more personalized treatments, which certainly present the chance for a better prognosis.in vivo could be DNA hypomethylation.While there is not much information currently available on the mechanisms responsible for inducing aberrant miRNA expression, there is a new possible explanation for their overexpression in OVCA. Iorio et al.  recentlyThe discovery of aberrant miRNA expression between control patients and those with OVCA holds great promise for improved patient care and more positive outcomes. Thus, identification of altered miRNA expression, as well as their targets, provides new opportunities for therapeutic strategies. MiRNA-based gene therapy targeting deregulated miRNAs will be the future tool for cancer treatment. Using data from miRNA profiling and knowledge of familial history of a certain diseases will help in developing miRNA-based personalized therapy. Detailed understanding of the characteristic miRNA abnormalities could contribute to novel approaches in early diagnosis and treatment of different diseases including cancer and can serve as a biomarker for cancer. miRNA expression profiling eventually assist physicians in providing patients with accurate diagnosis, personalized therapy, and treatment outcome.(miRNA): MicroRNA; (mRNA): messenger RNA; (RNAi): RNA-mediated interference; (RISC): RNA-induced silencing complex; (Pri): primary; (Pre): precursor; (RAN): Ras-related nuclear protein; (GTP): guanosine-5'-triphosphate; (ds): double stranded; (dsmiRNA): double stranded miRNA; (miRISC): miRNA-associated multiprotein RISC; (SMA): Spinal muscularatrophy; (SMN): survival of motor neuron; (miRNP): miRNA-containingribonucleoprotein particle; (ban): bantam; (polyQ): polyglutamine; (SCA 3): spinocerebellar ataxia type 3; (MHC): major histocompatibility complex; (TR): thyroid-hormone-receptor; (THRAP1): thyroid-hormone-receptor co-regulator TR-associated protein 1; (CLL): chronic lymphocytic leukemia; (EOC): Epithelial ovarian cancer; (OVCA): ovarian cancer.The authors declare that they have no competing interests.PPS drafted the manuscript. LEH contributed in writing part of the manuscript. SSK conceptualized, edited, and revised the manuscript. All authors have read and approved the final manuscript.Differentially expressed miRNAs in various diseases. The data provided represents the differential expression of miRNAs in various diseases. Also includes cited references for this file.Click here for file


Metabolic syndrome (MetS) encompasses a cluster of coronary heart disease and diabetes mellitus risk factors. In this study, we aimed to elucidate the factors underlying the clustering of MetS components in diabetic and non-diabetic individuals.Factor analysis was performed on 2978 (1652 non-diabetic and 1326 diabetic) participants. Entering waist circumference, homeostasis model assessment of insulin resistance (HOMA-IR), triglycerides, high-density lipoprotein-cholesterol (HDL-C) and systolic blood pressure (SBP), we performed exploratory factor analysis in diabetic and non-diabetic individuals separately. The analysis was repeated after replacing triglycerides and HDL-C with triglycerides to HDL-C ratio (triglycerides/HDL-C). MetS was defined by either adult treatment panel III (ATPIII), international diabetes federation (IDF) criteria, or by the modified form of IDF using waist circumference cut-off points for Iranian population.The selection of triglycerides and HDL-C as two distinct variables led to identifying two factors explaining 61.3% and 55.4% of the total variance in non-diabetic and diabetic participants, respectively. In both diabetic and non-diabetic subjects, waist circumference, HOMA-IR and SBP loaded on factor 1. Factor 2 was mainly determined by triglycerides and HDL-C. Factor 1 and 2 were directly and inversely associated with MetS, respectively. When triglycerides and HDL-C were replaced by triglycerides/HDL-C, one factor was extracted, which explained 47.6% and 38.8% of the total variance in non-diabetic and diabetic participants, respectively.This study confirms that in both diabetic and non-diabetic participants the concept of a single underlying factor representing MetS is plausible. Metabolic syndrome (MetS) encompasses a cluster of coronary heart disease and diabetes mellitus risk factors, including abdominal obesity, glucose intolerance, dyslipidemia and elevated blood pressure ,2. SubjePrevious studies have entered different numbers of variables ranged from 4 to 21 in factor analysis and extracted from 1 to 7 factors -13. SomeFactor analysis of the MetS has been the focus of little attention in the Middle Eastern ethnicity; furthermore, the clustering of MetS components is yet to be more explored in the diabetic patients. In this study, for the first time we performed factor analysis on the components of MetS in a large sample of Iranian diabetic and non-diabetic participants. MetS can be defined according to Adult Treatment Panel (ATPIII)  or InterA total of 3023 participants (aged 18-75 yrs), who were referred consecutively from September 2005 to December 2008 to one of the four Tehran University-affiliated health service centers located in east, west, south and center of Tehran, were studied. Participants were enrolled from individuals taking health examinations, or those who accompanied patients. Subjects who needed special concern beyond routine examination for their symptoms  along with those under insulin therapy were not included in the study. Diabetic participants were taking metformin, glibenclamide or both simultaneously for controlling their hyperglycemia. Due to missing data, 45 participants were excluded, resulting in a total of 2978 subjects. Oral informed consent was obtained from all individuals before study commencement. The study was conducted in accordance with Helsinki declaration and was performed in line with considerations, recommended by local ethics review committee of Tehran University of Medical Sciences.Waist circumference was measured at mid-distance between iliac crest and rib cage and was rounded to the nearest 0.1 cm. The participants were instructed to rest for at least 5 min before having their blood pressure checked twice with at least 5 min interval. Venous blood samples were collected following 12 h overnight fast. Fasting plasma glucose was measured by glucose-oxidase method. TG and HDL-C were determined by enzymatic methods . Insulin was measured by radioimmunoassay, using an antibody with no cross-reaction against pro-insulin and C-peptide . The intra and inter-assay coefficients of variation were lower than 4.3 and 3.4, respectively.The homeostasis model assessment of insulin resistance (HOMA-IR) was calculated as fasting insulin (U/l) × fasting plasma glucose (mg/dl)/405, as described by Matthews et al. . Diabete2 analysis. The relationship between individual variables was examined using bivariate Pearson's correlation coefficients. Since a large number of analyses were carried out, a P <0.01 was considered statistically significant. In order to improve the normality of skewed variables  natural log transformations were used in the subsequent analyses as appropriate. Factor analysis was performed in two separate models. In model 1 waist circumference, SBP, HOMA-IR, TG and HDL-C were selected for analyses. Exploratory factor analysis was performed using principal component analysis, a technique for reducing the number of original variables into fewer latent factors. The variables cluster on the basis of linear correlations that exist among them. Factors with an eigenvalue (the amount of variance attributable to the factor) of greater than one were extracted and transformed by varimax rotation method to enhance interpretation. Varimax method maintains the independence of extracted factors and simplifies the interpretation of analysis by maximizing factor loadings to one or minimizing it to zero. Varimax rotation cannot be performed if factor analysis extracts just one factor. Factor scores, which are estimates of individual factors with the mean and standard deviation equal to 0 and 1, respectively, were determined by regression method. The factor loading of a variable on a factor equals the correlation coefficient between that variable and the factor score. In accordance with several previous reports, factor loadings of ≥|0.40| were considered meaningful for interpretation; because this ensures that the variable shares at least 15% of the variance with the factor . Factor analysis was performed in diabetic and non-diabetic participants separately. Binary logistic regression analysis was used to assess the association of factor scores with MetS defined by either ATPIII or original IDF or by the modified form of IDF criteria.Data were analyzed using SPSS software . To extrapolate our results to the population of Tehran we carried out a complex sample survey analysis. The data were directly weighted for age (10-year strata) and sex distribution of diabetic and non-diabetic residents of Tehran, according to the results of an epidemiological study on patterns of diabetes, conducted by the Ministry of Health , and theConsidering that two of the variables entered in analyses are related to lipid profile (i.e. TG and HDL-C), in model 2 we repeated all aforementioned analyses after substituting ratio of TG to HDL-C for both TG and HDL-C.P <0.001)Sex-specific principal characteristics of our study population in diabetic and non-diabetic participants are presented in Table In non-diabetic participants, all variables entered in analysis were correlated with each other except HDL-C and SBP. However, in diabetic participants, SBP was merely associated with waist circumference Table .Entering waist circumference, HOMA-IR, TG, HDL-C and SBP as variables (components of MetS) in model 1, exploratory factor analysis extracted two factors in both non-diabetic and diabetic participants Table . In non-In model 2, substituting TG and HDL-C with TG to HDL-C ratio led to a single factor structure for both non-diabetic and diabetic participants, which explained 47.6% and 38.8% of variance, respectively , one factor was found, confirming previous observations on the monofactorial structure of MetS . FurtherAs previously mentioned, variations in sample collection, number and nature of selected components and also in methods used for interpretation of analysis preclude formal direct comparisons, but an overview elucidates some common patterns. In several previous studies insulin resistance loaded on the same factor with measures of obesity including weight, body mass index and waist circumference . Furthermore, measures of obesity were more likely to belong to the first extracted factor and to have the highest factor loadings . It has been demonstrated that visceral fat via its tendency to release free fatty acids, stimulates hepatic glucose production and accentuates insulin resistance . MoreoveMetS is believed to be related to insulin resistance which can be scored by measuring fasting insulin concentration -3. HowevInsulin resistance and hyperinsulinemia lead to hypertension via a number of mechanisms, including renal sodium and water retention, rennin-angiotensin-aldosterone system, plasma noradrenaline and sympathetic nervous system activity ,33. SincEthnic differences in the expression of MetS are linked to different appropriate waist circumference cut-offs for determination of abdominal obesity. It has been found that Asians in comparison to Caucasians are more prone to obesity related comorbidities even at lower waist circumference values . We receThe major limitation, relevant to our results interpretation, is use of cross-sectional data. However, consistent with our analysis most previous studies have used cross-sectional study design to evaluate the interrelatedness of MetS components. Cross-sectional analyses provides information at a single point in time, nevertheless progressive and reciprocal mechanisms are likely to be involved in the appearance of MetS. Therefore, longitudinal studies are suggested to improve better our understanding of MetS.This study revealed that in both diabetic and non-diabetic individuals the concept of a single underlying factor (pathophysiologic pathway) for the expression of MetS is plausible. Further, we showed that similar to ATPIII and original IDF definitions; modified IDF definition is also strongly associated with identified factors. Nevertheless, among components of MetS blood pressure is less influenced by the common factor and could be also mediated by other pathophysiologic processes.MetS: metabolic syndrome; SBP: systolic blood pressure; DBP: diastolic blood pressure; HDL-C: high density lipoprotein cholesterol; TG: triglycerides; ATPIII: adult treatment panel III; IDF: international diabetes federation; HOMA-IR: homeostasis model assessment of insulin resistance; OR: odds ratio.The authors declare that they have no conflict of interests.AE conceived the study, participated in its design, coordination and acquisition of data. AZ wrote the manuscript and performed the statistical analysis and participated in the acquisition of data. OK helped to draft the manuscript and interpret the data and participated in the acquisition of data. AM helped to perform the statistical analysis. HA helped to draft the manuscript and participated in the acquisition of data. All authors read and approved the final manuscript.


Thermococcus litoralis is a heterotrophic facultative sulfur dependent hyperthermophilic Archaeon, which was isolated from a shallow submarine thermal spring. It has been successfully used in a two-stage fermentation system, where various keratinaceous wastes of animal origin were converted to biohydrogen. In this system T. litoralis performed better than its close relative, P. furiosus. Therefore, new alternative enzymes involved in peptide and hydrogen metabolism were assumed in T. litoralis.Thermococcus litoralis. In silico analysis revealed that the region contained a putative operon consisting of eight genes: the fdhAB genes coding for a formate dehydrogenase and the mhyCDEFGH genes encoding a [NiFe] hydrogenase belonging to the group of the H2-evolving, energy-conserving, membrane-bound hydrogenases. Reverse transcription linked quantitative Real-Time PCR and Western blotting experiments showed that the expression of the fdh-mhy operon was up-regulated during fermentative growth on peptides and down-regulated in cells cultivated in the presence of sulfur. Immunoblotting and protein separation experiments performed on cell fractions indicated that the formate dehydrogenase part of the complex is associated to the membrane-bound [NiFe] hydrogenase.An about 10.5 kb long genomic region was isolated and sequenced from The formate dehydrogenase together with the membrane-bound [NiFe] hydrogenase formed a formate hydrogenlyase  complex. The expression data suggested that its physiological role is linked to the removal of formate likely generated during anaerobic peptide fermentation. In some of the heterotrophic species, capable to grow facultatively without S°, the fermentative utilization of sugars and peptides results in the formation of molecular hydrogen  hydrogenases were identified in furiosus -19. Basefuriosus ,21.T. litoralis  hydrogenase (Hyh1) was characterized from itoralis , and evihydrogen . In thisT. litoralis, which codes for a complex that is composed of subunits which show high sequence similarity to the components of the non-energy-conserving formate hydrogenlyase  system of E. coli  hydrogenases. The protein encoded by the ninth gene   clusters. It resembles the iron-sulfur binding subunits of different molybdopterin oxidoreductases like the iron-sulfur related protein of  (FHL-1) ,8. Thererogenase .mhyCDE genes code for highly hydrophobic proteins with various numbers of membrane spanning helices  responsible for the binding of the [NiFe] center . At the 2-evolving, energy-conserving, membrane-associated hydrogenases revealed an N-terminal extension, which was present only in the MhyF subunit and in the hydrogenase large subunits of the formate hydrogenlyase systems . This soli HycE .E. coli, were proposed to play a role in the electron transport [The deduced protein of 18.7 kDa is similar to the iron-sulfur cluster containing hydrogenase subunits. The easily recognizable two ferredoxin-type CxxCxxCxxxCP motifs suggested the presence of two [4Fe-4S] clusters, thus MhyG seems to represent an electron transfer subunit. The corresponding subunits of other hydrogenases like HycF in nGxCxxxGxmGCPP  motif characteristic for the [NiFe] hydrogenase small subunits  hydrogenases. It contains the [4Fe-4S] cluster binding CxxCxsubunits . There imhyH), an archaeal transcriptional termination signal could be found: a hairpin-loop forming palindrom followed by an oligo(dT) region  hydrogenase which belongs to the group of H2-evolving, energy-conserving membrane-associated hydrogenases. Remarkably, the operon could be found only in P. abyssi among the four closely related species  whose gin silico analysis predicted that few subunits (MhyCDE) are integrated membrane proteins. Western blotting experiments using anti-FdhB antibody revealed that the hydrophilic FdhB protein is also attached to the membrane, although large portion of FdhB could be detected in the soluble fraction. Protein purification and separation experiments, combined with activity staining and immunological detection, were performed to demonstrate that the membrane-bound hydrogenase was associated with the formate dehydrogenase subunits. Formate driven hydrogen evolution could also be detected, but this activity was not fully reproducible, probably due to protein sensitivity. Comprehensive in silico analysis made it unlikely, that except for formate, any other substrate of the FdhAB dimer  could be considered. Therefore, it was concluded, that the FdhAB-MhyCDEFGH proteins formed a membrane-bound formate dehydrogenase coupled hydrogenase (FDH-MHY) complex, although the subunits of the formate dehydrogenase seemed to be dissociable from the other part of the complex.The genomic context and transcriptional analysis of the genes indicated that the Fdh-Mhy proteins form a functional complex and There are a few reactions or pathways leading to formate formation in various microbes including the pyruvate , the metE. coli the formate hydrogenlyase is responsible for the removal of formate to prevent the cytoplasm from acidification  hydrogenase (formate hydrogenlyase) complex was demonstrated which was likely involved in the removal of formate generated during peptide catabolism. This might have an important practical consequence. In a two stage system converting keratin-containing biowaste to biohydrogen, T. litoralis showed better performance than P. furiosus  hydrogenase (part of FHL-2) in Escherichia coli, Hyh: cytoplasmic NADP-reducing [NiFe] hydrogenase in Thermococcus litoralis and Pyrococus furiosus, FNOR: ferredoxin:NAD(P) oxidoreductase, INR: initiator element, Mbh: membrane-bound [NiFe] hydrogenase of Pyrococcus furiosus type, Mhy: membrane-bound [NiFe] hydrogenase linked to Fdh, Nuo: NADH:ubiquinone oxidoreductase, MNH: multisubunit Na+/H+ antiporter, PFOR: pyruvate-ferredoxin oxidoreductase, TAT: twin-arginine translocation pathway.AOR: aldehyde:ferredoxin oxidoreductase, BRE: transcription factor B fdh-mhy operon, did the Western blot and Real-Time experiments and measured the enzyme activities. AT did the RT-PCR and helped to design the Real-Time PCR experiments. BB overexpressed and purified the FdhB protein for antibody production. Chromatography steps during protein purification were done by AV. GR took part in the isolation of fdh-mhy operon and participated in the design of the study and helped to draft the manuscript. KLK participated in the design and coordination of the study and helped to draft the manuscript.MT carried out the isolation of


Ras GTPases are lipid-anchored G proteins, which play a fundamental role in cell signaling processes. Electron micrographs of immunogold-labeled Ras have shown that membrane-bound Ras molecules segregate into nanocluster domains. Several models have been developed in attempts to obtain quantitative descriptions of nanocluster formation, but all have relied on assumptions such as a constant, expression-level independent ratio of Ras in clusters to Ras monomers (cluster/monomer ratio). However, this assumption is inconsistent with the law of mass action. Here, we present a biophysical model of Ras clustering based on short-range attraction and long-range repulsion between Ras molecules in the membrane. To test this model, we performed Monte Carlo simulations and compared statistical clustering properties with experimental data. We find that we can recover the experimentally-observed clustering across a range of Ras expression levels, without assuming a constant cluster/monomer ratio or the existence of lipid rafts. In addition, our model makes predictions about the signaling properties of Ras nanoclusters in support of the idea that Ras nanoclusters act as an analog-digital-analog converter for high fidelity signaling. Plasma membrane heterogeneity is a key concept in molecular cell biology due to its role in protein sorting and specificity of signaling Ras GTPases are small (21 kDa), lipid-anchored peripheral membrane proteins involved in signal transduction in vivo and in vitro experiments. Fluorescence resonance energy transfer (FRET) studies show that activation by EGF leads to significant decrease in Ras lateral diffusion, suggesting the existence of Ras Experimental evidence for the formation of nanoclusters (termed clusters from now on) is provided by et al. used GFP-Ras fusion constructs, which were treated with gold-labeled anti-GFP antibodies. The resulting immunogold point patterns were visualized with EM (immunoEM) to quantitatively describe Ras clustering  results in the formation of more rafts as expression increases, and supports the notion that lipidated molecules such as Ras can drive the formation of rafts in order to create signaling platforms in vitro membrane sheets, where no active, energy-driven processes can limit cluster size. Membrane sheets were fixed (and proteins immobilized) after membrane removal from cells Direct evidence for protein clustering in a membrane can be obtained from high-resolution electron microscopy (EM). However, Ras is too small and not electron dense enough to be observed directly. To circumvent this problem, Prior ustering  [7]. It Recent experiments even go further and probe the design principles of signaling by Ras clusters. Such studies suggest that, in the Ras-Raf-MEK-ERK pathway, Ras clusters act as an analog-digital-analog converter, where analog, continuous EGF input is converted into digital, fully active clusters. The number of fully active Ras clusters, not the activity of individual Ras molecules, translates into analog ERKpp output Here, we consider a physically-motivated model to study Ras clustering. The model mainly depends on a close-contact, attractive interaction between active Ras molecules . To test if our model produces digital-like nanoswitches, which are fully active even for small inputs, we identified clusters of two or more connected Ras molecules and calculated the cluster activity, i.e. the fraction of active Ras molecules in clusters. The bar chart in There has recently been immense interest in understanding the effect of noise in signal transduction olecules . Here, ii.e. the cluster/monomer ratio, is independent of expression level . This high turnover of clusters allows for high temporal precision. Third, Ras clusters act as digital nanoswitches, which may lead to noise reduction in the signal transmission step across the membrane due to coarse graining and averaging of rapidly fluctuating signals. In fact, Ras clusters have similarity to an analog-digital-analog converter known from engineering, transmitting analog EGF input into analog ERKpp output What is the role of Ras clusters beyond simple protein sorting? Harding The above listed properties of Ras clusters are supported by our model. According to Our model relies on the short-range attraction and long-range repulsion between Ras molecules. The physical origin of these interactions are yet to be determined. However, the attraction may originate from direct Ras-Ras interaction via hydrophobic, van der Waals, or electrostatic interactions i.e. the anti-GFP antibody can only bind a single epitope on GFP fused to a Ras molecule), immunoEM data can overestimate clustering. In contrast, two clustered Ras molecules in contact with each other could each interact with separate antigen-binding regions of the same antibody, since an antibody has two antigen-binding regions. In this case the cluster of two Ras molecules is unobservable by immunoEM, leading to an understimation of clustering in the gold point patterns. These issues would have to be addressed if immunoEM studies are to form the basis of an accurate quantification of Ras clustering.There are certain shortcomings of the immunoEM data, rendering the cluster analysis of immuno-gold data difficult. In our simulations, the addition of gold particles to completely random distributions of Ras molecules produced the following. If more than one gold-labeled antibody is allowed to bind a Ras molecule, provided there are no steric clashes between gold particles, the γ receptors, which are believed to associate with rafts or to form small clusters In conclusion, a comprehensive description of Ras clustering is an essential step in the understanding of Ras signaling properties, and of small, inner-membrane GTPases in general. For instance, we showed that clustering leads to robustness to noise, especially input noise  and 8. WRelevant experiments are described in e.g. EGF, except for the Ras activity mutants and lipid anchors, where it desribes an energetic bias in conformational state.A Ras molecule in the membrane can be in either one of two states, active (on) with energy in vivo FRET J between active Ras molecules, driving cluster formation. In order to limit cluster size, we introduce long-range repulsion r is the distance between two Ras molecules. For the repulsive interaction energy, we use a Gaussian function as previously applied for describing microphase separation of lipid mixtures T the absolute temperature.To describe clustering of active Ras molecules, as directly observed for K-Ras and H-Ras using  quickly . The fruin vitro membrane sheets, clustering is an equilibrium process. Models of such phenomena are therefore particularly amenable to Monte Carlo simulations, which include energetics as well as entropy M is the lattice size. On the lattice, each position is uniquely described by an index i  for the proteins mentioned in the text are: H-Ras P01112, K-Ras P01116, and N-Ras P01111.The primary protein accession numbers from the Swiss-Prot databank (


MicroRNAs (miRNAs), which are small, non-coding RNAs approximately 21-nucleotides in length, have become a major focus of research in molecular biology. Mammalian miRNAs are proposed to regulate approximately 30% of all protein-coding genes. Previous studies have focused on highly conserved miRNAs, but nonconserved miRNAs represent a potentially important source of novel functionalities during evolution.An analysis of the chromosome distribution of miRNAs showed higher densities of miRNAs on the X chromosome compared to the average densities on autosomes in all eight mammalian species analyzed. The distribution pattern did not, however, apply well to species beyond mammals. In addition, by comparing orthologous human and mouse miRNAs, we found that X-linked miRNAs had higher substitution rates than autosomal miRNAs. Since the highest proportion of X-linked miRNAs were found in mouse testis, we tested the hypothesis that testis miRNAs are evolving faster on the X chromosome than on autosomes. Mature X-linked testis miRNAs had an average substitution rate between mouse and human that was almost 25-fold higher than mature testis miRNAs on autosomes. In contrast, for mature miRNAs with precursors not expressed in testis, no significant difference in the substitution rate between the X chromosome and autosomes was found. Among mammals, the rapid evolution of X-linked testis miRNAs was also observed in rodents and primates.The rapid evolution of X-linked testis miRNAs implies possible important male reproductive functions and may contribute to speciation in mammals. MicroRNAs (miRNAs) are families of small, non-coding RNAs that are approximately 21-nucleotide in length. Non-coding RNAs have emerged as key post-transcriptional regulators of gene expression in metazoans and plants, and thus have become a major focus of research. By base pairing to mRNAs, miRNAs mediate translational repression or mRNA degradation -7.Until now, most research has focused on highly conserved miRNAs. Most current computational methods for the prediction of miRNA genes rely heavily on phylogenetic conservation of sequences, but nonconserved miRNAs represent a potentially important source of novel functions during evolution. In this study, we analyzed the genomic distribution of miRNAs and found higher densities of miRNAs on the mammalian X chromosome. Evolutionary analysis of miRNAs suggests rapid evolution of X-linked miRNAs, which may be related to their function in testis.Using miRNA data from the miRBase database we calculated the densities of miRNAs on chromosomes and found no miRNA on the Y chromosome in any species. Therefore, for subsequent distribution analysis of miRNAs on sex chromosomes, only the X chromosome was considered. By comparing the densities of miRNAs on the X chromosome and the average densities on autosomal chromosomes, we found a higher density of miRNAs on X chromosomes in mammalian species. In seven of eight mammalian species, the densities were greater than two-fold those on autosomes Fig. . A paireThe XX/XY system is one of the most common sex-determination systems and is found in the vast majority of mammals. To better examine the differences of miRNA densities between X chromosomes and autosomes, we calculated the density distributions in species beyond mammals, and found a ratio of 0.52 in mosquito and 0.94 in fruitfly. In addition, we calculated the distribution of miRNAs in chicken, which has the ZW sex-determination system, in which females have two different kinds of chromosomes (ZW), and males have two of the same kind (ZZ). We found no miRNA on the W chromosome, and a ratio of densities between the Z chromosome and autosomes of 0.64, a value less then 1, meaning that chicken has lower miRNA densities on the sex chromosome. Thus, it seems that mammals have higher densities of miRNAs on the X chromosome, but this phenomenon does not extend to all species. In fact, species other than mammals may even have reversed density distributions.The precursor miRNA substitution rates between human and mouse were calculated using the Kimura 2-parameter model. Comparison of substitution rates showed significantly higher rates for X-linked than for autosomal precursor miRNAs (p = 0.0054) . After dividing the precursor into four parts: terminal loop, upper stem region, lower stem region and basal segments, we found significant differences (p < 0.05) for the terminal loop, upper stem region and lower stem region. For miRNAs not expressed in testis, neither the entire precursor nor any of the four parts showed a significant difference between the X chromosome and autosomes Fig. .We did additional analyses to see if the phenomenon of rapid evolution of X-linked testis miRNAs existed in the primate and rodent branches of mammals. For orthologous miRNA sequences between mouse and rat, X-linked testis precursor miRNAs evolved faster than those on autosomes, with a significant difference p < 0.01). The upper stem regions and loops of testis precursor miRNAs also showed higher substitution rates on X chromosome than on autosomes (p < 0.01). In primates, the Kimura 2-parameter substitution rates between human and macaque were higher for X-linked testis precursor miRNAs than for autosomal testis ones (p < 0.01), and the substitution rates were also higher in the upper stem regions for X-linked testis miRNAs (p < 0.01). For miRNAs not expressed in testis, the Kimura 2-parameter distances of precursor miRNAs in both rodents and primates showed no difference between the X chromosome and autosomes Fig. . The pre. The uppTarget genes of X-linked miRNAs, in both testis and non-testis, were predicted by TargetScan, which uses sequence conservation information across mammals. Although some X-linked miRNAs have non-conserved seed sequences, for many miRNAs, the prediction of non-conserved targets will yield substantial false positives that could hinder meaningful functional comparison between target gene sets. Thus, only X-linked miRNAs with conserved sequences at nucleotides 2–8 were used for target gene analysis. Using this criterion, six X-linked testis miRNAs and 16 X-linked non-testis miRNAs were analyzed. Comparison of enriched biological process terms between target gene sets of X-linked testis miRNAs and X-linked non-testis miRNAs showed that the most enriched four terms exclusively in target gene set of X-linked testis miRNAs were cell cycle, cell cycle process, regulation of progression through cell cycle and regulation of cell cycle [see Additional file The mammalian sex chromosomes arose from autosomal progenitors approximately 300 million years ago. Before then, sex determination probably relied on environmental cues such as egg incubation temperature, as is the case in many extant reptiles . Ross etAnalysis of substitution rates of genome-wide orthologous precursor and mature miRNAs between human and mouse showed that the rates were higher on the X chromosome. With the available comprehensive testis miRNA data in mouse and the hints that mouse testis contains the largest number and highest proportion of X-linked mature miRNAs, we found the substitution rate difference between X and autosomes was evident only for testis-expressed mature and precursor miRNAs. The difference disappeared for mature and precursor miRNAs not expressed in testis. By dividing precursor miRNAs into four parts, differences were observed for upper stem parts, terminal loops and lower stem parts. In addition, the phenomenon of faster evolution for X-linked testis precursor miRNAs was also seen in rodents and primates by comparing orthologous miRNA sequences between mouse and rat, and between human and macaque. Differences for upper stem parts of precursor miRNA were significant in all comparisons between X-linked testis miRNAs and autosomal testis miRNAs. Upper stems are functional parts of precursor miRNAs. Their rapid evolution in X-linked testis precursor miRNA implies that they may play important roles in testis function. Chromosomal location alone does not explain the differences in substitution rate.Drosophila simulans, suggesting that a form of positive selection may be acting on sex chromosomes [Sex chromosomes and sex-linked genes have been a central focus of research in many areas of evolutionary biology, and there are conflicting evidences as to whether the evolutionary rate of the X chromosome is accelerated or reduced compared with that of autosomes . One theomosomes . Second,omosomes . These rIn this study, high substitution rates between orthologous X-linked miRNAs from human and mouse were observed, and a general pattern of rapid evolution was shown for mammalian X-linked testis-expressed miRNA genes. Additionally, Zhang et al.  have repDicer1, which is required for miRNA processing, specifically in germ cells were subfertile. During the initial wave of spermatogenesis, testes were approximately 50% smaller than those of wild type littermates, had a decreased number of germ cells and contained many Sertoli-cell only tubules [Spermatogenesis in testis involves massive and continuous cell division, from mitosis of spermatogonia to meiosis of spermatocytes. This leads to approximately 40 million sperm per testis daily in a sexually mature mouse . Gene on tubules . The losWe have shown that testis-expressed miRNAs on the X-chromosome are highly diverged compared with those on autosomes for mouse, rat, human and macaque comparisons. Sexual selection can influence sex gene-pool evolution by broad-sense sexual selection and may not be limited to secondary sexual traits and mating behavior . The hem[Homo sapiens, NBCI36), chimpanzee , macaque , mouse , rat , opossum , cow , dog , chicken , mosquito , and fruitfly .The genome coordinates of miRNAs on chromosomes were from miRBase Release 12.0 . Densiti[The mouse miRNA data used were from miRBase Release 11.0 . Rat ort,25. To f,27. The  was used were use.The orthologous sequences of two species were aligned by ClustalX. The Kimura 2-parameter model was used to calculate substitution rates (Kimura80 distance) of the precursor miRNAs and mature miRNAs . For preThe multi-tissue mouse miRNA expression data were from a mammalian miRNA expression altas based on small RNA library sequencing . Additio[Target gene analysis was performed using Targetscan 4.2 server Gene ontology enrichment analysis of target genes was performed using the program: DAVID 2008 , a Web-bA Kolmogorov-Smirnov Z test was performed to detect significant differences of the substitution rates (Kimura80 distances) between chromosomes. For comparison of miRNA densities between the X chromosome and autosomes, a paired Student's t-test was used. A p-value less than 0.05 was considered statistically significant. All data are given as mean ± standard error of the mean (SEM).miRNA: microRNA; SEM: standard error of the mean.XG, BS, ZZ and JS designed the experiments. XG performed the analyses. XG wrote the manuscript, which was read and approved by all authors.Genomic coordinates of predicted orthologous sequences of miRNA in different species. Excel spreadsheet presenting homology data on the mouse:human, mouse:rat, and human:macaque putative orthologs together with RNA secondary folding energies.Click here for fileGene ontology enrichment exclusively in target genes of X-linked testis miRNAs. Excel spreadsheet presenting gene ontology enrichment exclusively in target genes of X-linked testis miRNAs.Click here for file


AMP-activated protein kinase is a key enzyme in the regulation of energy metabolism. Its activation has pleiotropic effects in multiple tissues, including increased fatty acid oxidation, glucose uptake and glycolysis, as well as the inhibition of fatty acid and glycogen synthesis and gluconeogenesis, and stimulation of mitochondrial biogenesis. Recently, the AMP-activated protein kinase (AMPK) has also emerged as a regulator of appetite, contributing to the control of energy metabolism at both cell and the whole body levels. Pharmacological and genetic activation or inhibition of hypothalamic AMPK lead to increased or reduced food intake, respectively. AMPK appears to play a role in hypothalamic glucose and nutrient sensing and numerous studies have suggested a role for AMPK in mediating the orexigenic or anorexigenic effects of various endogenous and exogenous substances. AMPK is a heterotrimeric protein consisting of catalytic α and regulatory β and γ subunits; it is activated allosterically by an increase in the intracellular AMP/ATP ratio as well as by phosphorylation on Thr172 by upstream kinases ( (CaMKK) . CaMKKβ neurones . Recentlneurones . AMPK isneurones . AMPK acThe role of AMPK in the regulation of body weight and energy homeostasis is not limited to its actions in the peripheral tissues. In recent years, AMPK has emerged as a nutrient and glucose sensor in the hypothalamus , 9 and iet al. , a pharmacological activator of AMPK, into either the third ventricle or directly into the paraventricular nucleus of the hypothalamus significantly increased food intake . In a veet al.  showed t , whose signalling pathway in the hypothalamus partially overlaps with that of leptin, is given i.c.v. it not only reduces food intake of DIO mice, but also further suppresses hypothalamic AMPK activity, bypassing diet-induced leptin resistance  and long-term energy signals (chronic changes in diet-induced obesity (DIO). Fasting results in activation of AMPK whereas re-feeding inhibits AMPK activity in multiple hypothalamic regions in mice . Fastingor AICAR , 17, wheor AICAR . Long-teor AICAR . The lowor AICAR , 15. AMPor AICAR . In miceor AICAR , 19. Howsistance . Diabetisistance .et al. (11) studied the effect of leptin in the specific hypothalamic nuclei and showed that leptin inhibits AMPK activity in ARC and PVN. Furthermore, inhibition of hypothalamic AMPK is necessary for leptin’s effects on food intake and body weight, as constitutively active AMPK blocks these effects. A more recent study confirmed the importance of AMPK in the effect of leptin as AICAR was able to reverse the inhibitory effect of leptin on the electrical activity of glucose-inhibited neurones  neurones in the ARC  –34. Furtad libitum sucrose available to block the marked catabolism caused by high-dose corticosterone-pellets. Sucrose-drinking animals had lower hypothalamic AMPK activity compared to saline-drinking control rats, confirming the inhibitory effect of sucrose on AMPK activity. Corticosterone administration counteracted this effect of sucrose and increased hypothalamic AMPK activity to levels comparable with saline-drinking animals . Co. Co38).  animals a. The ef animals b and ana animals c. Metfor animals , was alset al.  inhibitor, which causes weight loss and anorexia, rapidly reduced the level of the phosphorylated AMPKα subunit in the hypothalamus . It alsoα-Lipoic acid, an antioxidant that reduces food intake, also inhibits AMPK activity in the hypothalamus . AICAR oAlthough there is evidence that thyroid hormones stimulate AMPK and acetyl-CoA carboxylase (ACC) expression in rat skeletal muscle , 48 and The role of AMPK as an appetite mediator has also been investigated in conditions known to affect food intake such as exercise and low temperatures.Exercise is known to influence appetite, but changes in energy metabolism during or after exercise are probably not coordinated by changes in hypothalamic AMPK as 1 h of strenuous exercise in rats did not elicit significant changes in hypothalamic AMPK activity despite an increase in plasma ghrelin . Other eInterestingly, AMPK has been shown to mediate cold-induced resistance to anorexigenic signalling in the hypothalamus . FeedingThe mechanisms through which AMPK influences feeding behaviour are not fully understood . Changesex vivo hypothalamus culture decreased expression of both AgRP and NPY, whereas pyruvate supplementation suppressed 2-DG-induced AgRP expression  pathway and the mammalian target of rapamycin (mTOR) pathway. Some studies show that modulation of AMPK by leptin , α-lipoiThe anorexigenic effect of leptin is mediated by inhibition of AMPK, probably in a coordinate manner with the signal transducer and activator of transcription (STAT3) and PI3K pathways . The actAnother possible downstream target of AMPK in the hypothalamus is the mTOR pathway. AMPK affects protein synthesis in the periphery through the mTOR-P70S6-kinase pathway . In the Several studies have investigated the role of AMPK in glucose sensing by specialised glucose-sensing neurones in the hypothalamus and in the hormonal counter-regulatory response to hypoglycaemia. Appropriate counter-regulatory response is crucial for recovery from hypoglycaemia and AMPK activation appears to mediate this function . InsulinRecurrent hypoglycaemia can lead to a phenomenon known as hypoglycaemia-associated autonomic failure . In thisIn addition to the effect of AMPK on the counter-regulatory response control, AMPK also mediates the effects of glucose, and possibly other hormones, on the electrical activity of hypothalamic neurones.et al.  found that alterations in external glucose levels did not alter the excitability of all ARC POMC and AgRP neurones. The different results may be explained, in addition to the different techniques and experimental setups, by the presence of distinct subpopulations of GE and GI neurones with different neuropeptide phenotypes, responses to hormonal stimuli and, in some cases, different glucose-sensing mechanisms (Two types of glucose-sensing neurones have been identified. Activity of glucose-inhibited (GI) neurones decreases in response to increased glucose concentrations whereas glucose-excited neurones increase their activity in response to increased glucose concentrations . GI neuret al.  showed tneurones . Forced neurones . Based o (et al.  showed t (et al. , 71, a m (et al. . POMC α2 (et al. . By cont (et al. . It is ichanisms . Future AMPK activity also influences NO production and neuronal activity in GI neurones. The AMPK activator, AICAR, increases both NO production and neuronal activity in GI neurones of the VMH . Glucoseet al. (One of the most interesting findings of the recent research into hypothalamic regulation of appetite is the neuronal link between the hypothalamus and the periphery. The hypothalamic regulation of peripheral metabolism that had previously been recognised seems now to extend to the AMPK as well . Minokoset al.  describeet al. .In leptin-over-expressing transgenic mice on a HFD, muscle AMPK phosphorylation and ACC phosphorylation are reduced compared with standard diet leptin-over-expressing transgenic mice and are comparable to HFD-non-transgenic mice. In these animals, leptin i.c.v., in addition to transgenic hyperleptinaemia, is not able to restore the impaired AMPK signalling because of the induced generalised leptin resistance. However, MT-II i.c.v. significantly augments AMPK and ACC phosphorylation, suggesting that MT-II is a potent AMPK activator in muscle, even in mice on a HFD . CentralInterestingly, hypothalamic AMPK has also been implicated in the regulation of muscle glycogen synthesis as i.c.v. AICAR treatment increased both insulin-mediated and non insulin-mediated glycogen synthesis . CentralHypothalamic AMPK, as described above, also mediates the counter-regulatory response to hypoglycaemia, increasing the release of peripheral hormones such as corticosterone, catecholamines and glucagon , 67, 68.These data emphasise the complexity of the regulation of whole body metabolism, and the role of AMPK being not only a peripheral or a central mediator, but also a key enzyme in coordinating the interaction between peripheral and central energy regulation.ad libitum feeding conditions , whereaabinoids , adiponeabinoids ]. A furtabinoids . Therefo