Acidithiobacillus ferrooxidans oxidizes sulfide ores and promotes metal solubilization. The efficiency of this process depends on the attachment of cells to surfaces, a process regulated by quorum sensing (QS) cell-to-cell signalling in many Gram-negative bacteria. At. ferrooxidans has a functional QS system and the presence of AHLs enhances its attachment to pyrite. However, direct targets of the QS transcription factor AfeR remain unknown. In this study, a bioinformatic approach was used to infer possible AfeR direct targets based on the particular palindromic features of the AfeR binding site. A set of Hidden Markov Models designed to maintain palindromic regions and vary non-palindromic regions was used to screen for putative binding sites. By annotating the context of each predicted binding site (PBS), we classified them according to their positional coherence relative to other putative genomic structures such as start codons, RNA polymerase promoter elements and intergenic regions. We further used the Multiple EM for Motif Elicitation algorithm (MEME) to further filter out low homology PBSs. In summary, 75 target-genes were identified, 34 of which have a higher confidence level. Among the identified genes, we found afeR itself, zwf, genes encoding glycosyltransferase activities, metallo-beta lactamases, and active transport-related proteins. Glycosyltransferases and Zwf (Glucose 6-phosphate-1-dehydrogenase) might be directly involved in polysaccharide biosynthesis and attachment to minerals by At. ferrooxidans cells during the bioleaching process.The biomining bacterium  Acidithiobacillus ferrooxidans is an acidophilic Gram-negative autotrophic γ-proteobacterium that obtains its energy through the oxidation of ferrous iron and reduced inorganic sulfur compounds. At. ferrooxidans, along with other bacterial, archeal and eukaryotic microorganisms forms structured biofilm communities in metal-rich low pH environments, where chemolithotrophic metabolism is the primary form of energy acquisition [At. ferrooxidans interacts with its energetic substrate is an important step towards rational design strategies to improve bioleaching efficiency [uisition . The biouisition . Severaluisition ,4. Howevficiency .N-Acyl homoserine lactone (AHL) mediated QS (AHL-QS) is the most intensively studied system. It has been characterized in great detail in Gram-negative bacteria. Molecular components of this system include LuxR-family transcriptional regulators, 18–20 nucleotide cis-acting elements called lux-boxes, AHLs and LuxI-family AHL synthases. LuxR-family proteins are AHL-responsive global transcriptional regulators that activate or repress transcription upon interaction with lux-boxes [Bacterial cell-cell communication is based on the regulation of gene expression of interacting bacteria through the exchange of low-weight organic compounds or small peptides that function as signals. Signaling mechanisms have a prominent role in the emergence of cooperative behaviors in bacterial populations and communities . Quorum ux-boxes . QS reguux-boxes  or as a ux-boxes ,11.At. ferrooxidans strain ATCC 23270 possesses a functional AHL-based QS system [luxI/luxR homologs termed afeI/afeR. Here, the QS locus comprises the divergently organized afeI and afeR genes and a third gene (orf3) located in the intergenic region between these two and with the same orientation as afeI. The protein product of orf3 is conserved among some bacterial species but its function is unknown. The synthase AfeI drives the synthesis of at least nine different AHL molecules with medium or large acyl side chains [afeI. This was demonstrated by using a heterologous biosensor construct in E. coli where AfeR promotes AHL-dependent transcription of afeI [afe-box regulatory element located upstream of afeI is an 18–20 bp palindromic sequence with nearly perfect dyad-symmetry. It overlaps the -35 region of a sigma 70 promoter consensus sequence, as it is in many other bacteria. However, the afe-box has the particularity of being the central fraction of a larger 30 bp palindrome with which it shares the same center of symmetry. Moreover, each half of the 30-bp sequence shows palindromy. Hence, two alternative centers of symmetry can be found in the total sequence [S system ,13. Thissequence .At. ferrooxidans cells to solid energetic substrates such as sulfur and pyrite [At. ferrooxidans, we have used a bioinformatic strategy to identify genes whose expression might be regulated by AfeR, as it has been described for other transcriptional regulators including CepR, a LuxR-family protein [afeI operator we constructed Hidden Markov Models (HMMs) to screen for putative binding sites for AfeR in the At. ferrooxidans ATCC 23270 genome. Results revealed that at least 75 genes corresponding to 2.6% of the identified ORFs from At. ferrooxidans could be direct targets for AfeR.Recently, it has been described that AHL molecules improve the attachment of d pyrite . As a st protein ,16. BaseafeI and orf3 genes of the At. ferrooxidans QS locus (afe-box) has an unexpected sequence complexity  were defined as “a fragment of the genome sequence where one or more HMM hit is found in either strand”. Thus, a total of 273 PBSs were identified. As expected, the A and B type PBSs were mainly overlapped. These two kinds of PBS were grouped in the “SP” set. The same kind of overlap occurred with the C and D HMM-models and therefore, these two kinds of PBSs were grouped in the “MP” set. The third set comprises the E model PBSs and thus it was named “LP”. Overlapping between the three kind of PBS sets was infrequent. Only five double sites (1 SP∩MP plus 2 MP∩LP plus 2 LP∩SP) and 2 triple sites (SP∩MP∩LP) were found. These overlapping sites were merged and each merge was considered a single PBS. Finally, the complete operation yielded a total of 265 unique PBSs.i.e. the DNA region between the stop codons of converging genes is not a “sense” IG region). The Type II set consists of the PBSs that are found 200 bp upstream and 50 bp downstream from a predicted start codon and are not part of an IG region. Type III set consists on all PBS that are not part of Type II or Type III sets  was calculated to determine if the binding site density was homogeneously distributed along the whole sequence or if it was biased towards IG regions. The 265 PBSs were further classified in three different sets according to their proximity to a predicted start codon . The TypIII sets . ApproxiIII sets . An arbiafeI upstream palindromic sequence as an input for this search. The results show that the most frequent and highest scoring motif was an 18-mer that corresponds to the MP based models present in each input sequence. The MEME program found the 18 bp motif in most of the MP-containing input sequences. On the other hand, the SP based PBSs were not detected as a MEME common motif. Interestingly, some of the SP containing input sequences possessed the MEME 18 bp motif near or overlapping the corresponding SP sequence. These new motifs were not detected as MP-based PBSs in the previous HMM search. Nevertheless, the MEME tool mainly confirmed the results obtained with the HMMs. Then, the gene functions associated with all these identified PBs were noted . All PBSs belonging to the Type III set were discarded as probable binding sites due to their location. Applying this filter yielded a total of 62 PBSs . Moreovere noted . The ideafeI palindrome. Two out of eight motifs were located in intergenic regions between converging ORFs. Two previously identified motifs were those located upstream the afeR gene and AFE_2100 (annotated as a virulence-associated protein). One new motif was found in the intergenic region upstream the AFE_1504 ORF (annotated as “nifW protein”). Also a new motif was located between the diverging ORFs AFE_2800 and AFE_2799, which are a glyoxalase family protein and an Ada family-DNA-3-methyladenine glycosylase II transcriptional regulator, respectively. The positions of PBSs that where found using the MEME algorithm are summarized in Selected PBSs that also shared the MEME 18 bp common motif, including the new ones located in the SP-containing inputs, were aligned and a consensus sequence was generated using MultAlin . Eight mlux-box consensus-based search, the upstream 250 nucleotides from the four most identical sequences to the product of afeI in the genbank database  were used as input for the MEME discovery tool . In all four sequences a 18 bp motif that corresponded to a palindromic sequence similar to the afe-box was found (Results not shown). The PSSM generated was put into the MAST tool and the intergenic region database from At. ferrooxidans was screened for common motifs. The result from this procedure showed that only the afeI 18-bp motif was found. On the contrary, when the same database was screened using the PSSM generated from the MEME analysis of the MP-based PBSs, the number of hits was broader (see results). This suggests that using lux-box like sequences for the generation of HMMs in the At. ferrooxidans search would have yielded fewer hits, and hence a narrower search.To evaluate the effectiveness of an alternative At. ferrooxidans are unknown. Here, we report results obtained from a bioinformatic approach to identify possible binding sites of the AfeR protein in the At. ferrooxidans ATCC 23270 genome sequence with the aim of defining a putative QS regulon for this bacterium.Functions regulated by QS in afeI gene as a template. Alignments of known lux-box type sequences to generate the HMMs were not performed because of the little overall conservation of these sequences [afe-box because different modes of binding are expected depending on the organization of sequence modules, as it has been experimentally tested for the fur binding site [lux-box is only a fraction of a larger structure suggests that non-AfeR transcriptional regulators might also bind to the afeI operator.HMMs were constructed based on alignments of hypothetical sequences that took the palindromic structure located upstream of the equences . The HMMing site . The faccis-acting regulatory motifs. Also, it is possible that this preference is due to the overlapping of the -35 element of a sigma 70 promoter from afeI with the modeled SP structure, thus implying that at least some of the SP hits may actually be -35 elements. Supporting the hypothesis mentioned above, the Bprom predictor found some Type I SP hits overlapping -35 elements but it also found hits overlapping -10 elements and hits with no overlapping at all. On the other hand, only MP model-based PBSs were found as common motifs when all PBSs were searched with the MEME tool, making MP PBSs more likely to be the correct regulatory motif for AfeR. Thus, as SP and MP based models did not show significant overlapping in the HMM search, the determinants for the AfeR interaction with DNA are likely to be enclosed within the central 18–20 nucleotides of the large 30 bp palindrome and that the SP PBSs may be binding sites for a distinct regulator. It is possible then that SP based PBSs may be part of a distinct regulon that overlaps the AfeR regulon in the transcriptional regulation of afeI. There are examples of multiple regulatory cis-acting elements upstream of various QS-controlled genes, including the regulatory zones of I and R genes [lux-box like elements as is the case of the binding site for the regulator RsaL, upstream of lasI from P. aeruginosa [luxI gene from V. fischeri [The search yielded a total of 265 PBSs. SP model-based PBSs were preferentially located on intergenic regions whereas MP model-based PBSs were not . This ca R genes ,23. Thesruginosa  or the Mlux-box like sequence is found upstream of QS-controlled genes. This is also the case for the lasB, hcn and pqsA genes from P. aeruginosa [Among the ORFs presented in the PBS table , five ofruginosa –27.afeI promoter in the presence of AHL [70 promoters. Some of them overlap -35 elements, others overlap -10 elements and others do not overlap either. Previously described LuxR family activators of transcription like TraR, LasR and LuxR itself generally bind a lux-box like element centered in position -43 (overlapping the -35 element of the σ70 promoter) in the presence of AHL. Some transcriptional regulators of the LuxR family bind their target sequences in the absence of and self-dissociate in the presence of AHL, as is the case of EsaR of Pantoea stewartii or ExpREcc of Erwinia carotovora [lux-box like sequence overlaps the -10 element of the σ70 promoter. The mentioned regulators can also function as activators of transcription in artificial genetic constructs where the position of the cis-acting element is changed to a -35 element overlap (maintaining the dependence on absence of AHL substrate) [lux-box like element position. Moreover, it has been demonstrated that genuine LasR binding sites are located as far as 383 bp from the translation start codon (phzA gene) [The type of regulation exerted over the AfeR targets remains unknown. AfeR can activate transcription from the e of AHL , but therotovora ,29. Bothbstrate) . This shzA gene) , thus maafeI upstream sequence nor perfect dyad symmetry. Dyad symmetry is not a prerequisite for the lux-box like elements to interact with their cognate regulator as demonstrated for various QS-controlled genes in P. aeruginosa [None of the PBSs found had similar palindromic complexity to neither the ruginosa .orf3 gene. The position of the afe-box located upstream of orf3 and afeR overlaps predicted -35 elements in both cases, suggesting an activator role for AfeR at this locus. Other LuxR-like proteins are also subject of self-regulation, as is the case for the positive feedback loop experienced by PhzR from Pseudomonas fluorescens [Pantoea stewartii [The gene functions associated to the 18 bp MEME motif have been related to several biological processes . One of orescens  and the tewartii .There were two other transcriptional regulators forming part of the predicted targets: first, a helix-turn-helix transcriptional regulator that also contains a LexA motif and second, a Sigma 54-dependent transcriptional regulator. This is consistent with the fact that AfeR is a global regulator of transcription that controls the expression of many genes indirectly. The S24/LexA-like domain is related to the bacterial SOS response; specifically it catalyzes its own proteolysis separating the DNA-binding domain and the rest of the protein. (CD number: cd06529) The Sigma 54-dependent transcriptional regulator has also an HTH DNA-binding motif and also an AAA-type ATPase domain (CD number: cd00009).In the same locus as sigma 54-dependent transcriptional regulator, but in the opposite orientation, there is a metallo-β-lactamase protein family gene. Interestingly, there is the second metallo-β-lactamase among the hypothetical regulon. These proteins belong to the same superfamily as the “quorum quenching” lactonase enzymes AiiA and AttM, frequently found in Gram-positive and Gram-negative bacteria, respectively . Quorum Another target related to β-lactam antibiotics is a protein that contains an ErfK/YbiS/YcfS/YnhG domain  which codes for a transpeptidase that functions as an alternative pathway for peptidoglycan cross-linking. This pathway provides resistance to β-lactam antibiotics because it functionally replaces the sensitive penicillin-binding protein (PBP).Pseudomonas aeruginosa. There, TonB mutants show an impairment in AHL production independently of its iron transport activity [We found four transport-related putative targets. The first two are the divergently organized genes encoding a TonB-dependent receptor and an ABC transporter (of the ABC_DR_subfamily_A). The TonB-dependent receptor is involved in the specific transport of substances across the membrane, of which iron-carrying siderophores are the best characterized ones (Conserved domain database (CD) number: cd01347). Nevertheless, a role in QS has been demonstrated for TonB in E. coli). TruB catalyzes the isomerization of specific uridines in tRNA. The third RNA-related gene is the aspartyl-tRNA synthase. Finally, the RibonucleaseT (RNAseT) protein in E. coli RNAseT is involved in tRNA turnover. It has a 3′-5′exonuclease activity, also related with DNA polymerase III. RNAseT is only found in gamma-proteobacteria (CD number: cd06134).Five genes related to RNA are also predicted targets. The first one is a TrmH-family protein, which is known to methylate rRNA. rRNA methylation in bacteria strongly correlates with resistance to ribosome-targeted antibiotics . A seconAgrobacterium tumefaciens [Several proteins with diverse activities where found to be part of this putative regulon. First, a conjugal transfer protein (TrbF), thought to be part of the pilus required for the transfer of DNA. DNA transfer is a major QS-regulated phenotype in efaciens . The reszwf a gene encoding for glucose 6-phosphate-1-dehydrogenase (G6PDH) has been also identified as part of this regulon. G6PDH is part of the pentose phosphate pathway and is directly related to the cellular pool of glucose 6-phosphate that is involved in the biosynthesis of EPS precursors UDP-glucose and UDP galactose [zwf leads to an approximately 90% reduction in alginate, one of the EPS produced by Pseudomonas aeruginosa [At. ferrooxidans is capable of synthesizing the EPS precursors UDP-glucose and UDP-galactose [zwf and afeI are increased in the presence of a tetrazolic AHL-analogue [Finally, two glycosyl-transferase activities were found among the putative regulon. These activities are usually related to polysaccharide biosynthesis. The two genes belong to different groups. The “Group 1” glycosyl-transferase protein transfer activated nucleotide-sugar molecules to several substrates, including protein, lipids and other sugar residues. The “Group 2” glycosyl transferase has the same activity but structurally it belongs to a different family (CD number: cd04186). It is possible that these proteins play a role in the biosynthesis of the exopolymeric substances (EPS) or the lipopolysaccharide. In addition, alactose . On the ruginosa . It is walactose  and EPS alactose ,41. Moreanalogue ,43 suggeafeI gene and the orf3 open reading frame were used to generate HMMs. First, four defined-length palindromes were chosen  .Fractions of the 30 bp palindrome located in the intergenic region between the indrome) . Second,www.softberry.com) Type I and Type II PBS sequences with additional 100 bp flanking each side ware used as input for Bprom. All Type I PBSs were further characterized. On the other hand, Type II PBSs were selected for further characterization only if a Bprom prediction occurred within the Bprom input sequence. Shared motifs were identified using the MEME motif discovery tool [At. ferrooxidans genome. The input for the MAST program was the position specific scoring matrix (PSSM) generated from the common motif sequences derived from MEME analysis. Multiple alignments of selected PBSs and consensus sequence (Available ORF sequences contain limited annotated information. For this reason, they were further annotated and organized in sets using ARTEMIS . The conery tool ,52. The ery tool  was usedsequence  were concis-regulatory elements. Among this set of genes, the afeR response regulator and the divergently organized conserved unknown gene orf3 were identified. For both genes, the cis-regulatory element overlaps the -35 promoter elements suggesting that auto-regulation of AfeR may take place, as it occurs in Vibrio fischeri and several other gram-negative bacteria.Our bioinformatic prediction allowed the identification of several gene functions associated with the presence of upstream putative quorum sensing At. ferrooxidans might be involved in biofilm formation and directly correlates with the increase of adhesion to pyrite and sulphur by At. ferrooxidans obtained by the addition of AHL molecules [At. ferrooxidans. However, it has been previously described that zwf is activated by QS in P. aeruginosa [zwf gene [At. ferrooxidans QS regulon.The identification of genes involved in the biosynthesis of exopolysaccharides strongly indicates that the QS pathway of olecules . Furtherruginosa . Moreovezwf gene , the biozwf gene . This su


Maintenance therapy with gefitinib notably improves survival in patients with advanced non-small cell lung cancer (NSCLC) and EGFR mutation-positive tumors, but the economic impact of this practice is unclear.A decision-analytic model was developed to simulate 21-day patient transitions in a 10-year time horizon. The clinical data were primarily obtained from the results of a pivotal phase III trial that assessed gefitinib maintenance treatment in patients with advanced NSCLC. The cost data were derived from the perspective of the Chinese health care system. The primary outcome was the incremental cost-effectiveness ratio (ICER) at a willingness-to-pay (WTP) threshold of 3 times the per capita GDP of China. Sensitivity analyses were used to explore the impact of uncertainty regarding the results. The impact of the gefitinib patient assistance program (GPAP) was evaluated.After EGFR genotyping, gefitinib maintenance treatment for advanced NSCLC with EGFR mutations increased the life expectancy by 0.74 years and 0.46 QALYs compared with routine follow-up at an additional cost of $26,149.90 USD . The ICER for gefitinib maintenance was $57,066.40 and $15,664.80 per QALY gained (at a 3% discount rate) without and with the GPAP, respectively. The utility of progression free survival, the hazard ratio of progression-free survival for gefitinib treatment and the cost of gefitinib per dose were the three factors that had the greatest influence on the results.These results indicate that gene-guided maintenance therapy with gefitinib with the GPAP might be a cost-effective treatment option. Lung cancer is the most prevalent malignant cancer in adults, with over 1.3 million deaths from the disease per year . LocallyThe role of maintenance therapy in patients who remained progression free after first-line chemotherapy has been well established by several Phase III trials -11. At pHealth resource allocation decisions are based increasingly on economic analyses that identify the therapies that provide the greatest health benefits at acceptable costs, especially in a health resource-limited setting. Because clinical trials rarely include economic health assessments, mathematical modeling is widely used to perform economic health analyses, particularly for extrapolating to timepoints beyond trial durations. The objective of our study was to compare the economic outcome of gene-guided gefitinib maintenance treatment with the routine follow-up following first-line platinum-based chemotherapy for advanced NSCLC with EGFR mutation from the perspective of the Chinese health care system. Although erlotinib and pemetrexed have been used for maintenance treatment, the current analysis would not include other treatment arms due to no clinical trials for directly comparing the clinical outcomes of gefitinib, erlotinib and pemetrexed.An economic model was constructed to analyze the ten-year clinical and economic outcomes of gefitinib maintenance therapies for patients with advanced NSCLC. Patients were assumed to either initiate observation with routine follow-up or to initiate maintenance treatment with gefitinib if the EGFR mutation screening was positive Figure A. Healthet al. [We assumed that the clinical characteristics of the hypothetical cohort were similar to those reported by Zhang L. et al.  All patiet al. ,22.The analysis was conducted from the perspective of the Chinese healthcare system. The costs are presented in 2012 US dollars. The outcomes calculated for each strategy included progression free life years, overall life years (LYs), quality-adjusted life years (QALYs) and the costs of advanced NSCLC care. The results are reported as the incremental cost-effective ratio (ICER) over the 10-year period calculated using the model. The costs and QALYs were each discounted at an annual rate of 3%.The transition parameters and proportions were based on a meta-analysis or randomized clinical trials to the greatest possible extent.Kaplan-Meier survival curves for PFS for each strategy were taken from the pivotal gefitinib maintenance clinical trials . A total2 and correlation coefficients are presented in Table t could be calculated by following formula: S(t) = P(T ≥ t) = exp(−λtγ). The transition probability at current cycel t could be calculated by following formula:After disease progressed, patients would receive either 2nd-line chemotherapy or supportive care. The proportion of patients receiving 2nd-line chemotherapy was derived from literatures -10. KaplThe costs were estimated from the perspective of the Chinese health care system. Indirect costs were not included in this analysis. The direct medical costs considered in the model were: the detection of EGFR mutation, maintenance and 2nd-line chemotherapy , concomitant medication during therapy, managing treatment-related SAEs, routine follow-up and laboratory tests.The cost of EGFR genotyping per patient was provided by the AstraZeneca Innovation Centre China, Shanghai laboratory. The estimated treatment costs were based on the following schedules: gefitinib (250 mg per day) would be administered to patients with progression free survival after initial chemotherapy until the disease progressed. After the cancer progressed, 2nd-line chemotherapy and supportive care would be available. Based on the reported clinical trials, nearly 56.6% (26%-72%) of patients would receive 2nd-line chemotherapy regardless of the first-line treatment . Of thosBecause it can be a challenge for patients to afford gefitinib in China, the Gefitinib Patient Assistance Program (GPAP) supplied by the pharmaceutical producer was introduced to make gefitinib available to eligible patients. Currently, the GPAP requires NSCLC patients to pay for six months of gefitinib, after which they receive donations of gefitinib until the end of their treatment. Therefore, the scenario analyses evaluated the importance of GPAP for gefitinib.The utility values of the progression free survival and survival with disease progression were derived from previously published studies, and 0.65 and 0.47 were assigned, respectively. The standard errors were estimated at 25% of the mean in our sensitivity analysis .Expected Cost and effectiveness(QALY and LY) accrued for the entire Markov process is the total number of cycles spent in each health state, each multiplied by the cost and effectiveness for that state.The median PFS and OS time of advanced NSCLC would not exceed one and two years, and most of patients would die within five years . In baseThe model results indicate that imitating gefitinib maintenance treatment increased the health benefits for patients who had completed standard first-line chemotherapy. Increased progression free LYs appeared after 1, 2, 5 and 10 years , and LYs increased by 0.16, 0.41, 0.69 and 0.74 years, respectively. The additional QALYs gained ranged from 0.12 at 1 year to 0.46 at 10 years Table . The incThe one-way sensitivity analyses showed that some model variables had a substantial impact on the results; these are presented in the tornado graphs in Figure A two-way sensitivity analysis incorporating the frequency of EGFR mutations and the cost of EGFR genotyping was performed. This analysis indicated that gefitinib maintenance was more cost-effective in the population with a higher rate of EGFR mutation-positive advanced NSCLC. The ICER was sensitive to rates from approximately 7% to 20%. A lower cost of detecting EGFR mutations would improve the ICER values of the Gefitinib strategy. However, the impact was small Figure .When no GPAP was supplied, the probabilistic sensitivity analysis showed a nearly zero cost-effective probability even at a threshold of $38,733.30 Figure . In the Reports of a clinical benefit from gefitinib maintenance therapy after first-line platinum-based chemotherapy in clinical trials caused great excitement among both oncologists and patients. However, the widespread and long-term use of gefitinib comes with a dramatically increased burden on health resources, which is a concern for health policy decision makers. The need for a precise economic assessment of gefitinib maintenance use in this clinical setting is becoming urgent.This work is the first study to address the cost-effectiveness of gene-guided gefitinib maintenance treatment after standard chemotherapy for patients with advanced EGFR mutation-positive NSCLC. Genotyping for EGFR mutations with the subsequent gefitinib maintenance treatment of patients with confirmed mutations yielded an average ICER of $57,066.40 per additional QALY gained against control strategy. This ratio is largely attributable to the higher costs associated with the acquisition of gefitinib, whereas the costs of EGFR genotyping and the costs of managing progressed disease had little influence. Finding of scenario analyses in Table Although gefitinib maintenance yielded greater health benefits, the ICER did not approach the willingness-to-pay thresholds of $16,349.10 and $38,733.30 . If the Gefitinib Patient Assistance Program were available to Chinese patients, the Gefitinib strategy might be a cost-effective alternative because the probability of cost-effectiveness reached nearly 51% at a threshold of $16,349.10  might be the best strategies to achieve a more favorable ICER.It is important to note that the current analysis did not evaluate the cost-effectiveness of gefitinib maintenance treatment for the whole cohort without EGFR genotyping. If all patients received gefitinib maintenance, the health outcome of 50% of the patients would not have improved because no statistically significant difference was found between the Gefitinib and Control strategies in patients who were EGFR mutation negative . The cosOther limitations of the study should also be considered. First, the present model did not include other EGFR-targeted agents used as maintenance treatments, such as erlotinib, to assess the incremental cost-effectiveness in comparison with gefitinib because no head-to-head trial data are currently available. Second, we did not conduct a budget impact analysis for the addition of gefitinib maintenance treatment on society. The annual incidence of lung cancer in China is approximately 300,000 cases . BecauseIn the Chinese setting, gene-guided gefitinib maintenance treatment for patients with advanced NSCLC and EGFR mutation-positive tumors after first-line chemotherapy is indicated as a cost-effective chemotherapy option compared to routine follow-up based on its superior PFS benefit and the use of the Patient Assistance Program.The authors declare that they have no competing interests.Jun Zhu and Te Li developed the economic model, performed the analyses and drafted the manuscript. Bin Wu contributed to the conception, design of the primarily model and interpreted the results. Xiaohui Wang and Ming Ye collected and reviewed data. Jian Cai provided clinical input, validated the model assumptions. Yuejuan Xu aided in defining economic setting. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/13/39/prepub


Podosomes are cellular “feet,” characterized by F-actin-rich membrane protrusions, which drive cell migration and invasion into the extracellular matrix. Small GTPases that regulate the actin cytoskeleton, such as Cdc42 and Rac are central regulators of podosome formation. The adaptor protein IRSp53 contains an I-BAR domain that deforms membranes into protrusions and binds to Rac, a CRIB motif that interacts with Cdc42, an SH3 domain that binds to many actin cytoskeletal regulators with proline-rich peptides including VASP, and the C-terminal variable region by splicing. However, the role of IRSp53 and VASP in podosome formation had been unclear. Here we found that the knockdown of IRSp53 by RNAi attenuates podosome formation and migration in Src-transformed NIH3T3 (NIH-Src) cells. Importantly, the differences in the IRSp53 C-terminal splicing isoforms did not affect podosome formation. Overexpression of IRSp53 deletion mutants suggested the importance of linking small GTPases to SH3 binding partners. Interestingly, VASP physically interacted with IRSp53 in NIH-Src cells and was essential for podosome formation. These data highlight the role of IRSp53 as a linker of small GTPases to VASP for podosome formation. Reorganization of actin filaments and membranes accompanies many cellular events, such as cell migration, where the leading edge extension and the rearward contraction coordinately occur on the opposite sides of the cell from each other. The leading edge is characterized by the formation of lamellipodia and filopodia, downstream of the functions of the small GTPases Rac and Cdc42, respectively Podosomes were first characterized in cells transformed with the Rous Sarcoma virus IRSp53 consists of the I-BAR (inverse BAR) domain, the CRIB motif, the SH3 domain, and the C-terminal variable region by splicing MENA and VASP belong to the Ena/VASP family proteins, which promote actin filament elongation The C-terminus of IRSp53 is spliced to create several isoforms. The shortest form (S) contains the PDZ-domain binding motif, where PDZ-domain containing proteins, such as Lin-7, interact for filopodium formation In this study, we analyzed the function of IRSp53 in podosome formation, and found that it connects small GTPases to VASP in the podosome formation by NIH-Src cells.NIH3T3 cells form podosomes when they are transformed with a constitutively active form of Src tyrosine kinase We then examined the specific effect of the IRSp53 siRNA on podosome formation, by restoring IRSp53 expression. The cells were sequentially transfected with the siRNA vector and the expression plasmid for each splicing isoform of human IRSp53 tagged with GFP . The resWe then used deletion mutants to test which domain of IRSp53 is important for podosome formation . The oveBecause the I-BAR domain is known to bind to Rac, actin filaments, and membranes We then tried to identify the molecules downstream of IRSp53 that participate in podosome formation. Since the role of VASP in podosome formation has not been characterized so far, we focused on VASP. The immunoprecipitation analysis revealed that VASP interacted with IRSp53 in NIH-Src cells . TherefoIn this study, we established the essential role of IRSp53 in podosome formation in NIH-Src cells. VASP appeared to be the actin regulatory molecule downstream of IRSp53. Interestingly, the membrane deforming ability of IRSp53 was not involved in podosome formation.Since IRSp53 is a multi-domain protein, the importance of each domain might vary, depending on the cell type. For podosomes in NIH-Src cells, C-terminal splicing of IRSp53 was not essential, suggesting that protein binding at the C-terminal region, which is variable due to splicing, is not crucial. Importantly, the overexpression of the ΔI-BAR mutant did not suppress podosome formation, and thus the role of the I-BAR domain in podosome formation might be marginal. However, it is possible that the ΔI-BAR mutant did not localize properly, and thus could not suppress podosome formation. In contrast, the deletion of the IRSp53 C-terminal region including the SH3 domain suppressed podosome formation, but it was much weaker than the RNAi of IRSp53 . TherefoThe strong inhibition of podosome formation by the overexpression of the I-BAR domain highlighted its role in podosome formation. IRSp53 was first proposed to function in actin filament bundling, through its I-BAR domain The I-BAR domain was first characterized as a Rac-binding domain When we consider actin filament organization and podosomes, it would be interesting if IRSp53 functioned with VASP, because podosomes consist of not only branched actin filaments generated by the Arp2/3 complex and N-WASP, but also bundled filaments in vivo.VASP and MENA play well-established roles in filopodia and lamellipodia formation. However, this is the first study to establish their roles in podosome formation. VASP and MENA are reportedly associated with invasiveness 5′-GGAGCTACAGTACATAGAC-3′, without changing the coding amino acids.Mouse NIH cells transformed with Src (NIH-Src cells) were cultured as described previously Transfection was performed with the Lipofectamine LTX and PLUS reagents (Invitrogen), according to the manufacturer's protocols. Two days after transfection, the cells were analyzed.The anti-IRSp53 antibody was affinity purified from rabbit serum 4 cells) were plated on a Transwell or BioCoat Matrigel Invasion Chamber (BD Biosciences) for 18 h. siRNA transfection was performed 48 h before plating on the chamber. The chambers were subsequently fixed in 3.7% formaldehyde in PBS for over 30 min. The chambers were then washed in PBS, and the invaded cells were stained with crystal violet. The non-invading cells were manually removed with cotton swabs. After washing the cells with PBS more than three times, the numbers of cells from three different sample points on the lower surface of the chamber were counted.The NIH-src cells  for 30 min. Cells were also stained with rhodamine–phalloidin (Invitrogen) to detect actin filaments. The cells were finally washed with PBS, mounted on glass slides, and examined with a fluorescence microscope (Olympus FV1000 or Zeiss LSM710).NIH-Src cells were washed with ice-cold PBS and lysed with either radioimmunoprecipitation assay buffer  or cell lysis buffer  supplemented with protease inhibitors and phosphatase inhibitors (Sigma). The cell lysates were subjected to immunoprecipitation with agarose bead–conjugated M2 antibodies to FLAG (Sigma) or anti-VASP antibody conjugated to protein A-agarose beads (Pierce), and the bead-bound proteins were analyzed by immunoblotting.t test, using data from at least three independent experiments. All images are representative of at least three independent experiments.All statistical analyses were performed using Microsoft Excel. Significance was assessed by the Student's


Low levels of self-esteem have been implicated as both a cause and a consequence of severe mental disorders. The main aims of the study were to examine whether premorbid adjustment has an impact on the subject's self-esteem, and whether lowered self-esteem contributes to the development of delusions and hallucinations.A total of 113 patients from the Thematically Organized Psychosis research study (TOP) were included at first treatment. The Positive and Negative Syndrome Scale (PANSS) was used to assess present symptoms. Premorbid adjustment was measured with the Premorbid Adjustment Scale (PAS) and self-esteem by the Rosenberg Self-Esteem Scale (RSES).Premorbid social adjustment was significantly related to lower self-esteem and explained a significant proportion of the variance in self-esteem. Self-esteem was significantly associated with the levels of persecutory delusions and hallucinations experienced by the patient and explained a significant proportion of the variance even after adjusting for premorbid functioning and depression.There are reasons to suspect that premorbid functioning is an important aspect in the development of self- esteem, and, furthermore, that self-esteem is associated with the development of delusions and hallucinations. Self-esteem, a global and complex concept, is comprised of both appraisal of self-worth based on personal achievements and anticipation of evaluation by others ,2. AlthoRegarding self-esteem as a consequence of mental illness, studies predictably show that stigmatization and self-stigmatization may lower self-esteem in persons with mental illness . Low selif and how strongly it will affect state self-esteem [Recent models of global self-esteem suggest that it is both a trait and a state measure . People f-esteem ,13. It af-esteem ,15.Studies have suggested that difficult childhood experiences such as childhood loss and social marginalization contribute to a cognitive vulnerability accompanied by a negative view both towards the person himself and towards others ,11,16. ITo our knowledge only one study has tried to examine the relationship between premorbid adjustment and self-esteem in patients with schizophrenia spectrum disorder . They foA relatively rich literature exists on the relationship between low self-esteem and symptom formation in severe mental disorders including psychotic disorders. One study showed that the contents of patient's delusions were consistent with patient's global self-esteem and suggested that low self-esteem accounted for the persistence of delusions . Other sOther studies have shown higher levels of self-esteem in patients with delusional disorder compared to depressed patients . HoweverSelf-esteem has been found to fluctuate over the short-term. It has been demonstrated that paranoid individuals display more fluctuations in their self-esteem, and that the fluctuation predicts the degree of subsequent increase in paranoid thinking . HoweverTo date the relationship between self-esteem, psychotic symptoms and premorbid adjustment in the early stages of psychosis has not been thoroughly explored. Previous studies of that nature have all been conducted with patients with chronic psychotic disorders where the effects of a long-term severe illness and secondary processes may significantly confound relationships. More studies are thus needed to explore the relationship between self-esteem and psychotic symptoms during the early phases of psychotic disorder. This is of importance as patients coming to their first treatment for a psychotic disorder are less influenced by stigmatization, treatment failures, and subsequent disappointments which may contribute to lowered self-esteem.The aims of the current study are thus to investigate the following questions in a large and well characterized group of patients with first episode psychosis:1) To what extent is premorbid adjustment ), related to self-esteem ), in this patient group?2) To what extent is self -esteem related to the level of hallucinations and delusions )?From February 2007 to October 2009, 113 patients from the main psychiatric treatment centres in Oslo and two neighbouring counties were consecutively included in the Thematically Organized Psychosis research study (TOP). The inclusion criteria were that they were within the age bracket of 18 to 65 years old and that they were coming to their first treatment for a schizophrenia spectrum disorder as defined in DSM-IV. Exclusion criteria were a history of organic brain disorder, a significant co-morbid medical condition or an IQ of less than 70.The diagnostic distribution was as follows; (N (%)): schizophrenia 68 (60.2%), schizophreniform disorder 7 (6.2%), schizoaffective disorder 11 (9.7%), brief psychosis 1 (0.9%), delusional disorder 7 (6.2%) and psychosis NOS 19 (16.8%).Patients were eligible for inclusion up to 52 weeks after the start of the first adequate treatment for their disorder and were not considered as First Episode Psychosis (FEP) patients if they had previously been treated with anti-psychotic medication in adequate dosage for more than 12 weeks, or until remission. Being psychotic was defined as having a rating of 4 or more on the PANSS items p1 (delusions), p2 (disorganisation), p3 , p5 (grandiosity), p6 (persecutory delusions) or (g9)  for more than one week. The mean age of the patients was 25.8 (SD 7.7). 37 (32.7%) were females and 76 (67.3%) were male. 82 (72.6%) were single, 24 (21.2%) were married or co-habiting, and 7 (6.3%) were divorced, separated or widowed. Mean years of education was 12.4 (SD 2.72) and median duration of untreated psychosis (DUP) was 78 weeks (range 0-1040) (N = 106). All patients gave written informed consent and the study was approved by the regional research ethics committee.Diagnosis was set according to the Structured Clinical Interview for Diagnostic and Structural Manual of Mental Disorders, fourth version (SCID I interview for the DSM -IV) . CurrentDepression was diagnosed according to the criteria in DSM-IV. We only measured major depression to avoid overlap with negative symptoms. The duration of untreated psychosis (DUP) was measured according to previously published criteria . PremorbAs the current study is part of a broad research initiative with an extensive interview protocol, most participants chose to divide the interview into 2-3 sections over 1-2 weeks. Significant efforts were made to make the assessments as close in time as possible.The patients were interviewed by trained psychologists and psychiatrists at the same time as the SCID-I was administered. The investigators had all completed general training and a reliability program with regard to the TOP research study. For DSM-IV diagnostics mean overall kappa with training videos was 0.77, and mean overall kappa for a randomly drawn subset of actual study patients was also 0.77 (95% CI 0.60-0.94). Inter-rater reliability, measured by the intra class correlation coefficient (ICC 1.1) was 0.82 (95% CI 0.66-0.94) for the PANSS positive subscale, 0.76 (95% CI 0.58-0.93) for the PANSS negative subscale and 0.73 (95% CI 0.54-0.90) for the PANSS general subscale.Correlations between demographic/clinical characteristics and self-esteem were calculated using Pearson's product moment co-efficients. To estimate how much of the variance in self-esteem was explained independently by premorbid functioning we performed a block-wise hierarchical multiple regression analysis with age and gender entered in the first block and premorbid adjustment in the second block. As academic adjustment in childhood versus academic adjustment in early adolescence, and social adjustment in childhood versus social adjustment in early adolescence were strongly inter-correlated (with r = 0.66 and r = 0.77 respectively), only results for early adolescence were entered to represent PAS and avoid co-linearity problems. The associations between global self-esteem and hallucinations and delusions, both general and persecutory, were analyzed similarly using Pearson's correlations and followed up with three block-wise hierarchical multiple regression analysis with hallucinations, delusions and persecutory delusions as the dependent variables. Demographic information was placed in the first block, premorbid adjustment in the second block, depression i.e whether the patient was in a major depressive episode or not, in the third block and self-esteem in the fourth. By entering self-esteem in the fourth we adjusted for the amount of variance explained by the variables in the first three blocks. Finally, we conducted various interactional analyses to explore whether self-esteem acted as a mediator or moderator of the relationship between premorbid adjustment and symptoms.Table As shown in table In the first hierarchical multiple regression analysis, with self-esteem as the dependent variable, the included variables explained 25% of the variance in self-esteem Table . Only geIn the second hierarchical multiple regression analysis performed, with positive psychotic symptoms as the dependent variable, self-esteem explained a significant amount of the variance in both hallucinations and persecutory delusions, even after adjusting for age, gender, premorbid adjustment and depression. In general, levels of self esteem did not explain a significant amount of the variance in occurrence of delusions (P1) Table .Finally, various interactional analyses revealed no significant interaction between premorbid adjustment and symptoms mediated or moderated by self-esteem.This study demonstrates both a statistically significant relationship between poor premorbid social adjustment and low levels of global self-esteem and between self-esteem and positive psychotic symptoms i.e hallucinations and persecutory delusions. The relationship between self-esteem and positive psychotic symptoms remained significant even after adjusting for the presence of a major depressive episode indicating that this effect is not mediated by the presence of depressive symptoms.The current study is one of the first to show a relationship between poor premorbid social adjustment and level of global self-esteem in psychotic disorders. The only other study exploring this relationship  did not Premorbid social adjustment as a concept incorporates issues including such factors as how you interact with your schoolmates, adjust to groups and friends and the presence of age-relevant sexual interest. Previous studies have shown that general cognitive abilities, exposure to bullying, social marginalization, abuse, neglect or the presence of neurodevelopmental factors are the strongest predictors of social adjustment -37. ManyFurthermore, studies of persons with auditory hallucinations have shown that voice hearers experience a subordinate relationship to their voices mirroring other social relationships. This suggests the existence of maladaptive inter-personal schemata serving both . These sWe also found self-esteem to be a predictor of both hallucinations and persecutory delusions in early psychosis, even though the explained variance was rather moderate. This is in line with previous studies ,21,28,40We further argue that poor premorbid social adjustment with social withdrawal and subsequent marginalization provides content for psychotic attribution due to a lack of correcting social feedback. This is supported by findings in studies of patients at high risk of developing psychosis . In lineOur findings are also supported by findings in the general population. Negative ideas about oneself and others have been found to be predictors of paranoid thinking in the general population . In addiHowever, there are studies showing that patient's self-stigma tends to be most affected during the early course of the disease, and that self-stigma and self-esteem are closely related . It may Gender was a significant predictor of self-esteem in this FEP sample, with women having significantly lower levels of self-esteem than men even after correction for differences in levels of depression. A vast body of literature from the general population indicates a small but significant gender difference in the same direction . There iThe present study has some limitations. This is a cross-sectional study where conclusions about directions of relationships cannot be ascertained, and where data on premorbid adjustment is necessarily gathered retrospectively. There may also be a recall bias regarding the scores for premorbid adjustment. To what degree self-esteem is affected before the development of psychosis is thus not possible to test directly using the current design. Furthermore, the present study, due to the limitations of the study protocol, did not allow for more sophisticated measures of self-esteem, such as the measurement of fluctuations in self-esteem, which would have been of interest in this group.The current study revealed both a significant association between premorbid adjustment and self-esteem and between self-esteem and positive psychotic symptoms. Future studies of self-esteem should consider examining how self-esteem changes over the course of illness from the prodromal stage. It would also be of interest to explore factors such as stigma and metacognition and their relation to self-esteem. There are several factors that in further studies could be explored as possible mediators in this context. Women with psychotic disorders report a high prevalence of sexual trauma  which isIt is our opinion that this study may have clinical implications. It is possible that psychotheraputic interventions, such as cognitive behavioral therapy, may increase self-esteem and lessen the likelihood of development of positive psychotic symptoms or decrease their severity. Furthermore, psychotherapeutic intervention may help patients to acquire a broader personal narrative which would benefit their self-esteem.The authors declare that they have no competing interests.KLR has made substantial contributions to conception and design, acquisition of data, analysis and interpretation of data and drafting the manuscript. JIR has made substantial contribution to conception and design, analysis and interpretation, drafting of the manuscript and have been revising it critically for important intellectual content. CFH has made substantial contributions to acquisition of data and have been involved in drafting and revision of the manuscript. EH has made substantial contributions to acquisition of data and have been involved in drafting and revision of the manuscript. OAA has made substantial contributions to conception and design, drafting the manuscript and have been revising it critically for important intellectual content. IM has made substantial contributions to conception and design, drafting of the manuscript and have been revising it critically for important intellectual content. All authors have given final approval of the version to be published.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-244X/11/136/prepub


Understanding the complexity of the cellular environment will benefit from the ability to unambiguously resolve multiple cellular components, simultaneously and with nanometer-scale spatial resolution. Multicolor super-resolution fluorescence microscopy techniques have been developed to achieve this goal, yet challenges remain in terms of the number of targets that can be simultaneously imaged and the crosstalk between color channels. Herein, we demonstrate multicolor stochastic optical reconstruction microscopy (STORM) based on a multi-parameter detection strategy, which uses both the fluorescence activation wavelength and the emission color to discriminate between photo-activatable fluorescent probes. First, we obtained two-color super-resolution images using the near-infrared cyanine dye Alexa 750 in conjunction with a red cyanine dye Alexa 647, and quantified color crosstalk levels and image registration accuracy. Combinatorial pairing of these two switchable dyes with fluorophores which enhance photo-activation enabled multi-parameter detection of six different probes. Using this approach, we obtained six-color super-resolution fluorescence images of a model sample. The combination of multiple fluorescence detection parameters for improved fluorophore discrimination promises to substantially enhance our ability to visualize multiple cellular targets with sub-diffraction-limit resolution. Fluorescence microscopy is a widely used imaging tool for cell and molecular biology, and one of the strengths of this technique is the variety of fluorescent probes which may be used to label the specimen. Synthetic dye molecules, fluorescent proteins, and inorganic fluorescent nanoparticles are available with a range of spectral properties and may be linked to their target with high specificity through numerous coupling strategies.The recent development of far-field super-resolution fluorescence imaging techniques,22Stochastic optical reconstruction microscopy (STORM) is a super-resolution fluorescence imaging technique based on sequential, nanometer-scale localization of individual photo-switchable fluorescent labels, which emit light at different times during image acquisition.15We previously proposed a multi-parameter detection method for multicolor STORM, based on the discrimination of photo-switchable fluorescent probes by both activation wavelength and emission spectrum.To characterize Alexa 750 fluorescence switching, antibodies were labeled with Alexa 750 and bound non-specifically to a glass coverslip at low density such that individual molecules were resolvable from each other. Alexa 750 was excited at a wavelength close to its absorption peak by using 752 nm light. In the presence of thiol, that is, with β-mercaptoethanol (BME) or β-mercaptoethylamine (MEA) in the imaging buffer, the 752 nm light excites fluorescence from Alexa 750 and also rapidly switches the molecules to a non-fluorescent state. Additionally, 752 nm excitation light can also cause the dye to reactivate back into the fluorescent state, albeit at a much lower rate. Hence, single-molecule Alexa 750 traces exhibited a blinking behavior with sporadic fluorescence bursts  a. Illumν) is defined as the fraction of time the dye spends in the fluorescent state under constant illumination with imaging light.ρ) times the blinking fraction (ν). When ρν approaches one per diffraction-limited volume, the images of individual activated fluorophores begin to overlap substantially, thus inhibiting their precise localization. In this manner the equilibrium blinking fraction sets an upper bound for the density of fluorophores which may be present on the sample before blinking interferes with STORM data acquisition.4Two properties of photo-switchable fluorophores are particularly important for applications in STORM imaging: 1) the fluorophore brightness, and 2) the equilibrium blinking fraction. The first property, fluorophore brightness, may be quantified in terms of the number of photons detected per switching cycle from single fluorophores. Fluorophores emitting a higher number of photons per cycle are easier to detect and their positions may be determined with a higher degree of precision,To measure the photon yield and blinking fraction for Alexa 750, we exposed the dye-labeled antibodies to 752 nm light and recorded time traces of their fluorescence emission. The photon yield and blinking fraction of Alexa 750 is plotted in −3 in BME buffer and 0.2×10−3 in MEA buffer, significantly lower than that of Alexa 647 , chromatic aberration does not affect the image since only one fluorophore is detected, and the brighter photo-switchable reporter molecule (Alexa 647), which yields substantially higher resolution, can be used to image both targets.The two methods for discrimination of photo-switchable fluorophores described above, namely identification by activation wavelength or by emission wavelength, are compatible and may be combined in a multi-parameter measurement to increase the number of simultaneously distinguishable probes in a multicolor STORM image. As an example of multicolor STORM using this multi-parameter detection approach, we obtained six-color super-resolution images of a model sample consisting of fluorophore-labeled streptavidin bound to a glass surface. The use of a model sample simplified the demonstration of the method, while avoiding the complications associated with the preparation of a cell sample specifically stained for six distinct targets. Streptavidin molecules were labeled with one of the following six dye pairs: Cy3-Alexa 647, Cy2-Alexa 647, Alexa 405-Alexa 647, Cy3-Alexa 750, Cy2-Alexa 750, or Alexa 405-Alexa 750. The labeled streptavidin samples were mixed and bound to a glass coverslip through a biotin linkage at a high density, such that individual molecules were not resolvable by conventional fluorescence microscopy. The resulting STORM image is shown in 45In summary, we applied multi-parameter fluorescence detection to expand the color palette of fluorescent probes available for super-resolution imaging applications. Using Alexa 750 in conjunction with Alexa 647, we obtained two-color STORM images with multiple emission channels. Compared to imaging with multiple activation channels, the use of two or more fluorescence detection channels for STORM offers a means of obtaining multicolor images with reduced color crosstalk, an important factor in cases where a small number of molecules of one component must be detected in the presence of a high concentration of molecules of a second component. The lower degree of crosstalk in the dual-emission channel scheme is due to the well separated spectra of the two dyes. The crosstalk between the Alexa 647 channel and the Alexa 750 channel was measured to be approximately 1 % meaning that, potentially, as few as one target molecule of a given cellular component could be unambiguously distinguished against a background of 100 molecules of a second labeled component. However, the multi-activation channel imaging scheme also has advantages over the multi-emission channel scheme, particularly in avoiding imaging artifacts due to chromatic aberration or errors in channel alignment, and allows high resolution in all color channels by pairing a bright reporter such as Alexa 647 with different activators.Finally, we demonstrated six-color super-resolution imaging using multi-parameter fluorescence detection. This approach substantially extends the number of simultaneously detectable probes, and may also be used to improve the accuracy of fluorophore identification. The multi-parameter detection concept may be generalized to any set of orthogonal spectral parameters, and for the implementation reported here we distinguished fluorescent probes by the fluorescence emission and the photo-activation spectra of the probes. The number of colors could be further increased by the addition of activation or emission channels, or by adding other detection parameters such as fluorescence lifetime.Donkey anti-rat antibodies (Jackson ImmunoResearch #712-005-153) and donkey anti-rabbit antibodies (Jackson ImmunoResearch 711-005-152) were labeled with one or two amine-reactive fluorophores. Each antibody was labeled with one fluorophore capable of photo-switchable fluorescence emission (the reporter fluorophore), and some were also labeled with an additional fluorophore (the activator fluorophore) which served to enhance the wavelength-dependent reactivation of the reporter fluorophore. Streptavidin (Invitrogen S-888) was labeled in a similar manner. The labeled probes are summarized in m, pH 8.5), and the labeling reaction was left to proceed at room temperature for 30 min. The labeled product was separated from unreacted dye by running the reaction mixture over a gel filtration column , and eluting in PBS. The labeled product was stored at 4 °C in PBS. Labeling of proteins with two fluorophores was completed in a single reaction step, by adding the reactive dye molecules together into the reaction mixture. A detailed labeling protocol is given in the Supporting Information.Amine reactive fluorophores were obtained from GE Healthcare  or Invitrogen . Dye labeling was carried out according to the manufacturer’s instructions, and has been described in detail previously.The degree of labeling of the antibodies was measured using a UV/Vis spectrophotometer. The labeling ratio was adjusted by varying the amount of each dye that was added. The average degree of labeling for the activator fluorophores  was approximately 3.0 fluorophores per antibody or streptavidin molecule, and the average degree of labeling for Alexa Fluor 647 was approximately 0.7 fluorophores per antibody or streptavidin molecule, as described previously.The microscope used for STORM imaging and single molecule fluorophore characterization has been described in detail previously.Cyanine dyes exhibit thiol dependent photo-switchable fluorescence, meaning that a thiol is required in solution in order for switching to occur.BME imaging buffer:m, pH 8.0)Tris (50 mm)Sodium chloride (10 mGlucose (10 % w/v)m, Sigma, M3148)β-mercaptoethanol (143 mEnzymatic oxygen scavenger system (1 % v/v)MEA imaging buffer:m, pH 8.0)Tris (50 mm)Sodium chloride (10 mGlucose (10 % w/v)m, Sigma, 30070)β-mercaptoethylamine, pH 8.5 (10 mEnzymatic oxygen scavenger system (1 % v/v)−1, Roche Applied Science, 106810) in PBS (200 μL), and centrifuging the mixture at 13 000 rpm for 1 min.The enzymatic oxygen scavenging system was added to the buffer immediately before use, and the stock solution was prepared by mixing glucose oxidase powder  with catalase . Alexa 647 or Alexa 750 were observed to initially switch off, and then to blink on and off stochastically. The fluorescence-versus-time trace of each molecule in the field of view was determined from the data. The number of photons per switching event was calculated by integrating the measured fluorescence for each event. Histograms of the photon counts were fit with an exponential function to determine the average number of detected photons per cycle for Alexa 647 and Alexa 750 in each buffer condition  and fixed with formaldehyde (3 %) and glutaraldehyde (0.1 %) at room temperature in PBS for 10 min. The fixing step was followed by quenching with sodium borohydride (0.1 %) in PBS for 7 min to reduce the unreacted aldehyde groups and fluorescent products formed during fixation. The sodium borohydride solution was prepared immediately before use to avoid hydrolysis. The fixed sample was permeabilized in a blocking buffer  for 10 min and stained with both of the primary antibodies against tubulin  and Tom20  for 30 min in a blocking buffer. The sample was then rinsed with a washing buffer  three times for 10 min each. The corresponding secondary antibodies labeled with photo-switchable probes were added to the sample  and left for 30 min at room temperature. The sample was then washed three times for 10 min each with a washing buffer. As a final step to preserve fluorophore binding, the sample was rinsed with PBS and then post-fixed for 10 min at room temperature with formaldehyde (3 %) and glutaraldehyde (0.1 %), and then stored in PBS at 4 °C before imaging.Green monkey kidney BS-C-1 cells were plated in LabTek II 8-well chambered coverglass (Nunc) at a density of 3×10The imaging procedure for multicolor STORM was slightly different depending on whether or not multiple fluorescence emission channels were used. For experiments using a single emission channel, the dataset was acquired in one step. Dual-emission channel data was acquired in two imaging steps—one for each channel. By exciting only one of the two fluorophores at any given time, leakage of fluorescence from the tail of the Alexa 647 emission spectrum into the Alexa 750 detection channel, and vice-versa, was reduced. Simultaneous imaging of the two channels would be possible by simultaneously exciting both fluorophores using two different wavelength laser lines, and by using a dual-view imaging setup to split the emission from the two fluorophores into two paths. For all experiments using multiple activation wavelengths for multicolor imaging, the appropriate activation light sources were pulsed in sequence to selectively activate different populations of fluorescent probes. For all STORM imaging experiments, samples were prepared in BME imaging buffer, with the exception of the single-emission channel dataset  g–i, forData analysis for STORM has been described in detail previously.Since the dual-emission channel multicolor STORM data is acquired by using two different optical detection paths, a method for image registration is required, in order to overlay the images and generate the final two-color STORM image. Because the dichroic mirrors and emission filters are different for the two channels, and also due to the difference in the spectral ranges detected, each channel may have small differences in magnification, image rotation, shear, and other aberrations. Furthermore, this alignment procedure must have a high degree of precision in order to avoid compromising the spatial resolution of the multicolor STORM image itself. We used fiducial markers  which were visible in both detection channels for alignment of the data sets. During data analysis for both datasets, the bead positions were localized with high precision over the course of the experiment, based on their images which were visible in the raw data. As a result, each of the two resulting STORM images contained tightly localized clusters of localizations corresponding to the beads fixed to the sample. The set of localizations collected from the Alexa 647 channel was transformed, using a polynomial warp transform to account for differences in magnification, rotation, shear, etc., and then the images were aligned using a rigid translation, based on the bead positions in the image. Control measurements verified that this procedure resulted in an image-registration precision of 5.6±2.5 nm. Further details of the alignment procedures and control measurements are given in the Supporting Information.Color crosstalk in STORM images can be reduced by using a statistical analysis equivalent to linear un-mixing in conventional fluorescence images, as described previously.A quantitative measurement of color crosstalk present in the STORM data is shown in the Supporting Information. For two color images of microtubules and mitochondria, crosstalk is measured by choosing four or five regions of the sample containing only microtubules or Tom20, and measuring the relative numbers of localizations in these regions for each of the two channels. Each region picked contained a minimum of one thousand localizations. The average degree of crosstalk across the image was determined by calculating the mean of the crosstalk values measured for each region. The reported error for the average crosstalk corresponds to the standard error of the mean. For analysis of the STORM image of labeled streptavidin, crosstalk was quantified by picking isolated clusters of localizations which appear in the data, most likely corresponding to single streptavidin molecules. These were measured to determine the number of localizations of each color within the clusters, yielding a value for the degree of crosstalk. Further details of the crosstalk-analysis procedures are given in the Supporting Information.


Phosphorylation is one of the major mechanisms by which the activities of protein factors can be regulated. Such regulation impacts multiple key-functions of mammalian cells, including signal transduction, nucleo-cytoplasmic shuttling, macromolecular complexes assembly, DNA binding and regulation of enzymatic activities to name a few. To ensure their capacities to replicate and propagate efficiently in their hosts, viruses may rely on the phosphorylation of viral proteins to assist diverse steps of their life cycle. It has been known for several decades that particles from diverse virus families contain some protein kinase activity. While large DNA viruses generally encode for viral kinases, RNA viruses and more precisely retroviruses have acquired the capacity to hijack the signaling machinery of the host cell and to embark cellular kinases when budding. Such property was demonstrated for HIV-1 more than a decade ago. This review summarizes the knowledge acquired in the field of HIV-1-associated kinases and discusses their possible function in the retroviral life cycle. The genetic information of human immunodeficiency virus type 1 (HIV-1) is carried by an RNA genome of approximately 9.3 Kb packaged into viral particles as a non-covalent dimer ,3. WhileIn addition to the difficulty associated with discriminating between host factors that are selectively or passively packaged into viruses, the identification of cellular proteins embedded in viral particles is technically complicated. The most critical aspect is the strict necessity to discriminate between virus-incorporated components and cellular factors contaminating viral preparations. The latter group includes proteins docked to the outside of cell-free virions. This group also comprises cellular proteins contained in microvesicles and exosomes with sizes and densities comparable to viruses, that co-sediment with viral preparations and represent a source of contamination even after the density gradient separation of viral particles ,10,11. AHZ321 isolate grown in HUT78 T lymphocytes [ELI viruses prepared from MT4 cells [NLAD8 grown in primary macrophages [Another technical feature to consider when studying HIV-1-associated cellular factors is the cell type and the viral isolate or strain used to prepare the biological samples. The array of packaged cellular proteins may differ greatly according to the host cell used to propagate the virus. This aspect has been well documented for membrane molecules embedded in the envelope of virions produced from various T cell lines. The acquisition of CD55 and CD59 complement decay factors , LFA-1  and MHC phocytes  and fromhttp://web.ncifcrf.gov/research/avp/), among which only a small fraction has been functionally characterized. A significant proportion of these molecules are glycoproteins expressed at the surface of the host cell that become incorporated into the lipid bilayer surrounding the retroviral particle, as extensively reviewed previously [The above mentioned strategies have led to the identification of a surprisingly large variety of HIV-1 associated cellular components  ,31,36,37The viral incorporation of nuclear proteins is typically illustrated by the selective packaging of histones  and a number of proteins that interact with nucleic acids. This group comprises the active histone deacetylase HDAC1 and the chromatin remodeling protein INI1/HSNF5, which is selectively incorporated into HIV-1 virions  ,42. ThisHerpesviridae, Poxviridae, Baculoviridae), the phosphorylation of viral proteins can be catalyzed by protein kinases encoded by the viral genome. The knowledge in this field has recently been summarized in a complete review [lck, cdc42, PKC and STAT1 for which only their presence in the virus has been reported [In this review, we focused primarily on the class of HIV-1-associated host cytosolic factors known as protein kinases. Phosphorylation is one of the major mechanisms through which the activity of protein factors can be regulated. In mammalian cells, up to 30% of all proteins may be modified by phosphorylation. Such regulation impacts multiple levels, including nucleo-cytoplasmic shuttling, the assembly of macromolecular complexes, DNA-binding capacity and enzymatic activation. The presence of kinase activities in viral particles was observed early. The first observations in the field demonstrated that high levels of protein kinase activity are packaged in the Rauscher murine leukemia virus  and vacce review . Regardie review -60, littreported . The kinThe MAPKinase ERK2 was the first protein kinase of cellular origin to be detected within HIV-1 viruses ,7. The vAttempts to identify the function of HIV-1-associated ERK2 have rather focused on its capacity to phosphorylate the retroviral matrix protein (MA) . A fractFinally, searching for ERK2 substrates has evidenced that the kinase also phosphorylates the L-domain-containing p6 protein . The comIn summary, the data accumulated since the initial description of ERK2 in HIV-1 particles indicate that the encapsidation of this cellular kinase is strictly required for optimal infectivity. Although its function is not clearly elucidated, virus-associated ERK2 could assist early steps of HIV-1 replication either by supporting the establishment of a functional reverse transcription complex or by regulating nuclear import of the preintegration complexes. Very recent data have shown that ERK2 interacts with the poly-proline motif located near the cyclophilin binding loop at the N-terminus of HIV-1 CA domain of Gag . This moA second well-documented example of a cellular kinase packaged within HIV-1 particles is the cAMP-dependent protein kinase (PKA). In recent years, our group has demonstrated that PKA is packaged into HIV-1 viruses . In the in silico to model the consequences of phosphorylation at the level of a p24 hexamer has revealed that negative charges generated by phosphate conjugation at each serine position favors inter-monomer repulsion or cleavage of important inter-monomeric bonds required for preserving the stability of the hexameric ring of p24 [in vitro assembly experiments of recombinant p24 with serine-to-aspartic acid mutations, mimicking constitutive phosphorylation. These results need to be considered in the context of the reversible processes required for the association of the retroviral core during the assembly of the viral particle and for the dissociation of the conical capsid once delivered into the target cell. Indeed, phosphorylation and dephosphorylation events are regulators of protein-protein interactions.In mature HIV-1 particles, monomeric p24 assembles into a lattice of hexameric and pentameric rings to form a conical core containing the retroviral genome and associated proteins. Our approach developed g of p24   particles has revealed that cell-associated capsid proteins are highly phosphorylated, while capsids assembled into cell-free virions are dephosphorylated [Togaviridae. In rubella virus, cycles of alternate phosphorylation of the capsid protein, during the early stages of replication, and dephosphorylation during the latter stages, timely regulate the assembly of the nucleocapsid and the packaging of genomic RNA [Data accumulated from various models have proven that kinases from viral or cellular origins act as regulators of the assembly and disassembly of viral particles by modulating the association or repulsion of proteins involved in the structures of the viral cores and in the packaging of viral genomes. This mechanism is particularly well documented for herpesviruses. Herpes simplex virus type 1 (HSV-1) tegument protein undergoes phosphorylation and dephosphorylation according to the stage of replication. Moreover, the solubility of the viral tegument is significantly enhanced in the presence of ATP-Mg and functional kinase activity -94. In torylated ,97. In torylated . From thorylated -102. Theorylated -105. Finomic RNA . Therefoin vivo  substitutions mimicking constitutive phosphorylation of potential target sites. Although it may be informative, this approach fixes the experimental system into a phosphorylated or unphosphorylated state and accounts neither for dynamic phosphorylation and dephosphorylation events nor for the stoichiometry of the reaction. Accordingly, each model elaborated using this strategy needs to be refined to account for the real proportion of molecules phosphorylated articles . These din vitro and in vivo with limited emergence of resistance [Finally, the above mentioned studies point to the necessity to question the physiological role played by the packaged kinases. Recently, a proteomic analysis of SIV virions revealed that ERK2, PKC and PKA catalytic subunit are packaged into particles . Moreovesistance . If usedIn summary, a limited number of studies have uncovered the nature and the function of cellular kinases packaged into HIV-1 particles. While the experimental approaches developed remain imperfect, these studies have clearly shown that HIV-1-associated kinases are required for infectivity and act as key regulators at various steps of the replicative cycle, including nuclear shuttling of the preintegration complex and capsid assembly/disassembly. These functions are regulated through phosphorylation of HIV-1-encoded proteins. Given the variety of cellular kinases detected in viral particles, this family of proteins is expected to regulate additional steps of the HIV-1 life cycle. Analyzing their function will certainly provide new information regarding the biology of HIV-1. In addition, the capacity of HIV-1-associated kinases to target proteins in the newly infected cells has also been proposed although not formally demonstrated. This issue needs to be examined carefully to determine the contribution of virus-associated kinases in HIV-1 induced pathogenesis.The authors declare that they have no competing interests.NC and LB wrote the manuscript and made the figures. CG contributed to the manuscript writing and editing. All authors read and approved the final manuscript.


Gastro-esophageal reflux disease (GERD) is one of the most frequent diseases of the upper gastro-enteric tract and incidence increases during lifetime . In eldeWe drove a prospective study in a period from March 2012 to March 2013, in which 13 elderly patients were treated with VLP Toupet fundoplication. Of these, 11  completed the follow up to six months, while 2  have not respected the post-operative protocol.The inclusion criteria for the study were:1. Patient age > 652. Long-term diagnosis of GERD3. Severe symptoms with conservative treatment4. Good LES basal tone and good esophageal motilityThe patients have been preliminarily submitted to a 24 hours pH-metry and to an esophageal manometry.The study foresaw a follow up of six months, that consisted in the repetition of 24 hours pH-metry and manometry one month and six months after the surgical intervention.The results of study shows that after the intervention there are appreciable improvements, because all patients refer a meaningful decrease of symptoms severity, without post-operative disphagia. No manometric alterations and improvements of 24h ph-matric values were present in all cases.Laparoscopic Toupet fundoplication seems to be a good alternative in selected elderly patients with severe symptoms of GERD, offering similar results of Nissen-Rossetti fundoplication .


By the end of 2011 Global Fund investments will be supporting 3.5 million people on antiretroviral therapy (ART) in 104 low- and middle-income countries. We estimated the cost and health impact of continuing treatment for these patients through 2020.Survival on first-line and second-line ART regimens is estimated based on annual retention rates reported by national AIDS programs. Costs per patient-year were calculated from country-reported ARV procurement prices, and expenditures on laboratory tests, health care utilization and end-of-life care from in-depth costing studies. Of the 3.5 million ART patients in 2011, 2.3 million will still need treatment in 2020. The annual cost of maintaining ART falls from $1.9 billion in 2011 to $1.7 billion in 2020, as a result of a declining number of surviving patients partially offset by increasing costs as more patients migrate to second-line therapy. The Global Fund is expected to continue being a major contributor to meeting this financial need, alongside other international funders and domestic resources. Costs would be $150 million less in 2020 with an annual 5% decline in first-line ARV prices and $150–370 million less with a 5%–12% annual decline in second-line prices, but $200 million higher in 2020 with phase out of stavudine (d4T), or $200 million higher with increased migration to second-line regimens expected if all countries routinely adopted viral load monitoring. Deaths postponed by ART correspond to 830,000 life-years saved in 2011, increasing to around 2.3 million life-years every year between 2015 and 2020.Annual patient-level direct costs of supporting a patient cohort remain fairly stable over 2011–2020, if current antiretroviral prices and delivery costs are maintained. Second-line antiretroviral prices are a major cost driver, underscoring the importance of investing in treatment quality to improve retention on first-line regimens. As of the end of 2009, over 5 million people were receiving antiretroviral treatment (ART) globally Increased international financing has enabled the rapid growth in the number of people receiving ART, though including all those who do not currently have access to treatment will require US$ 7 billion annually, as a minimum estimate based on the WHO's 2006 treatment eligibility criteria We use new data on Global Fund financing of ART programs to estimate the future funding required to enable the cohort of 3.5 million persons expected to be receiving ART in Global Fund-supported programs as of 2011 to continue receiving treatment through 2020. We also compute the health impact of continued provision of ART for this cohort, in terms of deaths averted/postponed and life-years saved, and explore key determinants of cost.We estimate the long-term costs and impact of Global Fund support by following the cohort of patients on treatment in 2011. New patients who will start treatment in Global Fund-supported programs over next years are not considered in the current analysis, for simplicity and in order to establish a minimum commitment. We estimate the future cost and health impact by projecting the number of patients that will still be alive and receiving treatment in future years by subtracting those that die of AIDS or non-AIDS causes. Although some patients may stop therapy voluntarily that does not reduce the obligation to continue them on treatment if they desire it.We have used as a starting point the number of patients on ART with Global Fund support in 2011. While the actual grant delivery results for 2011 will not be available until early 2012, the funding committed for ART treatment is already in place, and supported programs have each set their target for the number of people to receive treatment through ongoing grants and approved grant proposals for next calendar years.Patients are assumed to survive according to recent, internationally agreed best estimates of current rates of survival, as discussed below. Patients may also fail first line treatment and require more expensive second line. Although many patients live in countries where the provision of second line is inadequate we assume that all patients get second line when they need it, for the purpose of estimating the funds required to provide necessary treatment.Impact is measured in terms of deaths averted and life-years gained, as detailed below, comparing the actual patient cohort with a counterfactual scenario in which essentially all those patients on treatment in 2011 would die within a few years if they could not longer access treatment.For the 2011 cohort of 3.5 million patients, ART need in future years is estimated based on the retention and survival of those patients in each subsequent year. According to reports submitted to the World Health Organization (WHO) by 38 national AIDS programs in low- and middle-income countries in 2008, the proportion of patients (adults and children) remaining on treatment – i.e. not dying and not lost to follow-up – averaged 80% at 12 months after treatment initiation, and 75% after 24 months, 74% after 36 months and 73% after 48 months For the start year of projections, 2009, we used the WHO 2008 survey for 38 National AIDS Programs to determine country-specific proportions of patients on second-line regimens. For countries not participating in this survey, we applied regional weighted average proportions. Across all Global Fund-supported countries, the weighted average proportion was 2.5% of patients on second-line regimens. For subsequent years, we calculated the proportion of patients on second-line regimens using regional migration rates with an average of 1.9% of patients who survived on first-line regimens switching to second-line regimens each year We estimated the health benefits of Global Fund-supported ART by comparing survival of patients initiating and continuing treatment, with a counterfactual cohort for which continued treatment was not available from 2011 onwards.To calculate the health impact of continued ART, we assumed that all HIV-infected people starting ART have advanced disease and meet standard ART eligibility criteria, according to the WHO's 2006 Treatment Guidelines which propose ART for HIV-infected people with clinical stage III or IV disease, and/or CD4 below 200/µL Estimates of cost per patient-year of ART are shown in Prices for both first-line and second-line ARVs vary substantially by country but have declined over recent years. Based on country-reported procurements reported through the Global Fund's Price and Quality Reporting system and WHO's Global Price Reporting Mechanism, median prices across Global Fund-supported countries were $204 for first-line drugs and $1238 for second-line drugs as of 2009 In most programs ART patients are monitored with laboratory tests for CD4 counts, blood chemistry and hematology. Some programs also include viral load monitoring. Treatment protocols usually specify monitoring every three to six months. Our review of 15 published reports from low- and middle-income countries on the costs of laboratory tests found a median annual cost of $180 for monitoring each patient receiving ART  [13]––[9],Using review results we calculated non-ART care requirements, once HIV patients fail ART, at 5.5 out-patient visits, 9.7 in-patient days and $49 in non-ARV drug costs per patient-year. Based on WHO-CHOICE country estimates of the cost of hospital days and outpatient visits in each country All costs are expressed in 2008 US dollars.Cost estimates were repeated in sensitivity analysis that varied, univariately, the future prices of first-line and second-line ARVs, the mix of ARV regimens used and the rate of migration from first-line to second-line ARV regimens. Of the 3.5 million people receiving ART with Global Fund support in 2011, we estimate that 2.3 million will still be alive and receiving treatment in 2020.  The propThe health impact of continuing the 2011 cohort of patients on ART will be quite large since all those receiving ART today would die by 2020 if they stopped ART. The largest number of deaths  is averted in 2012, the year after the 2011 cohort reaches its full size . The annThe averted deaths correspond to 860,000 life-years saved in 2012, increasing to 2.3 million annual life-years saved by 2017 . CumulatThe future investment required to support those currently receiving ART will be influenced by a number of factors including changes in ARV prices, the distribution of patients over ARV regimens of differing prices, and patient laboratory monitoring practices. The median prices of the most commonly used first-line ARVs declined by an average 12% per year from 2006 to 2009 Considering the higher current prices there is more scope for faster declines in second-line ARV prices in coming years The average per-patient cost of first-line regimens may rise in the future as countries implement the revised 2009–10 WHO ART guidelines Countries that use routine viral load monitoring have higher migration to second-line regimens, around 6%, than those that do not routinely monitor viral load . If all Global Fund-supported countries had 6% annual migration to second-line regimens, then financing needs in 2020 would increase by $560 million, a 33% increase.The number of people receiving ART in low- and middle-income countries has expanded rapidly in recent years. Much work has focused appropriately on what is required to continue this scale-up to reach all those in need of treatment. This study examines a previously unexplored aspect of treatment, the financing required to continue supporting patients receiving ART.Investments by the Global Fund together with other partners have enabled and catalyzed a rapid expansion of HIV programs in low- and middle-income countries that is reducing mortality and new infections The costs of treatment in this analysis vary only by first and second line treatment. In reality costs may be different for different types of patients, pregnant women, children, TB patients. We have used an average per patient cost, which implicitly assumes that the distribution of patients by type will not change.Sensitivity analyses showed that program costs are sensitive to ARV prices and regimens used, in particular for second-line ARV regimens. A balanced use of lower-cost generic ARVs and more expensive innovator drugs will impact costs. Efforts to improve treatment quality to retain patients on first-line regimens will be critically important to manage future costs. Equally, considerable variations in the protocols and frequencies of laboratory testing and associated cost point to opportunities for efficiency gains in patient monitoring. Recent studies in resource-constrained settings suggest that CD4-based patient monitoring is generally cost-effective compared to clinical monitoring alone, but that viral load (HIV RNA) monitoring is not always cost-effective To further refine this type of assessment, we will need progressively better information on country-level variation and determinants of cost of ART delivery Also, program achievements in terms of patient retention and survival, on first-line and second-line regimens, deserves improved monitoring and assessment. In-depth studies and evaluations in countries with relatively advanced patient tracking systems suggest that routine national program records may overestimate actual retention and survival in several high-HIV countries, given elevated mortality among patients lost-to-follow-up, and (passive) over-counting of retention time among patients who dropped out from recent clinic or pharmacy appointments. On the other hand, patients transferring between facilities may be over counted as new initiations and undercounted in retention We estimate continued investment needs for a cohort of 3.5 million people receiving ARVs. The global need for treatment and financing of HIV is far larger than this number. The 2010 revised WHO HIV Treatment Guidelines While this study examined ART costs for existing patients through the year 2020, investment needs continue beyond this date, as by that year 2.3 million people (66% of the 2011 cohort) will still be alive and in need of continuing treatment Support for these patients will also include non-ART costs such as prevention and treatment of opportunistic infections including tuberculosis and malaria In conclusion, the rapid expansion of ART in low- and middle-income countries in recent years has produced dramatic declines in mortality and represents an important success in our efforts to improve global health. Sustaining and protecting gains will require sustained investment from international donors such as the Global Fund, as well as sustained contributions by the governments of supported country programs.Table S1Laboratory costs of ART, USD per patient-year.(DOCX)Click here for additional data file.Table S2Service delivery costs of ART, per patient-year.(DOCX)Click here for additional data file.