R = −0.80 across the entire human proteome). Notably, statistically significant anti-matching is seen only in the case of adenine. Our results provide strong evidence for the stereo-chemical foundation of the genetic code and suggest that mRNAs and cognate proteins may in general be directly complementary to each other and associate, especially if unstructured.Recently, the ability to interact with messenger RNA (mRNA) has been reported for a number of known RNA-binding proteins, but surprisingly also for different proteins without recognizable RNA binding domains including several transcription factors and metabolic enzymes. Moreover, direct binding to cognate mRNAs has been detected for multiple proteins, thus creating a strong impetus to search for functional significance and basic physico-chemical principles behind such interactions. Here, we derive interaction preferences between amino acids and RNA bases by analyzing binding interfaces in the known 3D structures of protein–RNA complexes. By applying this tool to human proteome, we reveal statistically significant matching between the composition of mRNA sequences and base-binding preferences of protein sequences they code for. For example, purine density profiles of mRNA sequences mirror guanine affinity profiles of cognate protein sequences with quantitative accuracy (median Pearson correlation coefficient  In the 50 years since the discovery of messenger RNA (mRNA) , the relRelated to this, we have recently shown that pyrimidine (PYR) density profiles of mRNA sequences tend to closely mirror sequence profiles of the respective cognate proteins capturing their amino-acid affinity for pyridines, chemicals closely related to PYR . These fSaccharomyces cerevisiae . An amino-acid residue and an RNA base were considered to be neighbors and form a contact if their centers of geometry were separated by less than a given cutoff distance. All the results reported in the main manuscript are given for the cutoff of 8 Å, whereas for testing purposes, this cutoff was also varied between 6 and 10 Å with a 0.25 Å step. We separately analyzed contact statistics for residues having at least one neighboring base , at least two neighboring bases  or include only the two closest neighboring bases .All available structures of protein–RNA complexes (both X-ray and nuclear magnetic resonance structures) were downloaded from the Protein Data Bank (PDB)  in Septerevisiae , Escherihia coli  and Thermophilus  ribosomeiodurans  and Haloi = 1, … ,20 for amino acids and j = 1, … ,4 for bases) were estimated using the following standard distance-independent contact potential formalism with the quasi-chemical definition of the reference state wheInteraction preference scales of amino acids were obtained separately for guanine (‘G-preference’), adenine (‘A-preference’), cytosine (‘C-preference’), uracil (‘U-preference’), PUR  and PYR (both C and U ‘PYR-preferences’).http://www.ebi.ac.uk/ena). The protein as well as RNA sequences with only canonical amino acids or nucleotides were chosen for analysis. The complete set of mRNA/protein sequences used herein is included in the Supplementary Data. The average content of codons when it comes to individual nucleobases or PYR or PURs for all 20 amino acids  was extracted from the thus-obtained cognate mRNA and protein sequences.The sequences of the complete human proteome (17 083 proteins) and coding sequences of their corresponding mRNAs were extracted from UniProtKB database (January 2013 release), with maximal-protein-evidence-level set at 4 (i.e. proteins annotated as ‘uncertain’ were excluded) and with only the reviewed Swiss-Prot  entries R) were calculated between nucleobase preferences and ‘codon content’ scales and between sequence profiles of nucleobase content for mRNAs and of different amino-acid preference scales for proteins from the complete human proteome set. Before comparison, the profiles were smoothed using a sliding-window averaging procedure; the window size of 21 residues/codons was used for all calculations.Pearson correlation coefficients  of the observed correlations was estimated using a randomization procedure involving random shuffling of the interaction preference scales. Each scale was shuffled one million times, and Pearson correlation coefficients (R) against codon content scales as well as for mRNA/protein profiles were calculated for each shuffled scale. The reported P-values correspond to the fraction of shuffled scales, which exhibit a higher absolute R than the original  in the case of codon content comparisons, or for which <R> is higher in absolute value than <Roriginal> in the case of sequence-profile comparisons.Statistical significance  for the six cases of direct correspondence between nucleobase preferences and nucleobase composition profiles  and for the G-preference versus PUR mRNA content one (Gprotein-PURmRNA). Each of these subsets contains 1707 proteins, for which average disorder values were assigned. Means and standard deviations of the 14 thus-obtained distributions of average predicted disorder were compared with those of the entire human proteome (background). The significance of the mean difference from the background was estimated for each of the analyzed subsets using the Wilcoxon signed-rank test. The gene ontology (GO) analysis was performed for the same seven top 10% best-matching protein subsets using DAVID functional annotation server  ≤10−10 were considered.The average disorder for each protein sequence in the human proteome was predicted using IUpred server . Fourteen server . The enthttp://www.pymol.org/) .The 3D structures of protein–RNA and amino acid/nucleobase complexes were visualized using PyMol (ol.org/) . ContactSupplementary Table S1). We use distances between centers of geometry of amino-acid side chains and nucleotide nitrogenous bases in combination with a fixed cutoff to define contacting neighbors  and use them to address the following questions: (i) how does the average composition of mRNA codons coding for a given amino acid relate to the preferences of this amino acid to interact with different nucleobases at protein/RNA interfaces and (ii) how does sequence density of different bases in mRNA-coding sequences relate to sequence profiles of amino-acid interaction preferences for these and other bases in cognate protein sequences?Using standard distance-independent contact potential formalism , we subsR of −0.84 . The observed signal for G is statistically highly significant as evidenced by randomization calculations . On the other hand, the interface statistics for adenine (A) and uracil (U) do not correlate with their average usage in codons. In particular, the A-preference of amino acids correlates inversely with the A-content (R = 0.59) or directly with the U-content of their codons (R = −0.51), whereas the U-preference exhibits relatively low correlations throughout  over the entire human proteome of −0.80 and a low P-value (<10−6) as determined by randomization  displaying typical  or best levels of correlation (Rmedian = −0.80 and P < 10−6). We also observe significant matching between C-preference profiles for protein sequences and both C- and PYR-density profiles of their cognate mRNAs with Rmedian of −0.55 and −0.47, respectively (Supplementary Table S2 for the full report of profile correlations) with Rmedian of −0.53. Finally, strong and significant level of matching is observed for PYR-binding preferences of amino acids and PYR mRNA profiles as well as PUR-binding preferences of amino acids and PUR profiles . This observation strongly suggests that close dense packing of nucleobases around amino acids may be required for specificity in cognate complex formation. Although interfaces may be dynamic and liquid-like, as we have suggested before, they may still need to be densely packed. Interestingly, if one reduces the 2+ set by including only the two closest bases in contact with a given amino acid (set ‘2’), the signal for G-preference/PUR-content even further improves by several percentage points (Supplementary Table S2).How sensitive is the level of matching to the choice of cutoff distance used to define contacting amino acids and nucleobases in protein/RNA complexes? To address this question, we have repeated the aforementioned analysis for a range of different cutoff values going from 6 to 10 Å in steps of 0.25 Å . Overalle points A, and thR coefficient . We have done this for the six cases of direct comparison whereby the same base type is used for both protein preference and mRNA profile density  and also for the case displaying the strongest signal in our analysis (Gprotein-PURmRNA). Importantly, in the case of Gprotein-GmRNA, Aprotein-AmRNA and Cprotein-CmRNA matching, we do observe a pronounced tendency for the top and the bottom 10% cohorts to be significantly enriched (top 10%) and depleted (bottom 10%) in disordered proteins (Supplementary Table S3), whereas in the case of Uprotein-Uprof matching, the situation is reversed. Interestingly, for PURprotein-PURmRNA, PYRprotein-PYRmRNA and Gprotein-PURmRNA matching, one observes slight disorder enrichment in both top and bottom cohorts. The most prominent shift of the distribution of predicted average disorder toward higher disorder as compared with background is observed for the top 10% cohort of proteins displaying strong matching between C-preference profiles of their sequences and the C-content of their cognate mRNAs . One might argue that this effect could just be related to compositional properties of such protein and mRNA pairs, whereby disordered proteins are simply encoded by C-rich sequences. However, the differences between nucleobase compositions of mRNAs from the Cprotein-CmRNA top 10% cohort and the complete proteome are minor, suggesting that the underlying explanation might be more complex .To further study the role of protein structural disorder in matching, we have analyzed the levels of the predicted disorder of the top and the bottom 10% of proteins when it comes to the degree of mRNA/protein profile matching as captured by Pearson Supplementary Table S4, we report the most significantly enriched biological functions  shared by proteins from the analyzed cohorts. In a striking agreement with our hypothesis, in most cases, we observe pronounced enrichment of terms related to nucleic-acid/protein interactions, including regulation of RNA metabolic processes, ribonucleoprotein complexes and transcription. The latter, in particular, allows one to speculate that protein tendencies to associate with cognate mRNA might be used by the cells to modulate gene expression pathways. What is more, PUR or PYR density profiles of mRNAs are identical to PUR or PYR density profiles of coding-strand DNA sequences (with Us being replaced by Ts). Although based on our statistical potentials, we cannot say anything about T-binding preferences of amino acids, it is possible that our results may be generalizable even to DNA-protein interactions as well as other RNA-protein interactions. One should also mention that depending on the particular type of matching, other biological functions also tend to be enriched. For instance, the Uprotein-UmRNA top 10% subset displays significant enrichment of membrane proteins, whereas Gprotein-PURmRNA top cohort seems to be populated by extracellular proteins and particularly those involved in the functioning of the innate immune system. Altogether, our preliminary GO analysis illustrates significant functional differences between proteins that strongly complement their cognate mRNAs and the rest of the human proteome, and these findings will be further explored in another manuscript.Which biological functions might be associated with a high level of complementarity between proteins and cognate mRNAs? To address this question, we have performed GO analysis for seven different top 10% subsets of proteins displaying strong matching with cognate mRNAs . In High levels of matching between base-binding-preference profiles of proteins and PYR- or PUR-density profiles of cognate mRNA-coding sequences, defined primarily by amino acid preferences to co-localize with G and C bases at RNA/protein interfaces, allow one to speculate that direct complementary binding interactions may be a key element underlying the whole mRNA/protein relationship when it comes to both its evolutionary development as well as present day biology B. This aMoreover, our results provide a clear evolutionary perspective concerning the physico-chemical origins of translation in line with the stereo-chemical hypothesis of the origin of the genetic code . In partImportantly, it should be emphasized that the stereo-chemical hypothesis of the code’s origin may differ from the cognate mRNA/protein complementary interaction hypothesis in terms of its evolutionary underpinnings. Direct templating of proteins from mRNAs in ancient systems  does not necessarily imply that modern proteins directly interact with their own mRNA (complementary interaction hypothesis). However, our findings support the possibility that the origin of the genetic code and potential complementarity between proteins and cognate mRNAs might have the same physico-chemical background. It is well possible that other independent influences have shaped both effects, and the two hypotheses leave ample room for such refinements. However, we would like to stress that in our view, the two hypotheses are inter-linked: cognate binding is on the one hand a reasonable consequence of the stereochemical hypothesis, but on the other hand, it also gives a potential biological rationale for the early development of the code to begin with, such as stabilization of RNA structures by bound polypeptides, as has been suggested before .There are a number of open challenges concerning the aforementioned proposal. First and foremost, the structural features of mRNAs and cognate proteins impose severe constraints on any putative complementarity between the two. Namely, with the contour length of the mRNA coding part being ∼4.5 times longer than that of a cognate protein, it is not clear what structural arrangements may be consistent with any complementary interactions. We would like to suggest that structures of such complexes may be dynamic and liquid-like with mRNA stretches enveloping and solubilizing cognate protein stretches . Second,These challenges notwithstanding, our findings provide strong evidence that the ability to interact with mRNA might be a widespread phenomenon in the cell involving not only cognate proteins but also other proteins based on similar principles. The potential significance of such physico-chemical complementarity between mRNAs and proteins potentially extends to all facets of nucleic acid and protein biology in the modern cell including transcription/translation regulation ,10,47,48Supplementary Data are available at NAR Online.


This feasibility study was designed to assess the ability to measure mitochondrial DNA (mtDNA) in cerebrospinal fluid (CSF) cells that contributed to minimal disease/persistent or residual disease (MD/PRD) from children with acute lymphoblastic leukemia (ALL). Increase in mtDNA copies in cancer cells has been suggested to play a role in MD/PRD. CSF as well as blood specimens from 6 children were assayed for MD/PRD and mtDNA copy numbers by quantitative real-time polymerase chain reaction. Of 7 MD/PRD-positive specimens, 6 had increased mtDNA copy numbers; while 11 MD/PRD-negative specimens had no increase in mtDNA copy numbers, p < 0.003. This is the first proof-of-concept study to measure mtDNA copy numbers in MD/PRD-positive CSF specimens from children with ALL. Increase of mtDNA copy numbers in MD/PRD childhood ALL cells and its significance as a mechanism for recurrence requires further investigation.Minimal residual disease; Acute lymphoblastic leukemia; Central nervous system; Cerebrospinal fluid; Mitochondria The presence of minimal disease/persistent or residual disease (MD/PRD) is associated with poor prognosis in childhood acute lymphoblastic leukemia (ALL) . In idenAssessing MD/PRD in the cerebrospinal fluid (CSF) provides additional challenges due to the relatively small number of cells. However, when central nervous system (CNS) relapse is diagnosed from analyzing CSF cells, the percentages of CSF lymphoblasts is relatively high compared to analyses of peripheral blood lymphoblast percentages. Thus on a per-cell basis, recognizing the opportunity to study mtDNA copies in CSF lymphoblasts provided the mechanism for the proof-of-concept study to determine if mtDNA copy numbers could be measured in CSF lymphoblasts from clinical specimens positive for MD/PRD. We posit that CSF and peripheral blood specimens, which are positive for MD/PRD, would have increased mtDNA copy numbers compared to MD/PRD-negative specimens.CSF cells and peripheral blood mononuclear cells (PBMC) were obtained from a tissue repository which had previously processed and stored CSF and blood from pediatric ALL cases without patient identifiers as per guidelines by the University of Hawaii Institutional Review Board. Cases were selected from the repository from which diagnostic and follow-up specimens were available for CSF; and two consecutive follow-up specimens available for PBMC; all from children diagnosed with pre-B-cell ALL. Age, gender, and limited clinical data were available for each case.The PBMC and CSF samples were previously assessed for MD/PRD , 11. Bri2 copies to 106 copies containing a specific portion of mtDNA and the genomic sequence were prepared to establish the standard curves. The mitochondrial primers  amplified the mtDNA region encoding the NADH dehydrogenase subunit 2, while the genomic primers  amplified the encoding region for Fas ligand. DNA from the specimens and appropriate positive/negative controls were set up in either duplicate or triplicate with each reaction containing iQ SYBR Green Supermix, 10 pmol forward primer and 10 pmol reverse primer with cycling parameters: 95oC for 1 min; 40 cycles of 95oC for 30 sec; 58.9oC for 40 sec; 72oC for 4 min; and 50oC for 30 sec. At the conclusion of the PCR, melt curve data were collected from a starting temperature of 65oC, with 0.5oC incremental increases every 5 sec for duration of 70 cycles. Copy numbers of mtDNA were obtained as previously reported with the average mtDNA copy number in PBMC or non-malignant cells ranging from 60-100 copies per cell [All MD/PRD-positive and MD/PRD-negative specimens from the repository were repurified by ethanol precipitation, reconstituted in Tris-HCl-EDTA buffer, and found to be adequate in quantity and quality by UV spectrophotometry. Using techniques previously established in the laboratory, a multiplex real-time PCR assay was performed on each DNA specimen to amplify a Fas ligand nuclear region and a region of the mtDNA . Brieflyper cell .The Mann-Whitney test was used to determine the associations between the mtDNA copy numbers/cell in the MD/PRD-negative and MD/PRD-positive specimens. Statistical analyses were performed using SPSS, Version 16 .Fourteen CSF samples from four children diagnosed with B-cell ALL and four additional PBMC specimens from two children with the same diagnoses were available from the tissue repository which comprised of diagnostic and follow-up specimens. Gender, age, and clinical course of the children are summarized in Analysis of the clinical course and CSF specimens from Patient 1 showed that the child remained in clinical remission with no clinical CNS relapse; and this was reflected in the MD/PRD-negative status of the CSF diagnostic and follow-up specimens. The diagnostic and follow-up mtDNA assessment of the MD/PRD-negative CSF specimens showed that the mtDNA copy number remained relatively stable and within the normal range measured in non-malignant cells (60 - 100 copies/cell) . SimilarThe CSF analyses from the next two patients showed parallel changes in MD/PRD status and mtDNA copy numbers/cell. Patients 3 and 4 both had clinical CNS disease at diagnoses; and both CSF specimens at diagnoses were MD/PRD-positive. While the diagnostic CSF for Patient 3 had high mtDNA copies/cell at diagnosis (191 copies/cell), in contrast, the diagnostic CSF from Patient 4 was low (63 copies/cell); even though both children had clinical CNS disease initially. Both Patients 3 and 4 developed CNS relapse within one year of diagnosis; and both patients had CSF specimens during the first year that were MD/PRD-positive and had high mtDNA copies/cell, 173 copies/cell at Visit 2 and 143 copies/cell at Visit 3, respectively.To assess the relationship of mtDNA copy numbers/cell in PBMC in children with ALL, Patients 5 and 6 were selected because both children developed relapse of their leukemia within one year of diagnoses . From boThe overall objective of this study was to determine the ability and feasibility of measuring mitochondrial DNA (mtDNA) copies per cell in CSF cells from children diagnosed with ALL in the context of the presence or absence of MD/PRD. The rationale for looking at CSF cells in childhood ALL cases in the context of mtDNA copy numbers for the current study focused on the fact that CSF lymphoblasts, when present, typically make up a higher percentage of cells in the CSF . The desIn conclusion, this study was designed as a proof-of-concept to test the feasibility of measuring mtDNA copy numbers from CSF specimens. Limitations of the study design center around the number of cases analyzed. Expanded studies are necessary to address the hypothesis that children with ALL who have MD/PRD-positive CSF specimens with high mtDNA copy numbers per cell may be at risk for CNS relapse. CNS relapse remains a major cause of morbidity in childhood ALL with treatment failure attributable to de novo or acquired resistance to a wide variety of cytotoxic drugs such as multi drug resistance , 16. Add


Listeria monocytogenes spreads within human tissues using a motility process dependent on the host actin cytoskeleton. Cell-to-cell spread involves the ability of motile bacteria to remodel the host plasma membrane into protrusions, which are internalized by neighboring cells. Recent results indicate that formation of Listeria protrusions in polarized human cells involves bacterial antagonism of a host signaling pathway comprised of the scaffolding protein Tuba and its effectors N-WASP and Cdc42. These three human proteins form a complex that generates tension at apical cell junctions. Listeria relieves this tension and facilitates protrusion formation by secreting a protein called InlC. InlC interacts with a Src Homology 3 (SH3) domain in Tuba, thereby displacing N-WASP from this domain. Interaction of InlC with Tuba is needed for efficient Listeria spread in cultured human cells and infected animals. Recent structural data has elucidated the mechanistic details of InlC/Tuba interaction, revealing that InlC and N-WASP compete for partly overlapping binding surfaces in the Tuba SH3 domain. InlC binds this domain with higher affinity than N-WASP, explaining how InlC is able to disrupt Tuba/N-WASP complexes.The bacterial pathogen  Listeria monocytogenes and Shigella flexneri and select species of the arthopod-borne genus Rickettsia, use an actin-based motility process to actively spread within human tissues  of Arp2/3, whereas IcsA stimulates Arp2/3 through recruitment of a human nucleation promoting factor known as N-WASP. Unlike ActA or IcsA, the Rickettsia surface protein Sca2 directly promotes assembly of actin filaments independently of Arp2/3 or other host factors. Sca2 may act as a functional mimic of a class of eukaryotic actin polymerization proteins called formins. Compared to the fairly detailed understanding of F-actin assembly by Listeria, Shigella, and Rickettsia, much less is know about how these pathogens produce protrusions that mediate cell-to-cell spread. This mini-review focuses on recent studies with Listeria that have shed light on the molecular mechanism of protrusion formation.Much progress has been made in understanding the mechanisms of F-actin assembly by Listeria  was found to be partly defective in in cell-to-cell spread in the polarized human cell line Caco-2 BBE1. Confocal microscopy analysis indicated that the ΔinlC mutant is unaffected in phagosomal escape or in the ability to produce F-actin tails. Instead the mutant is compromised in the formation of protrusions, producing these structures at only about 50% the efficiency of the wild-type bacterial strain.Recent work led to the identification of microbial and human proteins that regulate protrusion formation by Listeria proteins  results in a protein that folds normally, but is partly defective in binding the Tuba SH3-6 domain  that was about 5-fold higher than that of the isogenic wild-type strain. Importantly, this increase in LD50 was essentially identical to that observed for a mutant strain deleted for the inlC gene (ΔinlC). Compared to the wild-type strain, the inlC.K173A mutant strain exhibited lower bacterial loads in the liver. Histological analysis of livers indicated that the inlC.K173A strain produced smaller foci of infection than did the wild-type strain. These smaller foci are consistent with a role for InlC in cell-cell spread in vivo. Taken together, these results provide evidence that interaction of InlC with host Tuba is important for full virulence.A recent study took advantage of the The molecular and atomic details underlying the displacement of N-WASP from Tuba SH3-6 by InlC  helical conformation and binds in a class II orientation  involves the n-Src loop, the N-terminal residues of the RT-Src loop, the 310-helix and β-strand 5. This binding interface substantially overlaps but is not identical to that of the peptide binding groove. However, residues one to eight of the 12-reside peptide substantially clash with InlC in a superposition such that peptide binding to Tuba SH3-6 is eliminated by InlC binding , while in vivo an engineered Listeria strain carrying this mutation behaves identically to an inlC deletion mutant both in spread of bacteria and in protrusion formation  is needed for the resolution of protrusions into vacuoles containing Listeria (Chong et al., Listeria spread.Another critical unresolved question is what host physiological processes underlie Tuba-mediated regulation of The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Improvement of hand hygiene compliance in all-day setting is crucial and thus a multifaceted approach is generally advocated.In order to assess the influence of signal coloured hand disinfectant dispensers the number of dispenser activities was documented and analyzed in comparison to historical data and with respect to the number of health-care workers.In a 14-bed cardiology ICU all 25 standard dispensers were exchanged with signal-coloured dispensers . Dispenser activities were documented dispenser-specifically for 12 weeks and analyzed by week. The use of gown pocket dispensers was forbidden during the investigation. Health care workers had been trained in hand hygiene for the last three years continuously.A total of 81654 dispensers activities translating to 40827 hand rubs were documented with the majority (72%) occurring at patient-near dispensers . There was no time-dependency with dispenser activities ranging from 6119 to 8026 per week. Taking the number of patient days into consideration these activities led to 39 to 41 hand-rubs per patient day. This ranges between the 75. and 90. percentile in the national benchmark. Comparison with historical data revealed no difference. However, the nurse to patient ratio decreased during the study period by 8.5%. Taking an increased workload into consideration  revealed a 6% increase in staff-adjusted compliance.Signal-coloured dispensers seem to be a useful additional tool to improve and maintain hand hygiene compliance even in ICUs with an already good hand hygiene compliance.None declared


Timely access to facilities that provide acute stroke care is necessary to reduce disabilities and death from stroke. We examined geographic and sociodemographic disparities in drive times to Joint Commission–certified primary stroke centers (JCPSCs) and other hospitals with stroke care quality improvement initiatives in North Carolina, South Carolina, and Georgia.We defined boundaries for 30- and 60-minute drive-time areas to JCPSCs and other hospitals  by  using geographic information systems (GIS) mapping technology and calculated the proportions of the population living in these drive-time areas by sociodemographic characteristics. Age-adjusted county-level stroke death rates were overlaid onto the drive-time areas.Approximately 55% of the population lived within a 30-minute drive time to a JCPSC; 77% lived within a 60-minute drive time. Disparities in percentage of the population within 30-minute drive times were found by race/ethnicity, education, income, and urban/rural status; the disparity was largest between urban areas (70% lived within 30-minute drive time) and rural areas (26%). The rural coastal plains had the largest concentration of counties with high stroke death rates and the fewest JCPSCs.Many areas in this tri-state region lack timely access to JCPSCs. Alternative strategies are needed to expand provision of quality acute stroke care in this region. GIS modeling is valuable for examining and strategically planning the distribution of hospitals providing acute stroke care. Timely acute stroke care is necessary to substantially reduce disabilities that affect stroke patients ,2. For iExpeditious transport to hospitals capable of administering quality stroke care is an essential component of timely acute stroke care. Given that long delays can occur between onset of stroke symptoms and arrival at an emergency department -9 and thRecommendations for the Establishment of Stroke Systems of Care  were able to meet criteria for designation as a primary stroke center.We conducted a drive-time analysis to determine proportions of the population living within 30- and 60-minute drive times to primary stroke centers and to hospitals participating in stroke care quality improvement programs.We obtained 2000 population data by census tract for the tri-state region from the US Census . The stu2 analyses using SAS version 9.1 .We used Network Analyst 9.3  and StreetMap Pro 2007 road network software (ESRI) to create drive-time areas of 30 and 60 minutes to JCPSCs. We used the geographic information system (GIS) method of aerial interpolation to estimate the population residing within 30- and 60-minute drive-time areas. We first calculated the proportion of each census tract in 30- and 60-minute drive-time areas. Next, we used that proportion to calculate the percentage of the population residing within 30- and 60-minute drive-time areas by sociodemographic characteristics. We performed χWe obtained sociodemographic data from the 2000 US Census . We baseInternational Statistical Classification of Diseases, 10th Revision  were included in the analysis, we observed substantial increases in percentage of the population within 30-minute and 60-minute drive times, suggesting that these hospitals are well located to serve populations that do not have timely access to JCSPCs.These results highlight opportunities to strengthen stroke systems of care by including hospitals that may not meet the criteria for designation as a JCPSC but have the potential to provide acute stroke treatment services. The 2 main avenues for expanding the role of these hospitals are 1) establishment of an additional set of evidence-based criteria for acute stroke treatment center designation that complements but does not replace the JCPSC criteria (not unlike the multilevel trauma center designation) and 2) enhancement of telestroke networks.www.hfap.org/AccreditationPrograms/acute.aspx).An additional set of evidence-based stroke center criteria to complement the existing JCPSC criteria would enhance stroke systems of care by recognizing the vital role that smaller hospitals fill in treating stroke patients, especially in rural areas such as those in the tri-state region of North Carolina, South Carolina, and Georgia. Healthcare Facilities Accreditation Program is 1 example of an additional stroke center certification program that has certified 13 primary stroke centers nationwide  whether hospitals or facilities can appropriately be classified as stroke centers. Hospitals relying on remote survey techniques to self-report their stroke treatment capabilities may overestimate their stroke service capabilities . An addiTelestroke refers to the application of telemedicine in stroke care, in which consultation is performed by a remotely located expert through the use of high-quality videoconferencing. This type of remote evaluation of stroke patients is increasingly being used and strengthens state and regional stroke systems of care ,22. The Two other studies have estimated the percentage of the population living within a 60-minute drive time to stroke centers, with results similar to those of this study. In Canada, 67% of the total population lived within a 60-minute drive time to a hospital capable of administering tPA . In the The use of Network Analyst provides a more accurate representation of drive times than the more commonly used uniform distance buffers. However, drive-time analyses are limited because neither approach is able to account for changes in the elevation of the terrain or faster driving speed by emergency medical services. Consequently, the drive times in mountainous areas may be underestimated and may be overestimated in settings where emergency medical services are readily available and traffic patterns allow higher speeds. Another limitation of our study is the use of death certificate data for calculating stroke death rates. Geographic variations in reporting practices could introduce bias into the geographic patterns of stroke death rates. However, past studies have not documented a systematic geographic bias in the accuracy of reporting stroke deaths .In summary, many geographic areas in the tri-state region of North Carolina, South Carolina, and Georgia, an area of the country with a historically high stroke death rate, lack timely access to JCPSCs. The results of this study highlight the need for alternative strategies to expand provision of quality acute stroke care in the tri-state region, particularly to underserved populations. Application of the GIS technique used in this study visually portrays the population effect of selected scenarios for improving access to timely acute stroke care.


The aim of this report is to address treatment outcomes of patients with early-stage seminoma in a single institution with special reference to patients with history of surgical violation of the scrotum.Seventy four patients with pure seminoma were treated at King Hussein Cancer Center  between 2003 and 2010. All patients underwent orchiectomy. All but 3 patients received adjuvant radiotherapy. Patients who underwent surgical violation of the scrotum prior to referral were managed by further excision or irradiation of the scrotal scar. The follow-up ranged from 1 to 200 months .At the time of follow-up; all but one patient remain alive. The 3-year relapse-free survival for the entire cohort was 95.9%. Three patients developed relapse, all of whom received adjuvant irradiation following inguinal orchiectomy and initially harbored tumors larger than 4 cm upon pathological examination. Median time to relapse was 14 months . None were associated with elevated tumor markers prior to detection of relapse. All but one patient were successfully salvaged by chemotherapy.Our results confirm the excellent prognosis of patients with early-stage seminoma treated by orchiectomy and adjuvant radiotherapy in a developing country. Although all patients who developed relapse demonstrated adverse pathological findings upon initial assessment, no consistent predictor of relapse was found. Scrotal scar re-excision or irradiation in patients with prior history of surgical violation of the scrotum are effective measures in preventing local failure. Testicular cancer is the most common malignancy in men 20 to 40 years of age . More thDisappointingly, there is paucity of data assessing achievable outcomes of seminoma patients outside developed countries. The aim of this report is to address treatment outcomes in patients with early-stage seminoma in a developing country with special reference to patients who underwent surgical violation of the scrotum.Between January 2003 and December 2010; seventy four patients with histologically-confirmed pure seminoma  were treated at King Hussein Cancer Center (KHCC) . Records were electronically retrieved and retrospectively reviewed following acquisition of KHCC Institutional Review Board approval . Research conducted in this study was in compliance with the Helsinki Declaration. Exhaustive chart analysis was performed in an attempt to extract data pertaining to pathological characters, clinical stage, treatment, disease outcome and survival. Written informed consent was obtained from all patients. Patients were evaluated with thorough history, complete physical examination and computed tomography (CT) of the chest, abdomen and pelvis. Pathological diagnosis was confirmed by examining/re-examining surgical specimens obtained via ochiectomy by a staff pathologist at KHCC. Patients were classified according to the 2002 American Joint Committee on Cancer-International Union Against Cancer classification  and wereRadiation therapy was delivered via an appropriate energy Elekta linear accelerator . Conventional or CT simulation was performed in all patients. The para-aortic and dog-leg fields were treated via a two-field parallel-opposed technique to a total dose ranging from 2000 to 2500 cGy given over a period of 2 to 3 weeks (daily fractions in 5 consecutive days of the week). Standard, previously published, field designs were utilized . Figure Follow-up visits were conducted every 3 months for the first 2 years after radiotherapy and every 6 months thereafter. Clinical examination and analysis of AFP, LDH and β-HCG levels were recorded if available. CT scans of chest, abdomen and pelvis were performed twice a year for the first 2 years and annually thereafter for a total of 10 years. Acute and late radiotherapy-related side effects were recorded during radiotherapy and at each follow-up visit, respectively. Follow-up was in the form of a patient visit to a staff clinician at KHCC or retrieval of institutional-tumor registry and national data archives.The median age of our study population was 34 years . All patients underwent orchiectomy; 67 (90.5%) of which were performed via the inguinal approach. Seven patients were referred to our center following scrotal-approach orchiectomy. These patients were treated by further excision of the surgical scar (3 patients) or local-field irradiation of the scrotal scar delivered via an additional electron beam (4 patients). Fifty nine (79.7%) patients harbored T1 while 15 (20.3%) harbored T2 disease. None of the included patients had documented previous history of ipsilateral pelvic surgery. All but 3 patients received adjuvant radiotherapy via para-aortic fields in 63 (88.7%) patients or dog-leg fields in 7 patients (9.9%) (Table At the time of follow-up; all but one patient were alive. The 3-year relapse-free survival for the entire cohort was 95.9% Figure . Three pForty four testicular tumor cases were reported in the Jordanian national cancer registry in 2008. Although these neoplasms came short from ranking among the 10 leading cancer subtypes amongst males overall, testicular tumors ranked 7th and 5th amongst males aged 0–19 and 20–49 years, respectively [In this small series; we demonstrated the excellent prognosis for patients with early-stage seminoma treated by orchiectomy and adjuvant radiotherapy or active surveillance in a developing country. Unfortunately, there are no previously published reports assessing outcomes of seminoma patients in Jordan and as such we are unable to compare our results with those of another series. Furthermore, to the best of our knowledge, there is only one published study addressing testicular seminoma in a Middle Eastern country . This reRadical orchiectomy with ligation of the spermatic cord at the level of the internal inguinal ring is considered the standard treatment of seminoma . ScrotalPara-aortic radiotherapy is considered the standard adjuvant treatment of early stage seminoma with disease-specific survival rates approaching 100% ,13. The Although the para-aortic field has emanated as the standard radiation approach in seminoma patients , Power eDue to the overwhelming success of the treatment and the subsequent long-term survival, there has been growing interest in decreasing treatment-related morbidity in seminoma patients . Long-teSeveral reports have addressed the safety of surveillance in the management of stage I seminoma . AlthougAlthough no clear predictors of relapse were found, lymphovascular invasion, rete testis invasion and tumor size have all been reported to significantly increase the risk of relapse in patients with seminoma . In our Investigators have indicated that more than half of patients with relapse initially exhibit indicative symptoms and abnormal findings on physical examination highlighting the importance of patient education and meticulous medical examination . Less thOur results confirm the excellent prognosis for patients with early-stage seminoma treated by orchiectomy and adjuvant radiotherapy or active surveillance in a developing country. Although all patients who developed relapse demonstrated adverse pathological findings upon initial assessment, no consistent predictor of relapse was found. We have demonstrated the effectiveness of scrotal scar re-excision or irradiation in patients with prior history of surgical violation of the scrotum consequent to exploration or biopsy.The authors declare that they have no competing interests.JK, AS, FA conceived the study and drafted the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2490/12/10/prepub


However, whether D-ribose participates in glycation and leads to production of AGEs in vivo still requires investigation.D-Ribose, an important reducing monosaccharide, is highly active in the glycation of proteins, and results in the rapid production of advanced glycation end products (AGEs) Here we treated cultured cells and mice with D-ribose and D-glucose to compare ribosylation and glucosylation for production of AGEs. Treatment with D-ribose decreased cell viability and induced more AGE accumulation in cells. C57BL/6J mice intraperitoneally injected with D-ribose for 30 days showed high blood levels of glycated proteins and AGEs. Administration of high doses D-ribose also accelerated AGE formation in the mouse brain and induced impairment of spatial learning and memory ability according to the performance in Morris water maze test.These data demonstrate that D-ribose but not D-glucose reacts rapidly with proteins and produces significant amounts of AGEs in both cultured cells and the mouse brain, leading to accumulation of AGEs which may impair mouse spatial cognition. Non-enzymatic glycation of proteins by reducing saccharides such as D-glucose (Glc) and D-ribose (Rib) is a post-translational modification process D-ribose is a naturally occurring pentose monosaccharide present in all living cells including the blood and is a key component of many important biomolecules such as riboflavin  The rate of glycation depends upon monosaccharide concentration and anomerization rate and is inversely proportional to the number of carbon atoms in the reducing monosaccharide In vitro studies on the role of Rib in glycation have been carried out. Rib can glycate rat tail tendon collagen in vitro and the structure of the collagen is significantly altered by Rib-induced glycation in vitro, showing that ribosylation occurs much more rapidly than glucosylation. Ribosylated neuronal protein is much more cytotoxic than glucosylated protein. Nevertheless, little is known about whether Rib can rapidly induce glycation in cells and the AGEs produced by ribosylation can impair the cognitive function. Here, we treated cultured cells and mice with Rib and Glc to compare ribosylation and glucosylation for the production of AGEs. We found that Rib reacted rapidly with proteins and produced significant amounts of AGEs in cultured cells and mouse brain tissues, and that accumulation of AGEs impaired mouse spatial cognition. This finding implies that Rib-derived AGEs may be related to impairments of learning and memory ability.In this laboratory we have observed that glycation induces inactivation and conformational change of D-glyceraldehyde-3-phosphate dehydrogenase To investigate whether Rib leads to decreases in cell viability, SH-SY5Y human neuroblastoma (SH-SY5Y) cells and Human embryonic kidney 293T (HEK293T) cells were incubated with D-ribose or D-glucose at different concentrations. Cell viability was measured by MTT assays at 2 and 3 days after addition of the monosaccharide. After 2-day treatments, the viability of both cell lines decreased significantly in media containing 10 mM (P<0.05) or 50 mM (P<0.01) Rib, but it did not change markedly in the presence of Glc compared with the control . MTT assIt is known that AGEs have cytotoxicity in vitro, we investigated whether Rib is able to induce AGE formation in vivo.Having determined that Rib but not Glc is able to glycate proteins rapidly and produce high levels of AGEs in cultured cells D-ribose treatment does not cause significant dysfunction of the liver or kidneys. To compare the ability of Rib and Glc to elevate the level of AGEs in vivo, we intraperitoneally injected C57BL/6J mice with D-ribose or D-glucose  for 30 days. None of the sugar treatment groups showed any significant visual abnormalities and they gained weight normally within the period of treatment  -treated and Glc (both 0.2 g/kg and 2 g/kg) -treated mice spent significantly more time swimming in the target quadrant (where the platform was located) (no significant differences were observed among these four groups), than Rib (2 g/kg) -treated mice (P<0.01), whose time in each quadrant was more evenly distributed . These rin vitroAs a reducing saccharide, Rib reacts with protein amino groups to initiate a post-translational modification process widely known as non-enzymatic glycation Even though glycation of proteins with reducing saccharides has been widely studied, the formation of monosaccharide-induced intracellular AGEs has not previously been observed. Here, 10 mM Rib enhanced AGE formation in cultured cells, and diminished cell viability. This work is the first to show that Rib enhances the yield of AGEs in HEK293T and SH-SY5Y cells and primary cultured hippocampal neurons. Rib showed significantly higher cytotoxicity than Glc in cell culture. The high cytotoxicity of Rib may result from the rapid formation of AGEs as a result of ribosylation under these experimental conditions.Furthermore, this monosaccharide also enhances the yield of AGEs in both the hippocampus and cortex of the mouse brain after intraperitoneal injection. Impairment of spatial cognition was observed to be coincident with these increases in intracellular AGEs when Rib-treated mice were tested in the Morris water maze. Glc, however, was unable to elevate the yield of AGEs under our experimental conditions. This clearly demonstrates that an overload of Rib may result in a high level of AGEs in the brain and neurons. We would like to emphasize that Rib is much more effective than Glc in the glycation of proteins not only in vitro, but also in vivo. The intracellular AGE-enhanced cell model and the AGE-enhanced cognitive impairment mouse model successfully established here can be used for further investigation of the mechanisms behind the phenomena observed.As shown in the results of Western blotting  and 5, ain vivo, but also induce dysfunction of spatial cognition. This is the first time that Rib-induced cognitive impairment has been observed in mice. The spatial learning and memory ability of mice markedly declined after 30 days of Rib administration. However, those that were injected with the same concentration of Glc did not show significant impairments of spatial cognition compared with saline controls. This suggests that Rib overload for a relatively long period may be harmful to brain function. This treatment paradigm of inducing high levels of AGEs in the brain by administration of Rib can be used as a model for the study of dementia.High levels of Rib are not only able to enhance AGE formation in vitroHere, we believe that the decline in learning and the loss of memory are due to ribosylation and its resultant AGEs; glycation with Rib induces mouse spatial cognitive impairment. This viewpoint is based on the following observations and data. (1) Our previous work has shown that Rib is much more active in protein glycation than Glc AGEs were clearly evident in the hippocampus and cortex of Rib-injected mice . Glc, hoin vivo, and the mechanism by which Rib-induced AGEs impair spatial learning and memory, need further investigations.The relationship between AGE accumulation, cognitive decline and neurodegenerative disease is still under active investigation. Multiple studies have suggested that AGEs are directly neurotoxic to cultured neurons in vivo and cognitive dysfunction. Glycation affects the biological functions of proteins and crosslinking by glycation results in the formation of detergent-insoluble and protease-resistant aggregates. Thus, the effects of ribosylation on the human brain should be regularly examined during long term administrations of Rib as a drug. Our findings on the importance of ribosylation are relevant to medical professionals monitoring the therapeutic use of Rib.As a readily available source of energy, ribose is used to improve athletic performance and the ability to exercise by boosting muscle energy. It has also been used to improve symptoms of conditions such as chronic fatigue syndrome, fibromyalgia, and coronary artery disease In summary, we have shown that Rib rapidly reacts with proteins and produces AGEs in cells, inducing a decrease in cell viability. Administration of Rib to mice leads to the accumulation of a significantly high concentration of AGEs in the brain and subsequent impairment of spatial learning and memory ability. Administration of Rib can thus be used to establish a mouse model of dysfunction in spatial cognition.The handling of mice and experimental procedures were approved by the Animal Welfare and Research Ethics Committee of the Institute of Biophysics, Chinese Academy of Sciences (Permit Number: SYXK2010-128).2 incubator as described Human embryonic kidney 293T (HEK293T) cells To determine cell viability, we used the standard 3--2, 5-diphenyl tetrazolium bromide  test, with the slight modifications suggested by Mayo and Stein Male C57BL/6J mice (8–10 weeks) were obtained from Vital River Laboratory Animal Technology Co. Ltd. (China). After 1 week of acclimatization to the cages, mice were randomly divided into five groups and received intraperitoneal injections each day for 30 days with Rib at doses of 0.2 or 2 g/kg, or Glc at doses of 0.2 or 2 g/kg, or 0.9% saline (controls). All mice were maintained in animal facilities under pathogen-free conditions.After 30-days of injections, the Morris water maze (MWM) test was performed as described previously After behavioral testing, mice were sacrificed and their blood (about 0.7 ml per mouse) was collected as described previously Glycated serum protein (GSP) The level of AGEs or pentosidine was determined in cultured cells, brain tissues, and mice sera as described previously 2O2 in absolute methanol for 10 minutes to block endogenous peroxidase, and incubated in 10% normal goat serum in PBS at room temperature for 30 minutes. The specimens were incubated overnight at 4°C in anti-AGEs 6D12 monoclonal antibody solution diluted in PBS. After washing with PBS, sections were subsequently incubated with biotin-labeled secondary antibodies . The immunoreaction was detected using horseradish peroxidase-labeled antibodies  and red staining was visualized with an AEC system .As described Immunofluorescent staining was performed as described All values reported are means ± standard errors (SE) unless otherwise indicated. Data analysis was performed by one way analysis of variance (ANOVA) using Origin 7.0 statistical software. Differences with a probability level of 95% (P<0.05) were considered significant.


The organ of Corti and the vestibule in inner ears also suffer from degeneration caused by environmental stresses including noise and heavy metals, resulting in impairments of hearing and balance. In this review, we introduce impairments of hearing and balance caused by genetic and environmental factors and focus on impairments of SGNs and the vestibule in inner ears as the pathogeneses caused by these factors.Impairments of hearing and balance are major problems in the field of occupational and environmental health. Such impairments have previously been reported to be caused by genetic and environmental factors. However, their mechanisms have not been fully clarified. On the other hand, the inner ear contains spiral ganglion neurons (SGNs) in the organ of Corti, which serve as the primary carriers of auditory information from sensory cells to the auditory cortex in the cerebrum. Inner ears also contain a vestibule in the vicinity of the organ of Corti—one of the organs responsible for balance. Thus, inner ears could be a good target to clarify the pathogeneses of sensorineural hearing losses and impaired balance. In our previous studies with  It has been reported that about 250 million people worldwide suffer from hearing losses. About 30 % of people with congenital hearing loss are syndromic and the remaining 70 % are non-syndromic. In addition, most elderly people develop age-related (late-onset) hearing loss –3. In geInner ears have been analyzed in order to clarify the pathogeneses of sensorineural hearing losses. The inner ears contain the organ of Corti and stria vascularis (SV). The SV is essential for maintenance of endolymph potential. The organ of Corti contains two kinds of sensory cells [inner hair cells (IHCs) and outer hair cells (OHCs)] and plays an important role in mechanotransduction, by which sound stimuli are converted into electric stimuli. Auditory information from the sensory cells is transferred to spiral ganglion neurons (SGNs) as the primary carriers and is eventually transferred to the auditory cortex in the cerebrum , 2. The Ret and Ednrb) and by environmental stresses [low frequency noise (LFN) and heavy metals].Impairments of hearing and balance—both major problems in the field of occupational and environmental health—are caused by the intricate interplay of genetic, aging and environmental factors –3. HowevRET has been shown to be essential for the development and maintenance of the enteric nervous system (ENS)  mice [Ednrb/EDNRB cause syndromic hearing loss due to congenital defects of melanocytes in the stria vascularis of the inner ear [Ednrb homozygously deleted mice [Ednrb(−/−)-mice] developed congenital hearing loss , which is caused by mutations in the transcription factor Sox10 , cytokin mutated  and Ednr−)] mice  have beenner ear , 32–35. c-Ret-KIY1062F/Y1062F-mice developed severe congenital deafness with neurodegeneration of SGNs on postnatal day (P) 8-18, while c-Ret-KIY1062F/Y1062F-mice showed morphology of SGNs comparable to that in WT mice on P2-3 [c-Ret-KIY1062F/Y1062F-mice developed normally at least until P3 after birth, when Y1062 in c-Ret of SGNs from WT mice is unphospholylated. However, in c-Ret-KIY1062F/Y1062F-mice, phosphorylation of Y1062 is no longer maintained by P8–P18, when Y1062 in c-Ret of SGNs from WT mice exhibits significant phosphorylation [Ednrb(−/−)-mice with congenital hearing loss showed a decreased number of SGNs -mice did not show typical apoptotic signals and did not involve disturbance of hair bundles of IHCs and OHCs [Ednrb(−/−)-mice were improved markedly by introducing an Ednrb transgene under the control of the dopamine beta-hydroxylase promoter (Ednrb(−/−); DBH-Ednrb-mice). Neurodegeneration of SGNs was restored by introducing constitutively activated RET also in the case of c-Ret-mediated hearing loss. Thus, our results indicate that c-RET and EDNRB expressed in SGNs could be molecular targets in the prevention of hearing impairments.Phosphorylation of Y1062 in c-Ret has been shown to mediate several biological responses, including development and survival of neuronal cells , 37. In  on P2-3 . Phoshor on P2-3 . Thus, irylation . Furtherrylation . On the and OHCs , 36. TheExposure to noise is recognized as one of the major environmental factors causing hearing loss . Noise cOur studies provide direct evidence that c-RET and EDNRB expressed in SGNs are novel targets for hearing loss. These studies underline the importance of considering the activity as well as the expression of the target molecule in order to elucidate the etiologies of hereditary deafness. In addition, environmental stresses, including exposure to noise and heavy metals, can cause impairments of hearing and balance that are affected intricately by aging and genetic factors. Information obtained in previous studies prompts further investigation of the influence of environmental stresses on the impairment of hearing and balance with consideration of aging and genetic factors to develop new diagnostic, preventive and therapeutic strategies against impairment of hearing and balance.


At many endplates the large acetylcholine receptor plaque became fragmented into multiple smaller acetylcholine receptor clusters. 3) Between 19- and 25-months, the fraction of endplate acetylcholine receptors covered by synaptophysin fell 21%. By 28-months, half of the endplates imaged retained ≤50 µm2 area of synaptophysin staining. 4) Within aged muscles, the degree to which an endplate remained covered by synaptophysin did not depend upon the total area of acetylcholine receptors, nor upon the number of discrete receptor clusters. 5) Voluntary wheel-running exercise, beginning late in middle-age, prevented much of the age-associated loss of nerve terminal synaptophysin. In summary, a decline in the area of endplate acetylcholine receptor clusters at the onset of old age was followed by loss of nerve terminal synaptophysin from the endplate. Voluntary running exercise, begun late in middle age, substantially inhibited the loss of nerve terminal from aging motor endplates.Loss of connections between motor neurons and skeletal muscle fibers contribute to motor impairment in old age, but the sequence of age-associated changes that precede loss of the neuromuscular synapse remains uncertain. Here we determine changes in the size of neuromuscular synapses within the tibialis anterior muscle across the life span of C57BL/6J mice. Immunofluorescence, confocal microscopy and morphometry were used to measure the area occupied by nerve terminal synaptophysin staining and postsynaptic acetylcholine receptors at motor endplates of 2, 14, 19, 22, 25 and 28month old mice. The key findings were: 1) At middle age (14-months) endplate acetylcholine receptors occupied 238±11 µm There is increasing evidence to suggest that impaired motor innervation contributes to the weakness and impaired mobility of old age. Previous studies in humans have shown that from the age of 60 there were reductions in the number of motor units, with a compensatory expansion of the residual motor units The NMJ is a relay synapse. It is thought to depend upon its large size for proper function. Effective neurotransmission involves simultaneous release of quanta of acetylcholine (ACh) from many sites of exocytosis  onto postsynaptic membrane folds that contain tightly clustered acetylcholine receptors (AChR) A second feature of NMJs in aged rodents is the fragmentation/remodeling of postsynaptic specializations. In aged animals the endplate often consisted of many small AChR clusters Muscle usage can influence both pre- and postsynaptic elements of the NMJ over time. In young adult rats, 10 weeks of treadmill training produced an increase the size of the nerve terminal at the motor endplate A number of studies have investigated the effect of age on NMJ structure All mouse experiments described in this paper were conducted with the approval of The University of Sydney Animal Ethics Committee  in compliance with the NSW Animal Research Act 1985 and the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes 7th Edition NHMRC 2004.ad libitum. As a precaution against spread of pathogens sentinel mice, kept on the same rack, were sero-tested for pathogens quarterly by the animal facility veterinarian. The aging mice were inspected twice weekly by the authors and were euthanized if they developed an obvious tumor or sores that did not resolve. Over the course of the study 25% of the aging mice were found dead of natural causes. A further 9% required euthanasia  at 2-month of age, or as ex-breeders at 8-months . All mice were group housed in filter-top cages with wire-grid lids  and were fed rat and mouse chow (YSF brand) thanasia . When mithanasia  arrows tTo test the effect of exercise a low profile wireless running wheel (Med Associates Inc) was placed in the mouse cage. The number of wheel rotations was used to track average distance run over time, dividing the total distance run by the number of mice in the box. Overnight infrared video recordings showed that mice without access to a wheel engaged in some climbing on the underside of the wire cage grille. However sampling of the videos suggested that when aging mice were provided with a running wheel they spent about five-fold more of their time engaged in all exercise-type activities (mainly running), compared to aged mice without a wheel.At the time of sacrifice muscles were immediately dissected, embedded in OCT compound , snap frozen in liquid nitrogen and stored at −80°C. Cryosections were stained for immunofluorescence as described previously Confocal Z-stack images of NMJs were collected by scanning laser microscopy on the Leica SPE II confocal microscope . Endplates were imaged using an oil immersion 63X objective and a zoom factor of 2. The image size was 1024×1024 pixels and scanning was conducted at 600 Hz with the pinhole set to 1 Airy Unit. Z-stacks were collected with a spacing of 0.7 µm. To ensure retention of tonal detail, gain and offset were optimized for synaptophysin and AChR fluorescence at each individual endplate imaged? To reduce photomultiplier noise four scans were averaged. Precautions were taken to reduce the risk of subjective bias in sampling and analysis of NMJs. Within a given immunostaining experiment sections were collected from muscles of young versus old mice, or wheel versus non-wheel mice. Microscope slides were then coded by M.M. Image sampling and subsequent morphometry was then undertaken by A.C. blind to the age/treatment group of each slide. Each section was systematically scanned on the AChR fluorescence channel. All endplates that were relatively flat, presenting en face to the objective (AChR staining extending ≤15 µm in the Z dimension) were individually imaged and morphometrically analyzed.analyze particles’ command of ImageJ. Tiny suprathreshold areas consisting of fewer than 15 contiguous pixels  were excluded from the analysis. All remaining suprathreshold areas were summed to yield the total AChR area for the endplate. The minimum threshold method was also used to measure the area of synaptophysin-positive nerve terminal staining at the endplate. The total area of synaptophysin staining was assessed by summing all of the suprathreshold pixels that fell within the total (AChR) perimeter area using the ‘measure’ tool of ImageJ. At some endplates nerve terminal branches extended slightly beyond the AChR perimeter boundary. Such extensions were included in the measurement of nerve area. The ‘colocalization’ plug-in of ImageJ was used to create an overlay of the binary AChR and synaptophysin images, yielding the area of overlap.Two dimensional maximum intensity projection images were constructed from Z-stacks using the Leica software. Using NIH ImageJ software (rsbweb.nih.gov/ij/) a line was drawn to enclose all the AChR clusters comprising a single endplate. The area within this perimeter was defined as the total perimeter area. A minimum intensity threshold was then applied to the AChR staining such that the binary (suprathreshold) image included all AChR-rich structures in the endplate as perceived by eye from the full-tone image. This often yielded several discrete suprathreshold AChR clusters per endplate. The area of these AChR clusters was determined using the ‘Comparisons among the means for animals at multiple ages were tested by one-way ANOVA followed by Bonferroni's multiple comparisons post-test. Pair-wise comparison between the means for aged animals with and without a running wheel was by a two-tailed, unpaired Student's t-test. Some of the pooled data comparing endplates at multiple ages did not show a normal distribution so the non-parametric Kruskal-Wallis one-way ANOVA was used followed by Dunn's post-test. For all statistical tests significance was taken as P<0.05.2 while the average area of nerve terminal synaptophysin was 168±14 µm2 (mean ± SEM). Between 14- and 19-months (onset of old-age), there was a 30% fall in the average postsynaptic AChR area (At middle age (14-months) endplate AChR occupied 238±11 µm2 of synaptophysin-positive nerve terminal. At 2- and 14-months the distribution of endplate AChR area was also roughly Gaussian. The 19-month distribution revealed a transient shift to the left  rats, both pre- and post-synaptic specializations were significantly smaller than for young (8-month old) rats What is the significance of the fragmentation and shrinkage of postsynaptic AChR clusters? Previous rodent studies found that in old age the large endplate AChR plaque tended to become fragmented into a multiple smaller AChR clusters The most surprising result from the present study was the impact of voluntary running exercise. Early studies in which 28-month old mice were subjected to 3months of treadmill endurance exercise revealed reductions in the size of nerve terminals (stained with zinc iodide-osmium)


Finding where transcription factors (TFs) bind to the DNA is of key importance to decipher gene regulation at a transcriptional level. Classically, computational prediction of TF binding sites (TFBSs) is based on basic position weight matrices (PWMs) which quantitatively score binding motifs based on the observed nucleotide patterns in a set of TFBSs for the corresponding TF. Such models make the strong assumption that each nucleotide participates independently in the corresponding DNA-protein interaction and do not account for flexible length motifs. We introduce transcription factor flexible models (TFFMs) to represent TF binding properties. Based on hidden Markov models, TFFMs are flexible, and can model both position interdependence within TFBSs and variable length motifs within a single dedicated framework. The availability of thousands of experimentally validated DNA-TF interaction sequences from ChIP-seq allows for the generation of models that perform as well as PWMs for stereotypical TFs and can improve performance for TFs with flexible binding characteristics. We present a new graphical representation of the motifs that convey properties of position interdependence. TFFMs have been assessed on ChIP-seq data sets coming from the ENCODE project, revealing that they can perform better than both PWMs and the dinucleotide weight matrix extension in discriminating ChIP-seq from background sequences. Under the assumption that ChIP-seq signal values are correlated with the affinity of the TF-DNA binding, we find that TFFM scores correlate with ChIP-seq peak signals. Moreover, using available TF-DNA affinity measurements for the Max TF, we demonstrate that TFFMs constructed from ChIP-seq data correlate with published experimentally measured DNA-binding affinities. Finally, TFFMs allow for the straightforward computation of an integrated TF occupancy score across a sequence. These results demonstrate the capacity of TFFMs to accurately model DNA-protein interactions, while providing a single unified framework suitable for the next generation of TFBS prediction. Transcription factors are critical proteins for sequence-specific control of transcriptional regulation. Finding where these proteins bind to DNA is of key importance for global efforts to decipher the complex mechanisms of gene regulation. Greater understanding of the regulation of transcription promises to improve human genetic analysis by specifying critical gene components that have eluded investigators. Classically, computational prediction of transcription factor binding sites (TFBS) is based on models giving weights to each nucleotide at each position. We introduce a novel statistical model for the prediction of TFBS tolerant of a broader range of TFBS configurations than can be conveniently accommodated by existing methods. The new models are designed to address the confounding properties of nucleotide composition, inter-positional sequence dependence and variable lengths  observed in the more comprehensive experimental data now emerging. The new models generate scores consistent with DNA-protein affinities measured experimentally and can be represented graphically, retaining desirable attributes of past methods. It demonstrates the capacity of the new approach to accurately assess DNA-protein interactions. With the rich experimental data generated from chromatin immunoprecipitation experiments, a greater diversity of TFBS properties has emerged that can now be accommodated within a single predictive approach. Transcription factors (TFs) and their specific binding sites act to modulate the rate of gene transcription. They are central to key biological processes, such as organ development and tissue differentiation, nutrient and environmental stress responses and physiological signals. Delineating specific positions at which TFs bind to DNA is of high importance in deciphering gene regulation at the transcriptional level. Each TF binds a variety of DNA sites with sequence-specific affinity Classically, computational prediction of TFBSs is based on models called position weight matrices (PWMs) that reflect the preferred binding motifs associated to corresponding TFs by providing an additive score for any sequence. They approximate the true specificity of a TF and their parameters can be estimated through different methods  in Several efforts have explored more flexible models for the prediction of TFBSs. Bayesian hierarchical hidden Markov models (HMMs) have been used to model Recently, a new experimental technique has been developed to study sequences where proteins interact with DNA. This procedure is a combination of chromatin immunoprecipitation and massively parallel sequencing technologies - the well-known ChIP-seq procedure http://cisreg.cmmt.ubc.ca/TFFM/doc/ or directly through our web-based application at http://cisreg.cmmt.ubc.ca/TFFM/. TFFMs build upon the best properties of the established methods, while offering novel capacities within a unified framework. Every TF has its own DNA binding characteristics  which can be captured within the unified TFFM-framework.We introduce here a novel TFBS model and prediction system based on HMMs, hereafter referred to as TF Flexible Model (TFFM). Building upon previously developed models capturing dinucleotide dependencies and flexible lengths as described previously . In such models, each position Initially, we constructed standard first-order HMMs as TFFMs (denoted later as ion  see . In 1st-detailed TFFMs) mimicking the theoretical analysis made in In 1st-order TFFMs, starting a TFBS is given by a unique probability (representing the transition from the background to the TFBS) whatever the nucleotide found in the surrounding sequence. To allow for starting a TFBS depending on the nucleotide emitted in the background state, we implemented a more detailed and descriptive HMM template as TFFMs  were used to compare the two types of TFFMs with PWMs and DWMs. Sequences around the peak max positions (50 nucleotides on both sides) were extracted to construct the models and make predictions. The rationale for this is that ChIP-seq peak max positions represent where the maximum amount of ChIP-seq reads map on the genome of reference and TFBSs are expected to be strongly enriched in close proximity to the peak max position For each ENCODE ChIP-seq data set, the area under the curves (AUC) for the corresponding ROC curves  have been computed. To compare the predictive powers of the different methods, we focus on ChIP-seq data sets for which at least one predictive method achieves an Statistical significances of the differences in terms of discriminative power between the different methods has been computed for each pair of methods using a Wilcoxon signed rank test nt , see . Again, nt , see .To understand whether the TFFMs perform better than the PWMs because of the model or because of the training method (as both differ), we introduce a 0-order TFFM which is basically a PFM modeled by an HMM and trained using the Baum-Welch algorithm see . We usedThis analysis shows that the TFFMs perform better than PWMs and DWMs more often, with a statistically significant difference, and lead us to hypothesize that the TFFMs are, overall, better at capturing TFBS features found in the experimental data. To further evaluate this property, we analyzed how TFFM scoring correlates with the biological signal found in ChIP-seq data.An attractive feature of PWMs is that they can produce scores that are correlated with the energetic binding affinity between a protein and a DNA sequence In the previous section, we hypothesized that the signal values from ENCODE ChIP-seq peaks reflected the affinity of the TF protein to bind to DNA sequences. In For each predictive model, we computed the correlation between the predicted scores and the DNA-binding affinity values measured experimentally. Since some mutated sequences can no longer be bound by the Max TF (or with very weak affinity), it is interesting to focus on the sequences to which the TF can actually bind. Hence, we analyzed the correlation between predicted and experimentally measured DNA-binding affinity values by first focusing on the sequences lying in the top 10-percentile affinity values, then the top 20-percentile, and up to including all the sequences using 10-percentile steps. The results of higher interest, corresponding to stronger DNA-binding affinity values, are the top percentiles but all percentiles were computed for completeness. Using such a methodology, we expect the predicted scores obtained from the models to better correlate with high DNA-binding affinity values than with low values. To understand what characteristic(s) the TFFMs are capturing that is not represented by either the PWMs or the DWMs, we examined the DNA sequences obtaining the highest DNA-binding affinity values. We looked at the motifs for which the DNA-binding affinities are the highest by considering the top-scored 25, 50, 75, and 100 sequences see . Figure In In the previous sections, the TFFMs were used to model TFBSs with fixed length by taking into consideration the dinucleotide composition of the sequences. Another feature of the TFBSs that can be accommodated by TFFMs is flexible length.A subset of TFs bind to the DNA with different structural conformations, leading to TFBSs of different lengths et al. showed that JunD can bind to a core motif composed of either TGACGTCA or TGAC/GTCA where C/G stands for C or G. When applying MEME to the whole JunD K562 ENCODE ChIP-seq data set, we observed the enrichment of the two patterns. We used the data set exhibiting the greatest enrichment of the patterns to train and test the different models. We can see from the ROC curves in The first TF analyzed is JunD which has been previously shown to bind to motifs of flexible length using protein binding microarrays in mouse STAT TFs bind with a flexible length motif In the previous examples of flexible length binding motifs, we focused on motifs where a spacer was found between the two halves of a core motif. One can also consider motifs containing a flexible edge at the outer edges of a core motif. When analyzing MEME output for ENCODE ChIP-seq data, we observed that a MafK data set showed a weak motif on its edge separated by a 1 nt spacer from the core motif see . We hypoIn the previous sections, TFFMs have been used to predict specific TFBS positions. We extended the TFFM-framework to compute an integrated TF occupancy score across a DNA sequence using the TFFM scores. Using the TFFMs, the probability of occupancy (Pocc) of a TF within a defined DNA sequence is obtained by multiplying the TFBS probabilities at each position . This is a simpler approach than the physico-chemical models used in tools like GOMER ound see .In this report, we have introduced a flexible HMM-based framework for TFBS prediction. The new models are demonstrated to perform as well as classic methods for most data, while exhibiting improved performance for a subset of TFs. The new approach retains the desirable attribute of producing scores correlated with the binding energy of TF-DNA interactions. A new graphical representation is introduced to illustrate the properties of the models, complementing the classic and widely used sequence logos. In applications, the TFFM models have been shown to handle variable spacing between half sites, and to allow for the incorporation of flanking sequence properties into TFBS analysis. With a convenient software package and a breadth of opportunities for improvement, TFFMs are a suitable foundation for the next generation of TFBS prediction.The new TFFM-framework provides an opportunity for researchers to analyze more deeply the features of TF-DNA binding interaction by looking at local dinucleotide dependencies captured by the TFFMs and represented by the new logos. One can see the TFFMs as the probabilistic analog of the energetic BEEML models developed in For TFFMs, the greatest utility is in handling the growing subset of TFs with complex binding properties. Such complex binding characteristics of TFs may be decomposed into four categories The TFFM-framework creates new opportunities for innovation in TFBS bioinformatics analysis. Drawing from the initial studies here, it is apparent that refined approaches can be pursued for the identification of TFs capable of binding to motifs of variable width and the analysis of the role of TFBS flanking sequence on TF binding. While the number of cases of TFs tolerant of variable width binding sites has grown with access to high-throughput TFBS data, the TFFM-framework could be extended to enable a comprehensive survey of ChIP-seq data collections to identify additional cases. As observed in the analysis of MafK TFBS flanking sequences, TFFMs are sufficiently flexible to incorporate additional information represented in TFBS proximal sequences. There have been some indications that such sequences may specify interactions with co-factors cis-regulatory sequences. The global relationship of TFBSs and nucleotide variations is largely unknown Beyond the analysis of non-canonical TF binding motifs, there is a significant scientific opportunity to develop a new computational approach for the prediction of functionally significant DNA variations within http://cisreg.cmmt.ubc.ca/TFFM/doc/) to allow others to refine the approaches and make further innovations to broaden the use of TFFMs.A key to the long-term development and adoption of TFFMs is the access of researchers to both the binding models and the software for their generation. It is our plan to generate a collection of TFFMs trained on ChIP-seq data sets from ENCODE, as well as other sources compiled into the PAZAR repository The new TFFMs described in this report are designed to address the confounding properties of position inter-dependencies in site composition and variable lengths observed in experimental data. These two challenges have emerged as an increasing issue with the availability of large-scale ChIP-seq data, which reveals greater complexity of TFBSs than could be observed in the past. The TFFM graphical motif representation conveys properties of position inter-dependence, allowing researchers to visually analyze the features captured by the model. TFFMs have been assessed on human and mouse ChIP-seq data sets coming from ENCODE, revealing a higher discriminative power than established methods. TFFMs produce scores consistent with observed protein-DNA affinities measured experimentally and have the capacity to predict the impact of TF binding site mutations on TF-DNA binding affinities.The analysis of TFBS is a central challenge in bioinformatics. TFFMs provide a powerful and flexible framework within which a broad range of problems can be addressed. While many motif discrimination methods are available, it is our perception that TFFMs will emerge as a preferred approach for TFBS analysis.Comparisons between the different predictive methods were done using ChIP-seq data sets from the ENCODE project For assessing the performance of TFFMs allowing for flexible length motifs, we used the following ChIP-seq data sets: human ENCODE JunD TF from K562 cells by the University of Chicago, mouse ENCODE MafK TF from Ch12 cells by Stanford University, and STAT4 and STAT6 TFs from 1st-order HMMs used in 1st-order TFFMs are composed of a state modeling the background sequences surrounding TFBSs and one state per position ion  see . One canHMMs used in detailed TFFMs decompose each state of the 1st-order HMM with four corresponding states in the detailed HMM, each one emitting a nucleotide  with a probability equal to 1 see . Dinucleion  see .HMMs used in 0-order TFFMs are constructed with the same set of states as the ones used for the HMMs of the 1st-order TFFMs. The emission probabilities are different since no dependency between positions is captured. Hence, each state is associated to only four emission probabilities for the four nucleotides see .The TFFMs provide, at each position The different model predictive powers were compared using a 10-fold cross-validation methodology on human and mouse ChIP-seq ENCODE data sets.Given a ChIP-seq data set ftp://hgdownload.cse.ucsc.edu/goldenPath/hg19/encodeDCC/wgEncodeMapability/wgEncodeCrgMapabilityAlign36mer.bigWig). They were generated from contiguous 36mers, and keeping regions greater than 200 bp. These regions were split into 100 bp pieces. The background for each dataset was then drawn randomly by matching %GC composition of the foreground sequences.Ten background data sets .Train TFFMs on the 10 training sets from Apply the TFFMs on matching Compute the corresponding ROC curve.Apply MAST Construct corresponding PWMs  of a TFFM by summing the IC of all the positions computed as DNA-binding affinities between human Max transcription factor (isoforms A and B) and DNA sequences have been obtained experimentally by using the MITOMI method and reported in cor.test function of the stats R package By applying the 10-fold cross-validation methodology to the ChIP-seq data sets , we obtained a score for each one of the peaks (corresponding to the score of the best hit per peak). The ENCODE data associate a signal value to each one of the peaks. The signal value is a measure of the enrichment for the overall peak region . As the peak scores coming from ENCODE may be unevenly distributed, we computed the median of the distribution of prediction scores for the sequences within each 5-percentile of the peak scores. Hence, each peak score 5-percentile is associated to a predictive score corresponding to the median of their distribution within the percentile. A Spearman's rank correlation coefficient has been used to compare prediction scores and ChIP-seq peak scores for these latter data using the We compared experimental DNA-binding affinities from The detailed methodology used is as follows:Construct all 12 nt-long sequences conserving nucleotides GTG at positions 9, 10, and 11 (a nucleotide A is used at the very beginning for the 1st-order TFFM with no impact on the scores).Construct TFFMs, PWM (i.e. log-odds weight matrices), and DWM using an initialization from the top 600 peaks and trained on the whole ENCODE human Max K562 ChIP-seq data set.Compute the prediction scores on all the sequences using the different methods (only one strand is used for the score computations here).Score associations between experimental values and results obtained using the above methodology are given in cor.test of the stats R package Starting from the optimal binding site containing CACGTG, we also compared the changes in prediction scores between a mutated site and the optimal sequence with the changes using the experimental DNA-binding affinities for the same sequences. A Pearson correlation coefficient between the two scores is then computed using the function The first over-represented motif found by MEME in the JunD data set used for the flexible motif analysis is 14 nt-long with a G/C (G or C) at the centre of the core motif  and the Python port of the Template Toolkit (http://template-toolkit.org/python/index.html).The TFFM-framework is available at Figure S1Performances comparison between TFFMs and weight matrices. For the 94 ChIP-seq data sets obtaining an (EPS)Click here for additional data file.Figure S2ROC curves analysis. Comparison of the AUCs corresponding to the ROC curves analysis of the subgroup of 96 ENCODE ChIP-seq data sets for which at least one predictive method generated an (EPS)Click here for additional data file.Figure S3ROC curves analysis. Comparison of the AUCs corresponding to the ROC curves analysis of the subgroup of 94 ENCODE ChIP-seq data sets for which at least one predictive method generated an (EPS)Click here for additional data file.Figure S4Performances comparison between 0-order TFFMs, other TFFMs, and WMs. For the 94 ChIP-seq data sets used in (EPS)Click here for additional data file.Figure S5Statistical significance of the trend of correlation between ChIP-seq peak scores and the different predictive scoring methods. The statistical significance of the trend plot in p-value of getting a regression line (between ChIP-seq peak scores and TFFM or WM predicted scores) with an slope that far away from 0. We observe that the p-values are, overall, close to 0 and indicate that the average behaviour of the correlation given by a linear regression is far from an horizontal line.(EPS)Click here for additional data file.Figure S6Max sequence logo. Sequence logo corresponding to Max binding sites. Max binds to the DNA with the core site allowing for CACGTG nucleotides at positions (EPS)Click here for additional data file.Figure S7Sequence logos of the top DNA-binding affinity values. Sequence logos obtained from the top (A) 25, (B) 50, (C) 75, and (D) 100 scoring sequences using DNA-binding affinities measured experimentally.(EPS)Click here for additional data file.Figure S8Sequence logos for Max TF. Sequence logos obtained from the two TFFMs and the PWM after training on all the ChIP-seq data for human Max TF from the K562 cell line. (A) Summary TFFM logo for the 1st-order TFFM. (B) Summary TFFM logo for the detailed TFFM. (C) Sequence logo for the PWM.(EPS)Click here for additional data file.Figure S9Sequence logo for MafK TF. Sequence logo from the motif found over-represented with MEME. We observe a weak motif on the edge of the motif, starting at position 15, is separated by a 1 nt spacer from the core motif.(EPS)Click here for additional data file.Figure S10Sequence logos for JunD TF. Sequence logos of the top two motifs found over-represented with MEME. (A) The first over-represented motif found by MEME in the JunD data set is 14 nt-long with a G/C (G or C) at the centre of the core motif (see position 9). (B) The second most over-represented from MEME shows a CG (C followed by a G) at the centre of the core motif (see positions 7–8).(EPS)Click here for additional data file.Figure S11Sequence logos for STAT4 TF. Sequence logos of the top motif found over-represented with MEME.(EPS)Click here for additional data file.Figure S12Sequence logos for STAT6 TF. Sequence logos of the top motif found over-represented with MEME.(EPS)Click here for additional data file.Figure S13Discriminative power of the probability of occupancy. Using 1st-order and detailed TFFMs, the ratio between the AUC value obtained from the best site per sequence or the probability of occupancy (Pocc) and the best AUC obtained is plotted. (A) For the 97 ChIP-seq data sets for which at least one method obtains an (EPS)Click here for additional data file.Figure S140-order HMM schema. 0-order HMM schema used in 0-order TFFMs where the first state represents the background and the following states the consecutive positions within a TFBS. Each state emits a nucleotide with an independent probability for the previously emitted nucleotides.(EPS)Click here for additional data file.Table S1HMM-based tools for TFBS prediction comparison. Different HMM-based tools (names and references are given in the first and second columns) have been developed to predict TFBSs. The table compares different features of the tools. The third column indicates whether the models capture the dependencies between TFBS positions. The fourth column indicates whether the implemented HMMs allow for flexible length motifs. The fifth column indicates which tools implement the computation of probability of occupancy (Pocc) of a TF on DNA sequences. The sixth and seventh columns indicate whether the source-code of the tools is freely available for download and whether there is a corresponding online documentation. Finally, the last column indicates the tools for which a web-based application is available.(PDF)Click here for additional data file.Table S2Statistical significance for discriminative power differences between the predictive methods. The table contains the Benjamini-Hochberg corrected (PDF)Click here for additional data file.


A perspective by Chris Kenyon and Robert Colebunders discusses policy implications for use of first line antiretroviral therapies in resource-limited settings, emerging from a new research study conducted by Campbell and colleagues. PLoS Medicine:This Perspective discusses the following new study published in doi:10.1371/journal.pmed.10001290Campbell TB, Smeaton LM, Kumarasamy N, Flanigan T, Klingman KL, et al. (2012) Efficacy and Safety of Three Antiretroviral Regimens for Initial Treatment of HIV-1: A Randomized Clinical Trial in Diverse Multinational Settings. PLoS Med 9(8): e1001290. Thomas Campbell and colleagues report findings of a randomized trial conducted in multiple countries regarding the efficacy of antiretroviral regimens with simplified dosing.Choosing an optimal first line antiretroviral therapy (ART) in resource-limited settings (RLS) involves a careful balancing act. On the one hand, we would want to ensure that patients receive the most efficacious therapy available. On the other hand, the most efficacious regimens may be too costly for countries afflicted by the dual burdens of very high HIV prevalence and of resource scarcity. This dilemma is compounded by the recent emergence of good quality evidence to start ART earlier , which can improve life expectancy The World Health Organization (WHO) has thus advised that countries should consider a number of factors in addition to clinical efficacy when considering choice of ART. These include in-country ART costs, numbers of current and future individuals needing to start ART, and the country's national prevalence of chronic hepatitis B, tuberculosis, and anemia PLoS Medicine, Thomas Campbell and colleagues compared the efficacy and safety of three ART regimens for treatment naïve patients in the Prospective Evaluation of Antiretroviral Therapy in Resource-Limited Settings (PEARLS) trial In a new study published in this week's The trial found that ATV+DDI+FTC was inferior to EFV+3TC-ZDV in terms of treatment efficacy . EFV+3TC-ZDV and EFV+FTC-TDF were equally efficacious . This is in contrast to the only other randomized controlled trial to have evaluated the relative efficacy of these two regimens (GS-01-934), which found EFV+FTC-TDF to be more efficacious than EFV+3TC-ZDV How do we explain the discrepant findings between PEARLS and GS-01-934? The authors of PEARLS provide a plausible explanation relating to differences in how the primary efficacy endpoints were defined. GS-01-934 used as primary endpoint the US Food and Drug Administration (FDA) time to loss of virologic response (TLOVR). This includes all antiretroviral (ARV) substitutions as endpoints (failures). The ZDV containing arm had more adverse events than the TDF arm (22 versus 9), and this was the main determinant of the difference in primary endpoint between the two arms. Their explanation is given further credence by a reanalysis of PEARLS using the FDA-recommended TLOVR as the primary outcome. This transformed the PEARLS outcomes to concur with those of GS-01-934—the TDF regimen was superior.In our opinion, a cogent case can be made that including protocol-pre-specified drug substitutions in an efficacy endpoint that is labeled as virological suppression is misleading. Both the rates of virological suppression and drug side effects are important endpoints, but it is unhelpful and misleading to conflate the two into one category and then label this as virological suppression. Of further note, the analytical approach taken in GS-01-934 favoured the commercial funder of the trial, Gilead Sciences. Six of the 12 authors of GS-01-934 were employees of Gilead, who manufacture TDF, and according to the authors of GS-01-934, “the study was designed by and the data were analyzed at Gilead Sciences” The PEARLS study provides further evidence to back up the first two of four ART regimens listed by the WHO guidelines as acceptable first line ART regimens: AZT +3TC+EFV, TDF+3TC (or FTC)+EFV, AZT+3TC+NVP, and TDF+3TC (or FTC)+NVP PEARLS confirms the widely held view that TDF/FTC/EFV is an excellent first line ART regimen for use in RLS One of the most useful contributions that PEARLS makes to our knowledge of ART is the lack of heterogeneity of efficacy or toxicity of ART according to country, continent, race, and/or ethnicity. There have been numerous concerns raised in the literature that there may be increased risks of drug toxicity from particular ARVs in specific populations. It has been argued that there is a risk that both TDF Nevertheless, there are a number of limitations with the PEARLS trial that point the way for future research. One of these is the choice of protease inhibitor (PI)-based ART—unboosted ATV/DDI/FTC. A number of trials have shown the superiority of boosted- over unboosted-ATV, and this is now widely regarded as best practice