The objective of this study was to identify new viral biomarkers associated with acute on chronic liver failure (ACLF) by complete genomic sequencing of HBV. Hepatitis B virus mutations associated with ACLF were screened by Illumina high-throughput sequencing in twelve ACLF cases and twelve age-matched mild chronic hepatitis B patients, which were validated in 438 chronic hepatitis B patients  by direct sequencing. The results of Illumina sequencing showed that the mutations at 7 sites  of 12 ACLF patients were significantly higher than those of 12 controls. In the validation cohorts, a significantly higher ratio of genotype B to C was found in patients with ACLF than in patients with non-ACLF. Multivariate analysis showed that T216C, G1896A, C1913A/G and A2159G/C were independent risk factors for ACLF. C216 in any combination, A/G1913 in any combination, and G/C2159 in any combination had high specificity for ACLF. In summary, T216C and A2159G/C mutations were novel factors independently associated with ACLF. Combined mutations in hepatitis B cases could play important roles in ACLF development. Infection of hepatitis B virus (HBV) is one of the most serious and prevalent global health problems. More than 350 million people worldwide are chronically infected with HBV . HBV relHBV is a small enveloped DNA virus of the Hepadnaviridae family with approximately 3200 nucleotides. Since HBV replicates through the reverse transcription of pregenome RNA, mutations occur more frequently in HBV than in other DNA viruses . TherefoAll of the patients in present study were from the southeast of China. The diagnosis criteria were based on the Management Scheme of Diagnostic and Therapy Criteria of Viral Hepatitis issued by Chinese Society of Infectious Diseases and Parasitology and the Chinese Society of Hepatology . All parHBV DNA was extracted from 200 uL serum using a QIAamp DNA blood mini kit , according to the manufacturer's instructions. Primers pairs F1-R1, F2-R2, and F3-R3 were used to amplify 3 overlapping HBV full-length fragments . PCR reaThe error of Illumina high-throughput sequencing is higher than that of the Sanger method, therefore, a plasmid DNA containing full complete HBV served as an error rate control for measuring the accuracy of the Illumina high-throughput sequencing. The control plasmid and 24 samples were prepared simultaneously for Illumina high-throughput sequencing.Illumina reads from the control plasmid were mapped using the Bowtie software, with the default parameters. An initial analysis revealed that the error rate varied as a function of position within the read , so onlyThe overall error rate of plasmid control sequence was 0.49%, and the mean number of nucleotides with ≥4% mismatch error rate per one hundred nucleotides was 2.4. According to this result, 4% was empirically served as the limit to distinguish the authentic variants from technological artifacts .Semi-nest PCR was used to amplify the two target regions of HBV genome. Primer pairs F2-R4 were used as the first-round primers, and primer pairs F4-R4 served as the second-round primers for regions A. Primer pairs F5-R5 were utilized as the first-round primers, and primers-pairs F5-R5.1 served as the second-round primers for regions B . PCR wasHBV serum markers were tested by commercial enzyme immunoassay kits . HBV DNA was quantified by a PCR diagnostic kit  with a detection limit of 500 copies /mL. HBV genotypes were detected by a multiplex PCR assay .P value of < 0.05 was considered as statistically significant. The combined mutation frequencies of HBV were estimated by SHEsis software platform  [Chi-square test or Fisher’s exact test was used to determine the differences in categorical variables. Continuous variables with normal distribution were tested by one-way ANOVA among multiple samples and by Student’s t-test between two samples. Continuous variables with skewed distribution were compared using Kruskal-Wallis test among multiple samples and with the Mann-Whitney U-test between two samples. Serum viral loads with skewed distribution were adjusted to normal distribution by transformation into logarithmic function, and then tested by Student’s t-test or one-way ANOVA. Forward stepwise multivariate regression analysis was performed to determine the risk factors associated with ACLF development. All statistical tests were two sided, and performed using the Statistical Program for Social Sciences 13.0 (SPSS13.0). ruction) .P<0.05 or P<0.01).P<0.01). The nucleotide sequences have been submitted to the GenBank databases under the accession numbers JX681227-JX681664 and JX847884-JX848321. The mutation frequencies at seven sites in ACLF group enhanced significantly, as compared with ASC, CHB-M, and CHB-S groups (P<0.05). The mean values of substitutions at seven sites in ASC, CHB-M, CHB-S and ACLF groups were 0.88, 1.38, 2.34 and 3.74, which increased in a stepwise manner.The clinical and virological characteristics of 438 cases are showed in In ACLF patients with genotype B virus, the substitution rates of T216C, G285A, A1846T/G, G1896A, C1913A/G, A2159G/C, and A2189T/C were 70.6%, 60.3%, 66.2%, 76.5%, 63.2%, 47.1%, and 50.0%, respectively; while in ACLF patients with genotype C virus, the substitution rates were 8.3%, 13.9%, 44.4%, 63.9%, 41.7%, 41.7%, 47.2%, respectively.To avoid the potential influence on the results brought by age and sex differences among the 4 groups, we adjusted the age and sex in the flowing statistic analysis process. To clearly describe the associations of mutations in the S region and pre-core /core region of HBV genotypes B and C with ASC, CHB-M, CHB-S and ACLF, we firstly treated ASC as control . MutatioAs compared with non-ACLF cases, T216C, G285A, A1846T/G, G1896A, C1913A/G, A2159G/C, A2189T/C in genotype B and G285A, A1846T/G, G1896A, C1913A/G and A2159G/C in genotype C were significantly associated with an increased risk of ACLFsee .P < 0.05 or P < 0.01).Patients’ age, sex, HBV genotypes, HBV DNA level (log10 copies/ml), HBeAg status, and the presence of mutations at seven sites were included in the multivariate regression model . It was P < 0.01). Rather than a single mutation, combinations with any 2 or more of T216C, G1896A, C1913A/G and A2159G/C were significantly associated with the development of ACLF compared with non-ACLF  (P<0.01). We then evaluated the potential value of the presence of HBV mutations that were independently associated with ACLF for the indication of ACLF. As shown in The SHEsis software platform was used to estimate the combined mutations of hepatitis virus genome and mutations independently associated with ACLF were included in the analysis. Haplotype containing wild types at all of four mutation sites was inversely associated with ACLF  . The freFifty of the 104 ACLF patients survived for at least 3 months after the onset of liver failure, while 54 patients had a fatal outcome. Patients’ age, sex, HBV genotypes, HBV DNA level (log10 copies/ml), HBeAg status, ALT, TBIL, PTA, and the presence of mutations at the seven sites were included in the multivariate regression model. It was found that the higher TBIL  and lower PTA  were independently risk factors of the mortality of patients with ACLF.Up to now, many antiviral drugs, such as lamivudine, adefovir, Tenofovirand entecavir, have been used to treat hepatitis B, and one of the key issues for preventing the development of ACLF is early antiviral treatment of chronic hepatitis B (CHB) with acute exacerbation , 18. So The results of Illumina high-throughput sequencing in present study indicated that there were significant differences of the mutations at seven sites of HBV between twelve ACLF cases and twelve CHB-M cases. Of these seven mutations, A1846T/G, G1896A and C1913A/G were studied extensively, whereas T216C, G285A, A2159G, and A2189C were novel ACLF-related mutations identified in this study . To veriAs compared with the non-ACLF group, the AOR of T216C and A2189T/C mutation in ACLF cases with genotype B significantly increased, but AOR of T216C and A2189T/C mutation in ACLF cases with genotype C did not elevate significantly. It was suggested that HBV genotypes with distinct patterns of nucleotide substitutions were related to ACLF development. Multivariate regression analysis showed that T216C, G1896A, C1913A/G and A2159G/C mutations were independent risk factors for ACLF cases. Some previous studies also indicated that G1896A and C1913A/G mutations were in close relationship with ACLF . HoweverAlso it was found that the frequencies of T216C, G1896A, G1913A/G, and A2159G/C were >45% in the patients with ACLF, the combinative mutations at these sites could be utilized to diagnose clinically and to discriminate the HBV-infected patients with advanced disease. When the association of combined mutations of HBV with CHB progression was analyzed, we found that C216 in any combination, A/G1913 in any combination, and G/C2159 in any combination were specific for ACLF . Thus coTill now, the biological mechanism of CHB deterioration related to virus mutations has not been clear. Of the HBV mutations at nt.216, nt.1896, nt.1913 and nt.2159 sites independently associated with ACLF, only G1896A has been studied extensively and it can abolish HBeAg expression at the translational level . The C19In conclusion, the associations of HBV mutations with ASC, CHB-M, CHB-S, and ACLF were investigated systemically in present study. It was found that patients with CHB infected with genotype B virus were more prone to development of ACLF than those with genotype C virus. T216C, G1896A, C1913A/G and A2159G/C were factors independently associated with ACLF. A combined examination of different viral mutations could predict more precisely the progression of liver disease. These virus mutations, as molecular biomarkers, may be useful for preventing ACLF development and predicting prognosis of patients with hepatitis B.S1 FigAs shown in the plot, the first 20 base pairs of the read contain the lower amount of error.(TIF)Click here for additional data file.


Since early 2013, H7N9-subtype avian influenza virus (AIV) has caused human infection in eastern China. To evaluate AIV contamination and the public risk of infection, we systematically implemented environmental sampling from live poultry markets in Guangdong Province. Through real-time polymerase chain reaction assays and next-generation sequencing, we generated full nucleotide sequences of all 10 H6N6 AIVs isolated during sampling. Focusing on sequence analyses of hemagglutinin genes of the 10 H6N6 AIVs revealed that the viruses were low pathogenic AIVs with the typical hemagglutinin cleavage site of P-Q-I-E-T-R-G. The hemagglutinin, neuraminidase, and nucleocapsid genes of nine AIVs were of ST2853-like (H6-subtype) lineage, ST192-like (N6-subtype) lineage, and HN573-like (H6-subtype) lineage, respectively; whereas the other five genes were of ST339-like (H6-subtype) lineage. However, the polymerase PB2 and nucleocapsid genes of one strain (HZ057) were of GS/GD-like (H5N1-subtype) and ST339-like lineages. Phylogenic analysis revealed that all eight genes of the 10 viruses belonged to Eurasian avian lineage. Altogether, the 10 AIVs were reassortants of different genetic groups of exchanges with the same virus subtype, thus illustrating the genetic diversity and complexity of H6N6-subtype AIVs in Guangdong Province. Avian influenza viruses (AIVs) are single-stranded, negative-sense RNA viruses of the family Orthomyxoviridae that contain eight gene segments  have long been regarded as potential hotbeds for AIVs, whose transmission can be facilitated there via humans' direct and indirect contact with poultry. It has been reported that LPMs have become an important source for human infection with H5N1 highly pathogenic AIV  and thereafter analyzed the molecular evolution of 10 H6N6-subtype AIVs. Ultimately, with this study we have contributed fundamental data regarding controlling AIVs as well as epidemiological and molecular evidence of their evolution in Guangdong's LPMs.This study was approved by the Guangdong Provincial Center for Disease Control and Prevention (CDC) Experimental Animal Welfare Ethics Committee (permit number 2015–09).Surveillance of AIVs was conducted in LPMs in Guangdong for 12 months beginning in January 2013. Samples including poultry manure and material swabbed from defeathering machines, sewage, and chopping tools were randomly taken from LPM environments weekly. Samples were initially tested for influenza A virus with real-time polymerase chain reaction (PCR) in the local CDC. Positive influenza A samples were differentiated by HA subtype (H1–H18) via real-time PCR in local CDC laboratories and further verified by Guangdong Provincial CDC laboratories. Positive samples of H6-subtype were further tested by NA subtype (N1–N11), also with real-time PCR. In all, 10 positive samples of H6N6-subtype were purified and propagated in embryonated, 10-day-old specific-pathogen–free (SPF) chicken eggs and stored at −70°C . Reverse transcription and PCR amplification of all eight gene segments was performed using PathAmp™ FluA Preamplification Reagents . Products of PCR were purified with the Ampure XP purification kit  and quantified using an Ultrospec 2000 mass spectrophotometer . Full genome sequences of the isolated AIVs were sequenced using the Personal Genome Machine Sequencing 200 Kit v2 (American Life Technologies) and the Ion 316 Chip V2 on an Ion PGM System to sequence.Full sequences of the isolated AIVs were obtained by aligning the resulting sequences against representative sequences downloaded from GenBank , which included ST339-like and ST2853-like AIVs in internal gene trees. Unlike surface gene trees, AIVs in Group II rarely formed a monophyletic clade in internal gene trees. Meanwhile, AIVs circulating in natural hosts could also form the monophyletic Group III in all internal gene trees Figure .Phylogenetic analyses of internal genes showed that polymerase PB1 (PB1), polymerase PA (PA), matrix protein (M), and non-structural protein (NS) genes of all 10 viruses were of Eurasian lineage and ST339-like viruses, as well as shared 94.2–96.4, 95.3–95.5, 95.4–97.7, and 95.6–96.4% nucleotide identity with A/duck/Shantou/339/2000 (H6N2), respectively Figures . The polTen H6N6-subtype AIVs contained multiple basic amino acids (PQIETR↓G) at the cleavage site between HA1 and HA2, thereby indicating that the 10 AIVs were of low pathogenicity in domestic poultry. Amino acid residues T160, Q226, and G228 (H3 numbering) were retained in the receptor-binding pocket of HA1, thus indicating that the 10 H6N6-subtype AIVs preferred to bind to α-2,3-linked sialic acid AIV receptor , whereas the other nine AIVs had eight potential N-linked glycosylation sites in NA . Position 274 of the NA protein (NA of GS/GD number) was Y, which suggested sensitivity to the neuraminidase inhibitor oseltamivir  and their transmission among humans.RY, YK, ML, and CK conceived and designed the experiments. RY, YS, LZ, JW, and XZ performed the experiments. RY analyzed the data. RY, LL, XZ, HN, JYL, JL, ML, and CK contributed reagents/materials/analysis tools. RY wrote the paper.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The aim of this study was to characterize the prevalence and patterns of genotypic integrase inhibitor (INI) resistance in relation to HIV-1 clade.The cohort comprised 533 INI-naive subjects and 255 raltegravir recipients with viraemia who underwent integrase sequencing in routine care across Europe, including 134/533 (25.1%) and 46/255 (18.0%), respectively, with non-B clades .P = 0.009) and G140S/A occurred in 36/209 (17.2%) versus 1/46 (2.2%) subjects (P = 0.005). Intermediate- to high-level cross-resistance to twice-daily dolutegravir was predicted in 40/255 (15.7%) subjects, more commonly in subtype B versus non-B clades . A glycine (G) to serine (S) substitution at integrase position 140 required one nucleotide change in subtype B and two nucleotide changes in all non-B clades.No major INI resistance-associated mutations (RAMs) occurred in INI-naive subjects. Among raltegravir recipients with viraemia (median 3523 HIV-1 RNA copies/mL), 113/255 (44.3%) had one or more major INI RAMs, most commonly N155H , Q148H/R/K + G140S/A  and Y143R/C/H . In addition, four (1.6%) raltegravir recipients showed novel mutations at recognized resistance sites  and novel mutations at other integrase positions that were statistically associated with raltegravir exposure . Comparing subtype B with non-B clades, Q148H/R/K occurred in 42/209 (20.1%) versus 2/46 (4.3%) subjects (No major INI resistance mutations occurred in INI-naive subjects. Reduced occurrence of Q148H/R/K + G140S/A was seen in non-B clades versus subtype B, and was explained by the higher genetic barrier to the G140S mutation observed in all non-B clades analysed. A survey of genotypic resistance tests performed through routine care in a referral laboratory in the USA has shown similar integrase mutational profiles, and whereas the study population had unknown ART status and carried nearly uniformly HIV-1 subtype B,7 a more recent study has shown consistent findings in a large INI-experienced cohort.8Raltegravir was the first integrase inhibitor (INI) to receive approval for the treatment of HIV infection, marking an important therapeutic advance through the provision of a novel class of antiretroviral agents. Raltegravir inhibits the HIV integrase enzyme at the final integration step, the strand transfer reaction, which otherwise results in the covalent linkage of host and viral DNA.12In vitro, the activity of dolutegravir is not compromised by mutations occurring at integrase codon 143 or 155 in isolation, although whether mutations can affect susceptibility in combination with multiple others is the subject of ongoing research. Q148 mutations in combination with other mutations  reduce dolutegravir in vitro susceptibility by 10- to 20-fold.16 Findings from clinical studies are consistent with the in vitro susceptibility data, and indicate that the Q148 mutation pathway significantly markedly affects dolutegravir activity among INI-experienced patients, even when the drug is used twice daily.23Two further INIs, elvitegravir and dolutegravir, have recently been approved. Elvitegravir has a similar genetic barrier to and extensive cross-resistance with raltegravir.26 The CORONET project (Common Sequence Repository and Research Network for Integrase Inhibitors) is a European network for surveillance and collaborative research on INI resistance. A common repository is produced from genotypic resistance tests performed at multiple collaborating centres, which provide care for ethnically diverse cohorts. The aim of this analysis, the first of the CORONET database, was to determine the integrase sequence profiles of patients infected with diverse HIV-1 clades that were either INI naive or experiencing viraemia while receiving raltegravir-containing ART, and identify known and novel integrase mutations associated with raltegravir selective pressure, with a specific focus on the impact of HIV-1 clade on mutation patterns.Data on the genotypic pathways of INI resistance occurring with HIV-1 clades other than subtype B are limited to small cohorts. These studies have suggested that there may be important differences in the resistance profiles of subtype C, CRF01_AE and CRF02_AG, including a high prevalence of naturally occurring resistance in INI-naive subjects with CRF02_AG.http://128.40.115.16/hivrdb/public/default.asp) and the EuResist Database (http://www.euresist.org). Available clinical data included HIV-1 RNA load and ART status at the time of integrase sequencing. The populations undergoing sequencing were INI-naive patients and patients experiencing a viral load >50 copies/mL while receiving raltegravir-containing ART. Sanger sequencing was performed at each participating centre according to local practice and using in-house protocols. All sequences were re-analysed centrally. The current analysis is based upon all submissions made between 2008 and 2011. The Ethics Committee of the Royal Free Hospital in London approved the analysis of fully anonymous data; written informed consent was not required (06/Q0501/125 and MREC/01/2/10). Separate ethics approvals regulate the UK HIV Drug Resistance Database and the EuResist Database.Collection of integrase sequences started in 2008 from nine European laboratories and from the UK HIV Drug Resistance Database (http://hivdb.stanford.edu) and Rega HIV-1 subtyping tools (http://dbpartners.stanford.edu:8080/RegaSubtyping/stanford-hiv/typingtool/). Where results were inconclusive, the NCBI genotyping tool (http://www.ncbi.nlm.nih.gov/projects/genotyping) was used and sequences were characterized by phylogenetic analysis with PhyML. A maximum-likelihood tree was produced using a generalized time-reversible (GTR) model of nucleotide substitution with parameters estimated in PAUP* (bootstrap1000) and integrase reference sequences derived from the Los Alamos Database (http://www.hiv.lanl.gov).HIV-1 clades were assigned by submitting the integrase sequences to the Stanford University HIV Drug Resistance Database (http://hivdb.stanford.edu) and the International AIDS Society.27 Major RAMs comprised T66A/I/K, E92Q/G/V, F121Y, G140S/A/C, Y143H/R/C/K, S147G, Q148H/R/K and N155H/S/T. Minor RAMs comprised mutations at codons 51, 74, 95, 97, 114, 128, 138, 145, 146, 151, 153, 157, 163, 230 and 263. Cross-resistance to dolutegravir was classified according to the definition used in the VIKING clinical trials,18 modified to include the potential low-level resistance effects conferred by isolated Q148 mutations (in the absence of other INI RAMs) as indicated by the Stanford University HIV Drug Resistance Database. Based on available data from raltegravir-experienced patients,18 resistance was defined for twice-daily use of dolutegravir. Dolutegravir resistance categories were as follows: (i) no resistance, absence of Q148H/R/K; (ii) low-level resistance, Q148H/R/K in the absence of G140S/A/C, L74I and E138A/K/T; (iii) intermediate-level resistance, Q148H/R/K with one of the mutations G140S/A/C, L74I or E138A/K/T; and (iv) high-level resistance, Q148H/R/K with two or three of the mutations G140S/A/C, L74I and E138A/K/T.Integrase sequences were screened for INI resistance-associated mutations (RAMs) as defined by the Stanford University HIV Drug Resistance Database (February 2014) , subtype C (5% and 4%) and CRF02_AG (4% and 4%) . The most commonly observed major INI RAMs were N155H , G140S , Q148H , Q148R , E92Q  and Y143R  ; no patient was found to carry major INI RAMs. Among the 255 raltegravir recipients, the median viral load was 3523 copies/mL (IQR 555–27 793); 113/255 (44.3%) patients showed one or more major INI RAMs. In raltegravir recipients with versus those without major INI RAMs the median viral load was 3420 copies/mL (IQR 1195–20 899) versus 3834 copies/mL (IQR 376–32 342), respectively (P < 0.01) with raltegravir exposure.Minor INI RAMs that occurred at a frequency of >2% in raltegravir recipients are shown in Table P = 0.009), 161 (P = 0.004) and 170 (P = 0.009). These mutations occurred in subtype B  and CRF02_AG (I161L/T). They occurred commonly alongside other mutations, e.g. K159R + I161L occurred with Q148H + G140S, and E170A occurred with N155H and Q148H + G140S . In addition, the analysis revealed that raltegravir selective pressure was associated with significantly reduced diversity at integrase positions 24 and 167 (P < 0.001 for both).Comparing sequences from INI-naive and raltegravir-experienced patients, three integrase positions were newly identified statistically as being subjected to selective pressure, involving codons 159 (P = 0.009) and 36 (17.2%) versus 1 (2.2%) (P = 0.005), respectively (Table P = 0.02), occurring in subtypes C (n = 2), G (n = 1), A (n = 1) and CRF09 (n = 1). In patients with one or more major RAMs, the median viral load at the time of sampling was 3535 copies/mL (IQR 1472–25 704) versus 1820 (IQR 555–11 660) in subjects with B versus non-B clades, respectively (P = 0.33).Comparing subtype B with non-B clades, mutations at codons 148 and 140 occurred in 42 (20.1%) versus 2 4.3%)  and when restricting the comparison to subjects with intermediate- to high-level dolutegravir cross-resistance .Of the 255 raltegravir recipients, 211 (83%) had no predicted resistance to dolutegravir (for twice-daily use). Among those with any predicted dolutegravir resistance, 4 (1.6%) had low-level resistance, 36 (14.1%) had intermediate-level resistance and 4 (1.6%) had high-level resistance  and lack of detectable INI RAMs at virological failure.29 While we did not have raltegravir concentrations, we did not observe a significant difference in viral load levels between subjects with and without detectable INI RAMs, which might have indirectly suggested a difference in adherence levels.In this large European cohort with diverse HIV-1 clades, there was no evidence of major INI RAMs among INI-naive subjects. In contrast, nearly half of patients experiencing viraemia on raltegravir-containing ART showed one or more major INI RAMs. The prevalence of genotypic INI resistance measured in raltegravir recipients with virological failure was consistent with the resistance rates reported from clinical trials (32%–68%).30 Consistent with available data, there was a strong preferential association of N155H with E92Q, Y143H/R/C with T97A, and G140S/A with Q148H/R/K. Of these three mutually antagonistic genotypic patterns, Q148H/R/K and G140S/A were significantly more prevalent in subtype B than in non-B clades, occurring only in three subjects (with subtype C and subtype G). Strikingly, Q148H and G140S occurred in 13% and 16% of subtype B sequences, respectively, but were absent from all non-B clades analysed. This finding could be wholly explained through differences in codon usage between subtype B and non-B clades in INI-naive patients. In the vast majority of subtype B sequences, one nucleotide substitution was required to replace glycine with serine at integrase codon 140. In all the non-B clades examined  two nucleotide substitutions were required, thus raising the genetic barrier to the emergence of G140 mutants. As mutations at codon 140 play a key role in restoring the fitness of Q148 mutants,31 their occurrence can also influence the emergence of Q148H/R/K, thus explaining the reduced prevalence of Q148 mutants observed in non-B subtypes. Small studies previously proposed a higher genetic barrier for G140S and G140C in subtypes C, CRF01 and CRF0232 and more recently a large study of INI-experienced patients identified more Q148 mutations in subtype B versus non-B subtypes.8 Our findings thus confirm and extend these previous observations, indicating the unique propensity of subtype B to the development of the Q148 + G140 mutation pathway. Taken together, these findings show that theoretical predictions based on assessment of the genetic barrier to resistance translate into the actual acquisition of INI mutations in contemporary clinical practice.The three major recognized pathways of genotypic resistance to raltegravir  were represented in the raltegravir-experienced population. Interestingly, co-occurrence of these major pathways was not seen, possibly representing functional incompatibility of all three pathways in the context of a single virus. Indeed, the N155H and Q148H/R/K pathways have been shown to sequentially emerge in separate genomes within a given individual over time.33In vitro studies show that mutations at codons Y143 and N155 confer EC50 fold changes for dolutegravir consistently <3.15 Variable levels of resistance are seen with Q148 mutations depending on the presence of other INI RAMs.15 In the Phase 3 VIKING-3 trial, INI-experienced patients showed different virological responses to dolutegravir twice daily plus an optimized background regimen depending on the baseline mutation pattern.18 Virological suppression <50 copies/mL at 24 weeks was 100/126 (79%) in the absence of baseline Q148 mutations (regardless of the presence of Y143 and N155 mutants), 21/36 (58%) with Q148 mutations plus one minor INI RAM and 5/21 (24%) in the presence of Q148 plus at least two minor INI RAMs.18 Our findings are therefore of practical importance in relation to the likelihood of cross-resistance to dolutegravir in raltegravir-experienced patients. Q148H/R/K mutations were present in 17% of raltegravir-experienced patients and ∼16% of the raltegravir-experienced patients were classified as having intermediate- to high-level resistance to dolutegravir. These data are reassuring as they indicate that most raltegravir-experienced patients, including the large majority of subjects with non-B clades, will retain at least partial susceptibility to twice-daily dolutegravir.18 As clinical experience with dolutegravir in INI-experienced patients remains limited, a role for other mutation profiles in conferring dolutegravir cross-resistance cannot be excluded.Notably, the Q148 mutation pathway confers the highest level of resistance to available INIs, including a significant level of cross-resistance to dolutegravir.34In addition to recognized INI RAMs, non-classic amino acid substitutions at the major integrase resistance codons 92, 147 and 155 were detected in four raltegravir recipients with subtype B. The analysis also identified novel mutations as significantly associated with raltegravir selective pressure—K159Q/R, I161L/M/N/T/V and E170AG. Studies employing site-directed mutagenesis are necessary to investigate the phenotypic properties of these novel mutations and confirm a role in INI resistance. It should be noted that our observations do not exclude a role in resistance for rarer variants that would not be captured by a stringent statistical approach.42 the risk may increase with expanded use of INIs, indicating the need for ongoing surveillance. Importantly, the data also indicate that in the absence of INI selective pressure, major INI RAMs do not occur spontaneously in the diverse non-B clades analysed. Others have reported the presence of major INI RAMs in INI-naive patients infected with subtype CRF02_AG in a small cohort in Cameroon.26 We did not find evidence of major INI RAMs in 22 INI-naive patients infected with CRF02_AG in our study.The lack of major INI RAMs in INI-naive subjects suggests that transmitted INI resistance was rare in 2008–11. Although transmitted INI RAMs are still uncommonly described even among subjects with primary HIV infection,The study illustrates the ongoing importance of collaborative research between different centres in order to collect and meaningfully interpret data from large numbers of patients in routine care, and we hope that this study will prove useful in designing analyses of large depositories such as the Stanford dataset. However, the study has limitations. Policies and methods for resistance testing are likely to have differed across centres. The full treatment history and the duration of viraemia prior to the resistance test were not available and caution is therefore required in the interpretation of resistance rates. In subtype B, it has been shown that N155 mutants tend to be replaced by the more resistant and fitter Q148 + G140 variants with ongoing drug selective pressure. Thus, the resilience of non-B clades with respect to the emergence of these mutations should be considered relative and time-dependent rather than absolute.Merck to the Royal Free Charitable Trust. The funder approved the study design and had no involvement in the conduct of the study, data collection and analysis, and preparation of the manuscript. T. D. is a Wellcome Trust Research Training Fellow.The study was supported by a research award made by None to declare.JAC Online .Table S1 is available as Supplementary data at


While localized malignancies often respond to available therapies, most disseminated cancers are refractory. Novel approaches, therefore, are needed for the treatment of metastatic disease. CUB domain-containing protein1 (CDCP1) plays an important role in metastasis and drug resistance; the mechanism however, is poorly understood.Breast cancer cell lines were engineered to stably express EGFR, CDCP1 or phosphorylation site mutants of CDCP1. These cell lines were used for immunoblot analysis or affinity purification followed by immunoblot analysis to assess protein phosphorylation and/or protein complex formation with CDCP1. Kinase activity was evaluated using phosphorylation site-specific antibodies and immunoblot analysis in in vitro kinase assays. Protein band excision and mass spectrometry was utilized to further identify proteins complexed with CDCP1 or ΔCDCP1, which is a mimetic of the cleaved form of CDCP1. Cell detachment was assessed using cell counting.This paper reports that CDCP1 forms ternary protein complexes with Src and EGFR, facilitating Src activation and Src-dependent EGFR transactivation. Importantly, we have discovered that a class of compounds termed Disulfide bond Disrupting Agents (DDAs) blocks CDCP1/EGFR/Src ternary complex formation and downstream signaling. CDCP1 and EGFR cooperate to induce detachment of breast cancer cells from the substratum and to disrupt adherens junctions. Analysis of CDCP1-containing complexes using proteomics techniques reveals that CDCP1 associates with several proteins involved in cell adhesion, including adherens junction and desmosomal cadherins, and cytoskeletal elements.Together, these results suggest that CDCP1 may facilitate loss of adhesion by promoting activation of EGFR and Src at sites of cell-cell and cell-substratum contact.The online version of this article (doi:10.1186/s13058-016-0741-1) contains supplementary material, which is available to authorized users. The CUB domain-containing protein 1 (CDCP1) , has beeCDCP1 likely contributes to metastasis, in part, by allowing cancer cells to survive and metastasize in the absence of attachment. In the MDA-MB-468 breast cancer cell line, enforced CDCP1 expression induces cell detachment and growth in suspension even in the presence of a suitable adhesive substrate . CDCP1-mThis paper reports that CDCP1 forms a ternary complex with Src and the EGFR, and that this complex mediates Src activation and Src-dependent tyrosine phosphorylation of CDCP1 and EGFR . Furthermore, enforced expression of CDCP1 and EGFR cooperate to induce cell detachment from the substratum, and this effect is enhanced by stimulation of the cells with EGF. Together the results suggest that a novel CDCP1/EGFR/Src ternary complex activates several signaling responses that contribute to metastasis. These mechanisms include Src activation, CDCP1 tyrosine phosphorylation, and EGFR transactivation. Importantly, studies carried out with a new class of anti-cancer agents , which target epidermal growth factor receptor (EGFR) and its family members HER2 and HER3 , show thAnalysis of CDCP1-containing complexes using proteomics techniques revealed that CDCP1 associates with proteins involved in cell-cell and cell-substratum adhesion. These studies identified Galectin-1 and matrix metalloproteinase 14 (MMP-14) among the repertoire of proteins that preferentially associate with the full length or cleaved forms of CDCP1, respectively. The results suggest that the CDCP1/Src/EGFR complex is a novel, druggable target and that DDAs may be useful in abrogating the pro-metastatic functions of this signaling platform. Results presented here, along with previously published studies , 14, revCell lines were purchased from ATCC . EGF (GF001) was obtained from Chemicon International . Dasatinib (S1021), lapatinib (sc-202205), and GM6001 (CC1010) were from Selleckchem , Santa Cruz Biotechnology , and EMD Millipore , respectively. NSC624192, NSC624197, NSC333839, NSC624203, and NSC624205 were gifts from the National Cancer Institute’s Developmental Therapeutics Program. RBF3 was synthesized as described previously .6-Myc tagged CDCP1  and an id 31768 ) were frid 31768 . The Y70Forward: CCGCTGTGGGTATCTTCAATGACAACATC; reverse: GATGTTGTCATTGAAGATACCCACAGCGG. The Y734F CDCP1 mutant was prepared using the primers: forward: AATGACTCCCATGTGTTTGCAGTCATCGAGGAC; reverse: GTCCTCGATGACTGCAAACACATGGGAGTCATT. The Y762F mutant of CDCP1 was produced using the primers: forward: CTGCAGCCAGAGGTGGACACCTTCCGGCCGTTCCAGGGCACCATG; reverse: CATGGTGCCCTGGAACGGCCGGAAGGTGTCCACCTCTGGCTGCAG. These primers were also used to generate the 3YF  triple mutant. The mutant CDCP1 constructs were verified by DNA sequencing.The pLTR PKC™ plasmid encoding mouse PKC™ was obtained from Addgene (Addgene plasmid # 8419) and was used as a template for PCR to add a 5′ HindIII site and Myc tag and a 3′ EcoRV site using the following primers: forward: TTTTAAGCTTATGGAACAAAAACTTATTTCCGAAGAAGACCTTGCACCCTTCCTGCGCATCTC and reverse: TTTTGATATCTTAAATGTCCAGGAATTGC. The resultant PCR product was then cloned into the 5′ HindIII and 3′ EcoRV sites of pcDNA3. The PKC™/pcDNA3 vector was subsequently digested with HindIII and EcoRV and the PKC™ insert was cloned into the 5′ HindIII and 3′ EcoRV sites of pAdTrack-CMV. The PKC™/pAdTrack-CMV vector was recombined with pAdEasy1 in BJ5183-AD-1  cells using an electroporator set at 200 Ohms, 25 mF and 2.5 kV. Recombinant PKC™/pAdEasy1 was then linearized with Pac I and transfected into HEK 293 A cells using lipofectamine , according to manufacturer’s instructions. Adenovirus expressing PKC™ was subsequently amplified in HEK 293A cells.5, 34660], and Millipore Sigma .Affinity purification using anti-FLAG agarose , glutathione-agarose (Millipore Sigma), and TALON resin  was done as described –19. ImmuLabeling of cell surface proteins was performed using Sulfo-NHS-SS-Biotin  essentially as described previously . Briefly32P-ATP was omitted and phosphorylation of proteins in the assays was detected using the appropriate phosphorylation site-specific antibodies by immunoblot analysis.Kinase assays were performed as detailed  with theE-cadherin intracellular localization was assessed by confocal microscopy of cells stably expressing an E-cadherin-GFP fusion protein as described previously .2O. Solvent B consisted of 0.1 % v/v acetic acid, 96.9 % v/v ACN, and 3 % v/v H2O. The flow rate was 300 nL/min and LC-MS/MS analysis was carried out on a hybrid quadrupole-TOF mass spectrometer . The focusing potential and ion spray voltage were set to 275 V and 2400 V, respectively. The information-dependent acquisition (IDA) mode of operation was employed in which a survey scan from m/z 400–1200 was acquired followed by collision-induced dissociation (CID) of the four most intense ions.The protein bands were enzymatically digested with trypsin as previously described . The tryTandem mass spectra were extracted by AB Sciex Analyst QS 2.0 software. All MS/MS samples were analyzed using Mascot . Mascot was configured to search the IPI_bovine database (20120411 version with 30403 entries) for quality control sample of BSA digestion and the IPI_human database (20120411 version with 91464 entries) for experimental samples assuming trypsin digestion. Mascot was searched with a fragment ion mass tolerance of 0.50 Da and a parent ion tolerance of 0.50 Da. Iodoacetamide derivatives of cysteine were specified in Mascot as a fixed modification. S-carbamoylmethylcysteine cyclization of the N-terminus, deamidation of asparagine and glutamine and oxidation of methionine were specified in Mascot as variable modifications.Scaffold  was used to validate MS/MS-based peptide and protein identifications. Peptide identifications were accepted if they could be established at greater than 90.0 % probability as specified by the Peptide Prophet algorithm . ProteinEnforced CDCP1 overexpression in the MDA-MB-468 breast cancer cell line induces cell detachment from the substratum, cell proliferation and survival in suspension . Similar845 [This effect of CDCP1 overexpression is not universal, suggesting that MDA-MB-468 cells are particularly susceptible to CDCP1-mediated suspension growth. MDA-MB-468 cells express high EGFR levels in comparison with most other breast cancer cell lines. Thus, crosstalk may exist between EGFR- and CDCP1-dependent signaling. DDAs downregulate EGFR and its family members HER2 and HER3 and prior to EGFR downregulation, a shift in EGFR electrophoretic mobility is evident that parallels EGFR dephosphorylation on the Src kinase phosphorylation site Tyr845 , 25. ImmEGFR co-immunoprecipitated with adenovirally expressed, FLAG-epitope-tagged full-length and cleaved CDCP1 in MDA-MB-468 cells, and the coprecipitated EGFR exhibited tyrosine phosphorylation Fig. . In cont845, but did not alter the amount of EGFR that co-immunoprecipitated with CDCP1 , no differences in E-cadherin levels were observed between cell lines with or without EGF treatment Fig. . Thus, tThe cleaved form of CDCP1 and a deletion mutant, ΔCDCP1, which mimics the cleaved form of CDCP1, associate with E-cadherin . In ordeCDCP1 may exist as a dimer . Results734. E-cadherin [The ability of EGFR and CDCP1 to cooperate in Src activation resulting in cell-cell and cell-substratum detachment may reflect the ability of these proteins to induce E-cadherin relocalization from the plasma membrane to cytoplasmic vesicles. This hypothesis is consistent with previous reports demonstrating the ability of EGFR- and Src-dependent signaling to disrupt cadherin function –45. CDCPcadherin  and β1-icadherin  bind selEfforts to identify proteins that associate with the full-length, but not cleaved CDCP1 revealed a low-molecular-weight protein with this selectivity Fig. . HoweverThe CDCP1-associated protein Enigma/PDLIM7 identified in our mass spectrometry studies was selected for further study for three reasons. First, the function of Enigma has not been investigated extensively in epithelial cell lines. Second, studies in the heart indicate Enigma plays a key role in regulating cell signaling and the actinomyosin cytoskeleton –54. ThirCancer cell de-adhesion from the primary tumor, either in the form of individual cells or cell clusters, is recognized as a key event in metastatic spread. Clarification of the mechanisms that regulate this process is important for the development of approaches to effectively prevent metastasis by either blocking cell detachment or restoring anoikis. Dissemination of individual epithelial cancer cells necessitates disruption of cell-substratum adhesion, which involves focal contacts and hemi-desmosomes, and dissociation of cell-cell adherens, tight, and desmosomal junctions. CDCP1 associates with proteins involved in cell-cell and cell-substratum adhesion, including N- and P-cadherins and syndecans , E-cadheAn emerging theme from these studies  and thosEGFR transactivation by Src plays an important role in the cooperation between these two oncogenes during cell transformation by providing an EGFR “gain of function.” For example, Src phosphorylates EGFR on tyrosine residues that are distinct from EGFR autophosphorylation sites, and these transactivation sites couple to downstream PI3K activation more effectively than the EGFR autophosphorylation sites . OverallProteomics analyses indicate that CDCP1 multiprotein assemblies include proteins associated either directly or indirectly with the microfilament, intermediate filament, or microtubule cytoskeletal networks. These complexes, furthermore, contain proteins that mediate cell-cell and cell-substratum adhesion. Collectively, these results suggest that CDCP1 participates in the formation of protein complexes that control cell adhesion. CDCP1 complexes contain cell adhesion proteins that couple with cytoskeletal proteins , 16. SucThese results indicate a new function for CDCP1 in facilitating Src-dependent EGFR transactivation. Ternary complex formation among CDCP1, EGFR, and Src is paralleled by disruption of cell-cell and cell-substratum adhesion in human breast carcinoma cells, coincident with relocalization of E-cadherin from cell-cell junctions to cytoplasmic vesicles. The current findings suggest a mechanism whereby CDCP1, EGFR, and Src cooperate to induce cancer aggressiveness and dissemination, and may provide a rationale for therapeutic targeting of these proteins in combination. Alternatively, since DDAs disrupt the CDCP1/EGFR/Src ternary complex, in addition to effectively inhibiting the growth of HER2-positive malignancies , DDAs maCDCP1(Y734F), CDCP1 with a mutation of tyrosine 734 to phenylalanine; CDCP1, CUB domain-containing protein 1; DDAs, Disulfide bond Disrupting Agents; EGFR, epidermal growth factor receptor; GFP, green fluorescent protein; GST, glutathione S-transferase; HER2, human epidermal growth factor receptor 2; IDA, information-dependent acquisition; MMP14, matrix metalloproteinase 14; PAI-1, plasminogen activator inhibtor-1; SFKs, Src-family kinases; ΔCDCP1, mimetic of the cleaved form of CDCP1


Glioblastoma is the most common and lethal primary brain tumor. Tumor initiation and recurrence are likely caused by a sub-population of glioblastoma stem cells, which may derive from mutated neural stem and precursor cells. Since CD133 is a stem cell marker for both normal brain and glioblastoma, and to better understand glioblastoma formation and recurrence, we looked for dys-regulated microRNAs in human CD133+ glioblastoma stem cells as opposed to CD133+ neural stem cells isolated from normal human brain. Using FACS sorting of low-passage cell samples followed by microRNA microarray analysis, we found 43 microRNAs that were dys-regulated in common in three separate CD133+ human glioblastomas compared to CD133+ normal neural stem cells. Among these were several microRNAs not previously associated with cancer. We then verified the microRNAs dys-regulated in glioblastoma using quantitative real time PCR and Taqman analysis of the original samples, as well as human GBM stem cell and established cell lines and many human specimens. We show that two candidate oncogenic microRNAs, miR-363 and miR-582-5p, can positively influence glioblastoma survival, as shown by forced expression of the microRNAs and their inhibitors followed by cell number assay, Caspase 3/7 assay, Annexin V apoptosis/fluorescence activated cell sorting, siRNA rescue of microRNA inhibitor treatment, as well as 3′UTR mutagenesis to show luciferase reporter rescue of the most successful targets. miR-582-5p and miR-363 are shown to directly target Caspase 3, Caspase 9, and Bim. Glioblastoma multiforme (GBM) is a devastating brain tumor with an average survival time from diagnosis of 12–14 months in vivo and in vitro assays to establish that CD133 is a valid GSC marker for the human primary GBM cultures  used in this study GBMs are thought to arise from transformation of neural stem cells (NSCs) or de-differentiation of mature glioma cells with genetic lesions microRNAs (miRNAs) are small noncoding regulatory RNA molecules with profound impacts on many biological processes. MicroRNAs (miRNAs) are coded both inter- and intra-genically. They are excised from longer RNAs and processed in the nucleus as short hairpin pre-miRNA, and are further processed in the cytoplasm into a double-stranded, 22-nucleotide RNA. One strand of the miRNA is then preferentially incorporated into the RISC protein complex, and this ribonucleoprotein complex binds to messenger RNAs and prevents their expression–but this normal processing is dys-regulated in cancer Some miRNAs that have been analyzed in GBM and other cancers have come to be characterized as oncogenic, or able to drive tumor growth through mechanisms such as treatment resistance, escape from the immune system or dys-regulation of angiogenesis. miRNAs that have come to be understood as oncogenic or tumor-driving, with their expression usually increased in GBM, include but are not limited to miR-21, the miR cluster 17–92, miR-26a, and miRs-221 and -222 Tumor suppressor miRNAs are defined as those that decrease growth or invasion and are usually down-regulated in GBM. We and others have shown that miR-7 and miR-34a suppress well-known oncogenic targets, such as EGFR, c-Met, and Notch family members A recent work published miRNA expression levels in GBM stem cells compared to normal NSCs by identifying entire cell lines as pluripotent based on the ability of some of the cells to differentiate into multiple cell types Primary human GBM cultures enriched for GSCs termed GBM-2 , NCH644, and NCH441 were previously published Primary human GBM culture enriched for GSCs termed 0308, 0822, and 1228 were previously published GBM stem cell lines  were obtained from Dr. Jeongwu Lee GBM lines U373 and A172 were obtained from the American Type Culture Collection repository and was maintained as previously described Human specimen RNA from eleven GBMs and three normal human brain samples were obtained from surgical samples and cDNA was generated to evaluate miRNA levels in these specimens.Fluorescence-activated cell sorting (FACS) analysis was carried out on a LSR II . Cell sorting was carried out using a FACS-Aria-II (BD Biosciences). Antibodies human CD133 and human isotype control were all from Miltenyi Biotech . Data were analyzed using CellQuestand FACSDiva (BD Biosciences) and FlowJo software .Files S3, S4, and S5. Previously unstudied miRNAs that met these criteria were selected for experimental follow-up.All microRNA arrays were performed twice and initial analysis was performed by Beckmann Technologies, using the human microRNA version 2 (GPL7731) platform. Full analysis was performed for four samples: B4 human glioma CD133–, B4 human glioma CD133+, BLV4 human NPC CD133–, and BLV4 human NPC CD133+. Secondary analysis was performed for CD133+ NCH644 and NCH441 samples. Raw data and metadata were deposited in the NCBI GEO database, Accession numbers are: GSM1199638–GSM1199643 respectively. The microarray heatmap shown was created using Java Treeview. Raw intensity values were transformed by Beckman Coulter into log base 2 fold change intensity ratios for each CD133+ GSC sample compared to the NPC CD133+ sample, and each partial data analysis was provided as a separate Excel spreadsheet. miRNAs that demonstrated an initial p-value of less than.05 were then further selected if two GBM CD133+ samples demonstrated at least 0.7 log fold change in intensity ratios in the same direction (increased or decreased), as in other similar analyses where the 95% confidence interval falls around this line Select miRNAs were validated by quantitative PCR in a variety of tissue and cell types. Template RNA from the CD133+ NSCs and CD133+ GBM stem cell samples was prepared. Human specimen sample RNA was prepared using miRNeasy and miScript RT kits , according to the protocol directions. cDNA was diluted to 10 ng/microliter and 50 to 100 ng of cDNA was used per reaction. Primer assays  were used for each miRNA tested. 2 X Sybr Green reagent from ABI was used for qPCR. The control primer set was RN_U6B, recommended by Qiagen for miRNA analysis.Validated microRNAs were purchased as pre-miRNAs from Ambion , and used with the recommended Ambion pre-miRNA control. Anti-miRs and a scrambled Anti-miR control for validated oncogenic miRNAs were also purchased from Ambion. miRNA transfections were carried out using Oligofectamine  reagent as previously described Figure S6 in File S2. Western blots were repeated 2–7 times with multiple transfected wells per experiment.Protein expression of miRNA targets was evaluated by immunoblot as previously described Cell number was evaluated to determine the effect of forced miRNA or miRNA inhibitor transfection. Cells were counted three to five days after miRNA transfection. Ten microliters were analyzed using a hemocytometer.Caspase activity was detected as a means to evaluate apoptosis in cells expressing miRNAs or miRNA inhibitors. Cells were assayed for Caspase 3/7 activity three to four days after miRNA transfection using Caspase 3/7 Glo substrate solution as recommended . Luminescence was measured using a Glomax luminometer . Luminescence readings were corrected by cell number and also compared to protein concentration via Bradford assay . Values shown are RLU/cell number per mL, normalized to a percent maximal activity.Total apoptotic cell percentage was evaluated using a dual marker flow cytometry kit. Cells were stained four to five days after miRNA transfection using the PE-Annexin-V Apoptosis staining kit I , as recommended. Positive cells were sorted using a FACSCalibur and analyzed with FlowJo software.A broad caspase inhibitor was used to rescue the effect on cell numbers of miRNA inhibitor driven up-regulation of Caspase proteins. Cells were transfected with miRNA and then treated with 40 µM Z-VAD-fmk  or vehicle for three days prior to cell count.Luciferase reporting of miRNA target 3′-UTRs was used to determine specificity of the miRNA-target mRNA interaction. Caspase 3-3′-UTR-RenillaLuc was designed and initially tested by us, and other 3′-UTR-luc plasmids for BCL2L11  and Caspase 9  were used to define specific targets of the miRNAs. Cells were plated as described above and transfected with miRNA and then the appropriate plasmid. Plasmid transfection was carried out using Fugene HD  and was paired with a transfection control plasmid encoding beta-galactosidase . Forty-eight hours post-plasmid transfection, luciferase activity was assayed using Promega Renilla Luciferase Assay System or Promega Firefly Luciferase Assay system . Luciferase activity was corrected using beta-galactosidase activity and normalization with control-miRNA and empty vector controls.CGGCGGCAAATGATGATGTGGTGTCAA 3′. Reverse: 5′ GCCGCCGTTGACAAAACTAGAATACACCCACATCATCATTT. AGTCTTAAGTGGGGGG 3′, with wild-type sequence: 5′ CCCCCCACTT AAGACTGTGT ATTCTAGTTT TGTCAAACTG TAGAAATGATGATGTGG 3′.Primers encoding the entire seed match region of the 3′-UTR for BCL211, CASP3, and CASP9, were designed with 5–8 base pair substitutions introduced in the strongest putative binding region for the miRNA, and an Agilent Site Directed Mutagenesis kit  was used to introduce these mutations into the wild-type 3′-UTR of the targets. Two miR-363 binding sites, one for BCL211 and one for CASP3, were mutagenized, and the binding region for CASP3 for miR-582 was also mutagenized. The primer sets were as follows: For the 5′ CASP 3 site for miR-582: Forward: 5′CCCCCCACTTAAGACTGTGTATTCTAGTTT. CGCGCCGGGTGTTTCCTTTACCTTGTAAAATTTTGCTTATCAACTGAGCCAAATGTCTGT 3′, Reverse: 5′ CCGGCGCGACAGA CATTTGGCTCAGTTGATAAGCAAAATTTTACAAGGTAAAGGAAACAC 3′; with wild-type sequence: 5′ GTGCAATTGTGTTTCCTTTACCTTGTAAAATTTTGCTTATCAA CTGAGCCAAATGTCTGT 3′. For the miR-363 binding site in CASP 3′UTR, Forward: 5′ CGCGCCGCATTATGAGAGGCAATGTTGTTAAAAATTAGGAATAAATAAAAAT GGATACTG 3′, Reverse: 5′ CGGCGCGCAGTATCCATTTTTATTTATTCCTAATTTTTAACAA CATTGCCTCTCAT AATG 3′ with wild type sequence: 5′ GTGCAGTCA TTATGAGAGGCAATGATTGTTAAAAATTAGGAATAAATAAAAATGGATACTG 3′.For the miR-363 binding site in BCL2L11 (Bim) 3′UTR, Forward: 5′ Methods S1.Further methods are available in Using microarray analysis, we measured miRNAs in FACS-sorted CD133+  and – human GSC samples (B4) and CD133+ and – normal human neural stem cells (BLV4). We then further evaluated those miRNAs that were strongly increased or decreased only in CD133+ GSC relative to CD133+ NSCs Figure 1Eleven miRNAs were decreased in CD133+ GSCs, with two miRNAs strongly decreased in all three samples. miR-768-5p, miR-768-3p, and miR-598 are novel decreased miRNAs . miR-483-3p and miR-196a have been previously identified as being oncogenic in other cancer settings, but they were decreased in at least 2 of our GSC samples All other dys-regulated miRNAs in this study have been identified as dys-regulated in at least one other tumor type, but few except for the very well-known miR clusters have been identified previously in GBM. miR-34a has been extensively studied by our group and others as a tumor suppressor miRNA in GBM and other brain tumors Figure S1 in File S1). miR-582-5p and -363 were chosen for further analysis via quantitative PCR (qPCR) due to their novelty as potentially oncogenic miRNAs, and the strength of their relative fold changes  as well as immortalized astrocytes, by traditional qPCR and/or Taqman. The established oncomiR miR-221 was also measured in all of the cell lines to reconfirm the pattern of higher expression of oncomiRs in the GBM stem cell lines . miR-582-5p and miR-363 were strongly upregulated in all available GSC samples, most human GBM specimens, and the GBM stem cell lines.Levels of miR-582-5p and miR-363 were next assessed in three previously published GBM stem cell lines, which have been maintained in neurobasal medium with supplements and without serum  . These results support an anti-apoptotic mechanism of action of miR-363 and miR-582-5p.Evaluation of the biological effects of miR-582-5p and miR-363 through forced expression of their specific inhibitors (stabilized short antisense inhibitor molecules) in GBM neurosphere stem cell lines and serum-maintained GBM lines confirmed their oncogenic potential. anti-miR-363 caused growth-inhibition of the GBM stem cell line (0822) and the established cell line U373 . In keeping with this, the 0822 GBM stem cell line treated with anti-miR-582-5p expressed higher Caspase 3 and Caspase 9 as compared to anti-miR scrambled control (3B in  and S6 in File S2). GBM cell lines treated with miR-363 expressed less Bim and Caspase 3 protein, whereas GBM lines treated with anti-miR-363 expressed more Bim protein . The increases in target protein expression with anti-miR delivery indicate that these miRs are potent physiological regulators of Caspase 3 and Bim, respectively. A qPCR analysis of putative miR-582-5p targets confirms Caspase 3 and Caspase 9 mRNA expression sensitivity to this miR, and indicates that Caspase 10 may also be a target . miRNA target mRNAs and protein levels are not always consistently regulated, however, and we were unable to show consistent response of Caspase 10 at the protein level , and for CASP3 and CASP9 for miR-582-5p  and Caspase 3 by TargetScan seed match analysis, and miR-582-5p was predicted to target Caspase 3 and Caspase 9  regulates apoptosis by binding to anti-apoptotic members of the Bcl-2 family, including Bcl-2, Bcl-xl and Mcl-1, or by activating pro-apoptotic members Bax and Bak Figure 6This report also demonstrates that up-regulation of apoptotic pathway target proteins is sufficient to increase apoptotic activity in the case of both these novel miRNAs. While specific up-regulation of full-length Caspase-3 has been shown to make T-cells susceptible to apoptosis The strong up-regulation of both miR-363 and miR-582-5p across many GSC samples and their co-targeting of intrinsic apoptotic pathway components suggest that GSCs have a susceptibility to mitochondrial membrane destabilization and apoptosis. GSCs may attempt to evade apoptosis by increasing expression of these miRNAs. Several current anti-tumor therapies induce apoptosis in cancer cells using antibody delivery of toxic molecules, antibody binding to death receptors such as TRAIL followed by apoptotic induction, and inhibition of histone deacetylases (HDACs) leading to apoptotic induction File S1Contains the files: Figure S1: Taqman qPCR of several possible miRNAs increased or decreased in CD133+ GBM primary cultures compared to CD133+ NSCs. miRNA-specific reverse transcription, followed by Taqman primer and probe amplification of miRNA targets is shown as Taqman Fold Change for miRNAs with little or no prior association to GBM. Figure S2: Further quantification of miR-582-5p, miR-363, and known oncogenic miR-221 in GBM cell lines. miR-582-5p fold change in GBM established cell lines T98G, U87, and U251, relative to level of the miR in normal astrocytes (A). miR-363 fold change in GBM established cell lines T98G, U87, and U251, relative to level of the miR in normal astrocytes (B). Level of miR-221 in many GBM stem and established cell lines, normalized to the cell line with the lowest level, A172 (C). Figure S3: Densitometry of immunoblots. Bim, Caspase 3, and Parp protein in several cell lines was quantified using an Adobe Photoshop pixel intensity histogram and normalized to corresponding alpha-tubulin protein bands from the same lanes in immunoblots of control scrambled miR, miR-363, control scrambled anti-miR, and anti-miR-363 treated cell lysates (A). Caspase 3 and Caspase 9 protein in 2–7 lysate samples was quantified using Image J gel analysis tool by calculating the area under the curve for each band, normalized to alpha tubulin from the same lane, and averaged for all quantified blots (B).(EPS)Click here for additional data file.File S2Contains the files: Figure S4: Reduced apoptosis with miR-582-5p forced expression in established GBM cell lines. Caspase 3/7 luciferase activity (A) and Annexin V staining (shown as % apoptosis (B) in immortalized astrocytes and U373, an established GBM tumor cell line, respectively, in the presence of miR-582-5p or control miRNA. Student’s t-test was performed to generate p-values, with ****, p<.0001. Figure S5: Further miR-363 and miR-582-5p data showing targets related to apoptosis. mRNA levels of predicted targets were measured after forced expression of control scrambled anti-miRNA (black bars) or anti-miR-582-5p (grey bars) to determine whether the miR had a possible direct effect on mRNA levels of targets (A and B). Additional data showing normalized empty vector vs. Caspase 3 3′UTR reporter activity response to miR-363 (C) in U87, an established GBM cell line. *, p<.05. Figure S6: Western blots that were cut to show representative lanes are shown in full. In the case of U373 and 0822 cells, single lanes cut from the larger blots of Caspase 3 and alpha-tubulin are shown in (EPS)Click here for additional data file.File S3Supplemental Microarray File 1: A Microsoft Excel document showing tables of p-values and log-fold intensity ratios for CD133+ GSCs as compared to CD133+ NSCs for selected miRNAs for the heatmap in (XLSX)Click here for additional data file.File S4Supplemental Microarray File 2: Raw intensity values for all miRNAs and samples tested in a Microsoft Excel document, also all raw data files are available online at GEO under accession numbers GSM1199638–GSM1199643.(XLS)Click here for additional data file.File S5Supplemental Microarray File 3: A Microsoft Excel document showing selection criteria for miRNAs in the microarrays.(XLSX)Click here for additional data file.Methods S1A Microsoft Word document detailing some protocols and reagents.(DOC)Click here for additional data file.


This crystal structure showed a unique curling conformation at the flap regions that was not found in the previously reported ligand-free protease structures. Molecular dynamics simulations indicated that the curled flap conformation altered the flap dynamics. These results suggest that the preference for a unique flap conformation influences DRV binding. These results provide new structural insights into elucidating the molecular mechanism of DRV resistance and aid to develop PIs effective against DRV-resistant viruses.Darunavir (DRV) is one of the most powerful protease inhibitors (PIs) for treating human immunodeficiency virus type-1 (HIV-1) infection and presents a high genetic barrier to the generation of resistant viruses. However, DRV-resistant HIV-1 infrequently emerges from viruses exhibiting resistance to other protease inhibitors. To address this resistance, researchers have gathered genetic information on DRV resistance. In contrast, few structural insights into the mechanism underlying DRV resistance are available. To elucidate this mechanism, we determined the crystal structure of the ligand-free state of a protease with high-level DRV resistance and six DRV resistance-associated mutations (including I47V and I50V), which we generated by  At present, more than 20 antiviral drugs are clinically available to treat patients infected with human immunodeficiency virus type 1 (HIV-1). PIs, such as darunavir , suppress HIV-1 replication by inhibiting the functions of the viral protease (PR). DRV is a second-generation HIV-1 PI and is one of the most potent anti-HIV-1 drugs, owing to its high antiviral activity and high genetic barrier to the generation of resistant viruses  structure information of PR variants with high-level resistance to DRV. Nonetheless, few reports have published 3D structures of PR variants with high-level resistance to DRV  values were obtained from three independent experiments.The susceptibilities to the PIs were evaluated using an in-house drug susceptibility assay with the R5-MaRBLE cell line as described elsewhere  of the purified RNA was reverse transcribed using a PrimeScript II High Fidelity One Step RT-PCR Kit . Subsequently, an inner gag-PR region (681–3348) was amplified by nested PCR using PrimeSTAR GXL DNA Polymerase (Takara Bio Inc.). The primer sets used for the amplification were as follows: reverse transcription PCR, 5′-ATCTCTAGCAGTGGC GCCCGAACAG and 5′-TAC TTCTGTTAGTGCTTTGGTTCC and nested PCR, 5′-CTCTCTCGACGC AGGACTCG and 5′-TAA TCCCTGCATAAATCTGACTTGC.Viral RNA was extracted from the cultured system as follows. First, virus particles in the cell culture supernatant were collected by centrifugation at 20,000 × g at 4°C for 1.5 h. The collected particles were suspended in 300 μL of RNAgents Denaturing Solution . Then, the RNA was purified by phenol-chloroform extraction. The gag-PR region (699–2580) was inserted into the pNL4-3 clone vector using a GeneArt Seamless Cloning and Assembly Kit . First, we amplified the target gag-PR region with PrimeSTAR GXL Polymerase. The primer set used for amplification was as follows: 5′-CGGCTT GCTGAAGCGCGCACAGCAAGAGGCGAGGGGCGGCGACTG and 5′-TTA CTGGTACAGTCTCAATAGGACTAATGGG. The amplified PCR product was ligated with pNL4-3 without gag-PR.A DNA fragment of PR region (2253–2549) was amplified by nested PCR using KOD DNA Polymerase . The primer set used for amplification was as follows: 5′-ATATACATATGCCTCAGAT CACTCTTTGG and 5′-TG GTGCTCGAGTTACTAAAAATTTAAAGTGCAGCC. Subsequently, the PCR product was inserted into pET-41a(+)  using NdeI and XhoI restriction enzymes and a DNA Ligation Kit ver. 2.1 (Takara Bio Inc.). Mutagenesis was performed to obtain an inactive D25N PR mutant.The full-length The enzymatically-inactive PR was expressed, purified, and then refolded by using a method similar to that in the previous report by Dr. Schiffer's group . Then, initial crystallization conditions of PR were screened using following crystallization screening kits: Wizard I , Wizard II , and JCSG-plus Screen MD-137 . The final optimized crystallization condition after iterative optimization cycles was at 0.84 M Lithium chloride, 0.84 mM Sodium citrate, 17%(w/v) PEG 6000, 10% Glycerol, and 5% Ethylene glycol. The crystal was obtained by the hanging-drop vapor-diffusion method. The drops were incubated at 20°C.The purified PR was concentrated to a final concentration of 2.6 mg mL1KJ7) . The data were integrated with the HKL2000 program  . FS5929 contained one minor DRV resistance mutation L33F) (Table 9. FS59293F (Tablere Table .gag-PR of FS5929R1 (rFS5929R1) and FS5929 (rFS5929) based on the HIV-1 NL4-3 backbone. The susceptibility assays showed that rFS5929R1 displayed a >175-fold increase in DRV resistance compared with that of WT NL4-3, whereas FS5929 was susceptible to DRV (1.9-fold increase) (Table I47) and rFS5929R1 without I50V (rFS5929R1I50) exhibited a >175-fold and 96.9-fold increase in DRV resistance, respectively, whereas the rFS5929R1 without the two mutations (rFS5929R1I47∕I50) exhibited a 20.4-fold increase. The results suggest that the two major mutations, especially I50V, largely contribute to the DRV resistance in rFS5929R1.Next, we focused on FS5929R1 and analyzed its resistance to DRV. We generated recombinant viruses carrying the in vitro selection with DRV scarcely affected the susceptibilities to TPV, ATV, and SQV.We also measured the resistance to seven other PIs: amprenavir (APV), lopinavir (LPV), ritonavir (RTV), nelfinavir (NFV), tipranavir (TPV), atazanavir (ATV), and saquinavir (SQV). The rFS5929 and rFS5929R1 viruses exhibited 16.0- and >35.9-fold resistance, respectively, to the DRV-analog APV. Both viruses exhibited high-level resistance to LPV (>54.9 fold), RTV (>28.8 fold), and NFV (>83.1 fold). Interestingly, both viruses were susceptible to TPV (< 0.5 fold) and exhibited 3- to 15-fold resistance to ATV and SQV, suggesting that the mutations induced by the To investigate the mechanism underlying the DRV resistance of FS5929R1, we determined the crystal structure of the ligand-free state of the inactive FS5929R1 PR homodimer at a 1.8 Å resolution Figure , as seenin vitro selection . Interestingly, the regions included the two major DRV resistance mutations (I47V and I50V) and one minor mutation (V32I), which were induced by the n Figure . Hence, To assess the stability of the unique curling conformation at the mobile flap regions of the FS5929R1 PR Figure , we perfbis-tetrahydrofuran (bis-THF) moiety and the central hydroxyl group  harbored 6 DRV resistance-associated mutations in its PR region. The drug susceptibility tests with the recombinant virus variants suggested that the PV Table . The twoPV Table .Notably, the crystal structure of the ligand-free FS5929R1 PR showed an open-form configuration that was similar to those of the two previously reported ligand-free structures of PRs with high-level resistance to DRV (PDB IDs: 4NPU and 3UF3). However, the FS5929R1 PR exhibited a unique curling conformation at the flaps Figure . The higIt is also plausible that the preference for the unique conformation in FS5929R1 PR would reduce the PR dimerization inhibition activity of DRV, which is the second inhibitory mechanism of DRV  with the accession code Conceived and designed the experiments: WS and YI. Performed the experiments: MN, HO, KS, MF, MMaejima, YK, TM, JH, MMatsuda, AH, and YI. Analyzed the data: MN, HO, KS, MF, MMaejima, and YI. Contributed reagents/materials/analysis tools: YY, AS, NW, WS, and YI. Wrote the paper: MN, HO, and YI.This study was supported by the Japan Society for the Promotion of Science (JSPS) fellows (MN) under grant number 15J12567, by the JSPS (YI) under grant number 15H04740, and by the Japan Agency for Medical Research and Development (AMED) (YI and NW).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer YTY declared a shared affiliation, though no other collaboration, with one of the authors MF to the handling Editor, who ensured that the process nevertheless met the standards of a fair and objective review.


The first committed step of chlorophyll a biosynthesis is the insertion of a Mg2+ ion into the tetrapyrrole intermediate protoporphyrin IX, catalyzed by Mg-chelatase. We have identified a Synechocystis sp. PCC 6803 strain with a spontaneous mutation in chlH that results in a Gly195 to Glu substitution in a conserved region of the catalytic subunit of Mg-chelatase. Mutant strains containing the ChlH Gly195 to Glu mutation were generated using a two-step protocol that introduced the chlH gene into a putative neutral site in the chromosome prior to deletion of the native gene. The Gly195 to Glu mutation resulted in strains with decreased chlorophyll a. Deletion of the PS II assembly factor Ycf48 in a strain carrying the ChlH Gly195 to Glu mutation did not grow photoautotrophically. In addition, the ChlH-G195E:ΔYcf48 strain showed impaired PS II activity and decreased assembly of PS II centers in comparison to a ΔYcf48 strain. We suggest decreased chlorophyll in the ChlH-G195E mutant provides a background to screen for the role of assembly factors that are not essential under optimal growth conditions.Biogenesis of the photosystems in oxygenic phototrophs requires co-translational insertion of chlorophyll  The reaction center (RC) and core antenna proteins of both PS II and PS I require the co-translational insertion of chlorophyll  folding . In addihronized . There aofactors . Assembl antenna . The pre antenna . In addi antenna . Part ofsubunits .a biosynthesis pathway is comprised of at least 15 enzymatic steps, many of which are shared with the biosynthetic pathway for heme (a is the insertion of Mg2+ into protoporphyrin IX (PP IX) to form Mg-PP IX; this reaction is catalyzed by Mg-chelatase  and the Ala to Val substitution in the gun5 mutant corresponds to the Ala942 residue. Both Pro595 and Ala942 are located in the central cage-like structure of the protein and may interfere with the chelation reaction due to spatial hindrance , 0.3% sodium thiosulfate and appropriate antibiotics. Liquid cultures were grown photoautotrophically or photomixotrophically (with 5 mM glucose) in BG-11 liquid medium with appropriate antibiotics, as previously described . The slr0168 gene site has been used as a neutral site for integration of genes into the chromosome previously with no reported effect on phenotype (Δslr0168:specR plasmid was transformed into wild-type Synechocystis 6803 (GT-O1) to create the Δslr0168 neutral site control strain . The slr1055 (chlH) gene was amplified from the GT-O1 and GT-O2 wild types (Supplementary Table S1). These amplicons and the Δslr0168:specR plasmid, were digested with HindIII and ligated to produce the ChlH-G195G:specR and ChlH-G195E:specR plasmids, respectively. The resultant plasmids were transformed into Synechocystis 6803 strains (GT-O1 or GT-O2) to introduce either the unmodified chlH (in the GT-O1:G195G and GT-O2:E195G strains) or the mutant chlH gene (in the GT-O1:G195E and GT-O2:E195E strains) into the putative neutral site . In the second step, a ΔchlH plasmid was constructed using overlap-extension PCR in which a chloramphenicol-resistance cassette was located between the sequences up- and downstream of the native chlH gene . This plasmid was transformed into the strains which contained a copy of the chlH gene in the neutral site to produce strains which each contained only one copy of chlH, either the GT-O1 (Gly195) or GT-O2 (Gly195 to Glu) variant. A summary of the mutants created in this study is presented in Table Generation of sion PCR , in whichenotype . A HindIld types , using pycf48 (Δslr2034) plasmid has been described previously  of 1.5 in BG-11 supplemented with 25 mM HEPES-NaOH (pH 7.5) for physiological measurements. Chlorophyll a was quantified by extraction in methanol and measurement at 663 nm  as previously described . Proteina, respectively . The GT-O2 strain exhibited a reduction in the peaks at 435 nm and 685 nm corresponding to chlorophyll a absorbance but not the 625 nm phycobilin peak . Six unique genome sequence variants are present in the GT-O2 strain compared to GT-O1 cells . This Gly195 residue was conserved in ChlH proteins representing all five sections of cyanobacteria, and is part of a conserved protein region . Although chiefly monomeric in solution, the crystal structure of ChlH obtained by Supplementary Figure S2). Dimerization of ChlH subunits is mediated by polar interactions, including six hydrogen bonds and a salt bridge between the Glu199 and Lys1018 residues of opposing monomers. The Gly195 residue is in close proximity to Glu199 ; the Glu199 residue is conserved in many cyanobacteria, but is substituted for an Asp in the cyanobacterium Gloeobacter violaceus PCC 7421. The Lys1018 residue was found in the aligned cyanobacterial ChlH protein sequences except that of G. violaceus PCC 7421 .The ChlH Gly195 residue is located in the α6 helix of the N-terminal ‘head’ domain I  or mutant chlH gene (encoding the Gly195 to Glu mutation) was introduced at a putative neutral site (slr0168) in the Synechocystis 6803 genome . Once the introduced chlH gene had been fully segregated, the native gene was deleted and a chloramphenicol-resistance cassette was inserted in place of this copy of chlH . This method was used to generate strains containing only the GT-O1 copy of chlH, encoding Gly195, in both the GT-O1 and GT-O2 backgrounds. Similarly, strains were generated containing only the GT-O2 copy of chlH, encoding the Gly195 to Glu substitution, in both the GT-O1 or GT-O2 backgrounds. Mutants in the GT-O2 background enabled us to determine the impact of restoring the Gly195 to this background, as well as, including an additional control strain where the Glu195 was introduced into ChlH in the neutral site. The sequences of the oligonucleotides used for the construction and verification of these strains are in Table S1. In addition, a Δslr0168 control strain was produced which contained only the spectinomycin-resistance cassette in the neutral site and which retained the native chlH gene at its wild-type locus.To determine the effect of the ChlH Gly195 to Glu mutation 3 genome . A spectslr0168 control strain were similar (data not shown). Introduction of unmodified chlH, under its own promoter, to produce the GT-O1:G195G strain using the slr0168 site resulted in a whole-cell spectrum similar to that observed with GT-O1 cells, whereas introduction of the ChlH Gly195 to Glu mutation to produce the GT-O1:G195E strain resulted in decreased chlorophyll a absorption at both the 435 and 685 nm maxima compared to the GT-O1 strain . Conversely, the introduction of unmodified chlH into the GT-O2 background resulted in the GT-O2:E195G strain which had increased chlorophyll a maxima that were similar to the GT-O1 wild type . In addition, the GT-O2:E195G strain showed an increased absorption in the 475–520 nm range compared to the GT-O2:E195E strain suggesting elevated carotenoid levels. Furthermore, the spectrum of the GT-O2:E195E strain was similar to spectra from the GT-O2 and GT-O1:G195E strains, suggesting that the presence of the Gly195 to Glu variant in ChlH results in a decrease in chlorophyll a absorbance . The quantification of methanol-extracted chlorophyll a showed that the Gly195 to Glu mutation in ChlH resulted in a ∼35–40% reduction of chlorophyll a accumulation in the GT-O2 and the GT-O1:G195E and GT-O2:E195E strains, in comparison to the GT-O1 and the GT-O1:G195G and GT-O2:E195G strains in samples normalized to an OD730 nm of 1.0 . The Gly195 to Glu mutation had little effect on the rate of photoautotrophic growth of the GT-O1:G195E strain, although these cells reached a slightly lower final OD730 nm in stationary phase ; this is consistent with that observed in the GT-O2 wild type (slr0168 control strain was indistinguishable from the GT-O1 wild type and the GT-O1:G195G strain (data not shown). Using flow cytometry and electron microscopy we did not detect any difference in size between strains containing Gly195 or Glu195 (data not shown), therefore the OD730 nm should represent a similar cell number in all strains compared.The whole-cell spectra of the GT-O1 wild type and Δild type . The phoa. The Ycf48 assembly factor is associated with the RC pre-complex; however, the absence of this factor does not completely block PS II assembly ; this is consistent with other ΔYcf48 strains characterized . The GT-O1:G195G:ΔYcf48 mutant had a whole-cell absorption spectrum with decreased absorption maxima at 625 and 685 nm compared to the GTO1:G195G strain. The GT-O1:G195E:ΔYcf48 strain’s whole-cell spectrum was the most altered, exhibiting a decreased 685 nm maxima and an increased carotenoid shoulder compared to the GT-O1:G195G:ΔYcf48 strain . Levels of chlorophyll a measured for each strain at the same OD730 nm showed a 16% decrease in the GT-O1:G195E strain relative to the GT-O1:G195G strain . Deletion of Ycf48 resulted in a small decrease in chlorophyll a levels in the GT-O1:G195G:ΔYcf48 strain, whereas in a GT-O1:G195E:ΔYcf48 strain there was a ∼26% decrease in chlorophyll a compared to the GT-O1:G195G strain.Whole-cell absorption spectra of photomixotrophically grown cells showed a slight decrease in the 685 nm absorption of chlorophyll Figure 4D). In the GT-O1:G195G:ΔYcf48 strain, oxygen evolution was decreased, to 89 ± 12 and 71 ± 5% of the GT-O1:G195G strain in the presence of bicarbonate and DMBQ, respectively. Removal of Ycf48 in a ChlH-G195E background resulted in rates of oxygen evolution that were reduced, to 38 ± 1 and 17 ± 3% of the rates for the GT-O1:G195G strain in the presence of bicarbonate and DMBQ, respectively.Oxygen evolution was measured in mixotrophically grown cultures. The rates of oxygen evolution were similar in the GT-O1:G195E and GT-O1:G195G strains, when measured in the presence of 15 mM bicarbonate (measuring whole-chain electron transport) or 200 μM of the artificial quinone 2,5-dimethyl-1,4-benzoquinone (DMBQ) (measuring PS II-specific electron transport) . It was previously reported that removal of Ycf48 resulted in a considerable decrease in the variable fluorescence level and this was observed in the GT-O1:G195G:ΔYcf48 strain . In the presence of 40 μM 3--1,1-dimethylurea (DCMU), which blocks electron transfer beyond QA of PS II, variable fluorescence was similar in the GT-O1:G195G and GT-O1:G195E strains, reduced in the GT-O1:G195G:Ycf48 strain and further decreased in the GT-O1:G195E:ΔYcf48 strain . Variable fluorescence in the presence of DCMU provides an indication of the numbers of assembled PS II centers and these data are consistent with decreased centers in both strains lacking Ycf48, with the lowest number of centers in the GT-O1:G195E:ΔYcf48 strain.The activity of the acceptor side of PS II was analyzed by measuring variable chlorophyll 8 strain . The varFigure 5A). The 685 nm emission peak is attributed to unassembled pre-complexes and the CP43 core antenna protein, while the emission at 695 nm arises from the CP47 core antenna and is indicative of fully assembled PS II RCs . A similar decrease in the emission at 725 nm was observed in the GT-O1:G195G:ΔYcf48 strain and this strain also displayed a decreased emission at 685 nm with a further decrease at 695 nm. This is consistent with impaired assembly and/or repair of PS II in the absence of Ycf48; however, the decrease in fluorescence emission from PS I suggests an additional effect on PS I assembly due to the removal of this assembly factor. Deletion of Ycf48 in a ChlH-G195E background resulted in a complete absence of the 695 nm emission in the GT-O1:G195E:ΔYcf48 strain, suggesting a decreased number of PS II centers; this strain also showed a decreased PS I emission maxima at 725 nm, compared to the GT-O1:G195G:ΔYcf48 strain.Reduced PS II-specific oxygen evolution and variable fluorescence in the GT-O1:G195G:ΔYcf48 and the GT-O1:G195E:ΔYcf48 strains are consistent with decreased numbers of assembled PS II centers in these strains. Low-temperature (77 K) fluorescence emission spectroscopy was performed on photomixotrophically grown cultures using 250 nM sodium fluorescein as an internal standard. Introduction of the ChlH Gly195 to Glu mutation had little effect on the 685 and 695 nm fluorescence emission in the GT-O1:G195E strain with a 440 nm excitation light . In the GT-O1:G195G:ΔYcf48 strain, an increase in the fluorescence emission at 685 nm was observed; this emission originates from the ApcE terminal emitter of the phycobilisome antenna and indicates an increased level of uncoupled phycobilisomes in this strain . In the GT-O1:G195G:ΔYcf48 strain the D1 and D2 proteins detected in PS II dimers and monomers were reduced, and the unassembled D1 slightly increased. However, only low levels of PS II monomers and only trace amounts of dimers were detected in the GT-O1:G195E:ΔYcf48 strain, consistent with the very low levels of PS II activity, while a low level of the RC47 complex was present. In addition, an antibody to the PsaA subunit of PS I revealed that the ratio of PS I monomers to trimers in the GT-O1:G195G:ΔYcf48 appeared to increase and that trimers were greatly reduced or destabilized in the GT-O1:G195E:ΔYcf48 mutant .To further analyze the PS II complexes in the ChlH mutant strains, thylakoid membranes were analyzed using BN-PAGE and western blotting. In the GT-O1:G195G and GT-O1:G195E strains, the D1 and D2 core subunits were detected at similar levels in dimeric and monomeric PS II complexes, and in a CP43-less assembly/repair intermediate, RC47  and the ChlH-Ala942 to Val change  have both been studied in the Synechocystis ChlH subunit in vitro . We also observed that PS I assembly was destabilized in our GT-O1:G195E:ΔYcf48 strain, perhaps as an indirect consequence of perturbing nascent PS II assembly and repair complexes. A similar effect on PS I assembly in an A. thaliana mutant lacking HCF136 – the homolog of Ycf48 – has also been reported (During PS II biogenesis and repair Ycf48 is transiently associated with pD1-containing pre-complexes prior to D1 processing and subsequent formation of the RC47 assembly intermediate , 2012. Ireported .Given the close relationship between chlorophyll biosynthesis and the assembly and repair of PS II  and PS ITC performed the experiments and wrote the first draft of the manuscript. TS and JE-R designed the experiments, wrote, and edited the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Background. Chemical spills are on the rise and inhalation of toxic chemicals may induce chemical acute lung injury (ALI)/acute respiratory distress syndrome (ARDS). Although the pathophysiology of ALI/ARDS is well understood, the absence of specific antidotes has limited the effectiveness of therapeutic interventions.Objectives. Surfactant inactivation and formation of free radicals are important pathways in  ALI. We tested the potential of lipid mixtures with advanced surfactant protein B and C (SP-B and C) mimics to improve oxygenation and lung compliance in rabbits with lavage- and chemical-induced ALI/ARDS.Methods. Ventilated young adult rabbits underwent repeated saline lung lavages or underwent intratracheal instillation of hydrochloric acid to induce ALI/ARDS. After establishment of respiratory failure rabbits were treated with a single intratracheal dose of 100 mg/kg of synthetic surfactant composed of 3% Super Mini-B (S-MB), a SP-B mimic, and/or SP-C33 UCLA, a SP-C mimic, in a lipid mixture (DPPC:POPC:POPG 5:3:2 by weight), the clinical surfactant Infasurf®, a bovine lung lavage extract with SP-B and C, or synthetic lipids alone. End-points consisted of arterial oxygenation, dynamic lung compliance, and protein and lipid content in bronchoalveolar lavage fluid. Potential mechanism of surfactant action for S-MB and SP-C33 UCLA were investigated with captive bubble surfactometry (CBS) assays.Results. All three surfactant peptide/lipid mixtures and Infasurf equally lowered the minimum surface tension on CBS, and also improved oxygenation and lung compliance. In both animal models, the two-peptide synthetic surfactant with S-MB and SP-C33 UCLA led to better arterial oxygenation and lung compliance than single peptide synthetic surfactants and Infasurf. Synthetic surfactants and Infasurf improved lung function further in lavage- than in chemical-induced respiratory failure, with the difference probably due to greater capillary-alveolar protein leakage and surfactant dysfunction after HCl instillation than following lung lavage. At the end of the duration of the experiments, synthetic surfactants provided more clinical stability in ALI/ARDS than Infasurf, and the protein content of bronchoalveolar lavage fluid was lowest for the two-peptide synthetic surfactant with S-MB and SP-C33 UCLA.Conclusion. Advanced synthetic surfactant with robust SP-B and SP-C mimics is better equipped to tackle surfactant inactivation in chemical ALI than synthetic surfactant with only a single surfactant peptide or animal-derived surfactant. Despiteactivity , doubts activity .Chemical spills are frequently in the news and the lung has come under attack from chemical spills and even bioterrorism . The lunα-helices with three intramolecular disulfide bridges  (2-RIPCCPVNLKRLLVVVVVVVLVVVVIVGALLMGL-COOH), which is highly homologous to that of human SP-C, has an N-terminus with a pair of vicinal Cys residues linked to palmitoyl moieties via thioester bonds, thereby producing a true “proteolipid” . When incorporated into lipids, Fourier transform infrared (FTIR) spectroscopy also showed that dipalmitoylated SP-C is principally α-helical, with its long molecular helix axis parallel to the phospholipid acyl chains  , and beleolipid” . These pl chains . Additiohelicity .2-FPIPLPYCWLCRALIKRIQAMIPKGGRMLPQLVCRLVLRCS-COOH) that reproduces the topology of the N- and C-terminal domains of SP-B by joining the N-terminal (residues 1–25) and C-terminal (residues 63–78) α-helices with a custom β-turn that replaces SP-B residues 26–62. Thus, S-MB is a ‘short-cut’ version of SP-B, in which the neighboring N- and C-terminal α-helices adopt a helix-turn-helix motif that is cross-linked by disulfide bridges at Cys-8 to Cys-40 and Cys-11 to Cys-34. S-MB has been shown to be highly surface active in vitro and in vivo, and this may be partially due to its positively charged amphipathic helices binding to anionic surfactant lipids  was adapted from the native porcine sequence (see above), in which the vicinal palmitoylcysteines were replaced by serines, ten valine residues were swapped with leucines and a cationic amino acid was moved from the N-terminus closer to the other positively charged residues  on synthetic SP-C peptides has led to the creation of highly surface active peptide mimics of SP-B and SP-C, i.e., Super Mini-B (S-MB)  and SP-Ct lipids . The funive SP-C . Specifiresidues . Surfaceresidues . Recent residues .in vitro methods can rapidly provide information about surface activity of experimental surfactant preparations, whole-animal studies are still necessary to test potential treatment modalities in chemical lung injury. Surfactant deficiency induced by repeated saline lung lavages , a bovine lung lavage extract, was a generous gift of Ony Inc. . Young adult New Zealand White rabbits, weighing 1.0–1.3 kg, were obtained from I.F.P.S. .Peptide synthesis reagents were purchased from Applied Biosystems , high performance liquid chromatography (HPLC) solvents from Fisher Chemical Co. , and all other chemicals from Sigma Chemical Co.  and Aldrich Chemical Co. . Dipalmitoylphosphatidylcholine (DPPC), palmitoyloleoyl-phosphatidylcholine (POPC) and palmitoyloleoylphosphatidylglycerol (POPG) were from Avanti Polar Lipids . The clinical surfactant Infasurfα-helicity was determined by FTIR spectroscopy. Disulfide connectivities for S-MB  were confirmed by mass spectroscopy of enzyme-digested fragments (trypsin and chymotrypsin digestion) (S-MB peptide  and SP-Cgestion) . We renaSynthetic surfactant preparations were formulated by mixing synthetic phospholipids, consisting of 5:3:2 (weight ratio) DPPC:POPC:POPG, with 3% S-MB, 3% SP-C33 UCLA, or 1.5% S-MB + 1.5% SP-C33 UCLA. All surfactant preparations were formulated at a concentration of 35 mg phospholipids/ml. The composition of the synthetic phospholipid mixture was based on the lipid composition of native lung surfactant . InfasurAdsorption and dynamic surface tension lowering ability of all surfactant preparations were measured with a captive bubble surfactometer at physiological cycling rate, area compression, temperature, and humidity . We routThe animal studies were reviewed and approved by the Institutional Animal Care and Use Committee of the Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center (Research Project # 12507). All procedures and anesthesia were in accordance with the American Veterinary Medical Association (AMVA) Guidelines.® Pro, ADInstruments Inc., Colorado Springs, CO, USA). Respiratory function was followed by measurements of arterial pH and blood gases and dynamic lung compliance at 15 min intervals. Dynamic lung compliance was calculated by dividing tidal volume/kg body weight by changes in airway pressure (peak inspiratory pressure minus positive end-expiratory pressure) (ml/kg/cm H2O). Maintenance fluid was provided by a continuous infusion of Lactated Ringer’s solution at a rate of 10 ml/kg/h.Young adult New Zealand white rabbits (weight 1.0–1.3 kg) received anesthesia with 50 mg/kg of ketamine and 5 mg/kg of acepromazine intramuscularly prior to placement of a venous line via a marginal ear vein. After intravenous administration of 1 mg/kg of diazepam and 0.2 mg/kg of propofol, a small incision was made in the skin of the anterior neck for placement of an endotracheal tube and a carotid arterial line. After placement of the endotracheal tube, muscle paralysis was induced with intravenous pancuronium (0.1 mg/kg). During the ensuing duration of mechanical ventilation, anesthesia was maintained by continuous intravenous administration of 3 mg/kg/h of propofol and intravenous dosages of 1 mg/kg of diazepam as needed; muscle paralysis was maintained by hourly intravenous administration of 0.1 mg/kg of pancuronium. Heart rate, arterial blood pressures and rectal temperature were monitored continuously (Labchart2) was >500 torr at a peak inspiratory pressure <15 cm H2O in 100% oxygen (FiO2 = 1.0), the rabbits underwent repeated saline lung lavages until the PaO2 dropped below 100 torr  or received intratracheal HCl until the PaO2 dropped below 200 torr . Edema fluid appearing in the trachea was removed by suctioning. When the PaO2 was stable at <100 torr in lavaged animals (n = 39) or the PaO2/FiO2 ratio had reached stable values <200 torr  within 30 min in HCl-exposed animals (n = 40), an experimental or positive (Infasurf) or negative  control surfactant mixture was instilled into the trachea at a dose of 100 mg/kg body weight and a concentration of 35 mg/ml. Group size was 7–8 in the lavaged and 8 in the HCl-exposed rabbits. All rabbits were ventilated using a Harvard volume-controlled animal ventilator . Airway flow and pressures and tidal volume were monitored continuously with a pneumotachograph connected to the endotracheal tube and a pneumotach system . Animals were sacrificed 2 h after surfactant administration with an overdose of pentobarbital. End-points were gas exchange , pulmonary mechanics (dynamic lung compliance), and bronchoalveolar lavage fluid proteins and lipids.After stabilization on the ventilator, lung injury was induced by saline lung lavage or intratracheal administration of hydrogen chloride (HCl). Lung lavaging results in loss of active surfactant, whereas HCl instillation leads to epithelial and endothelial damage, lung hemorrhages and copious edema formation resulting in surfactant dysfunction. When the partial pressure of oxygen in arterial blood  collected during the first lung lavage with 30 ml of normal saline to induce surfactant deficiency (lavaged rabbits only) and the first postmortem lavage (both lavaged and HCl-treated rabbits) are shown in t-tests and analyses of variance (ANOVA). Student’s t-test was used for comparisons versus control values. Between groups comparisons at various time-points were done by one-way ANOVA and time courses were analyzed with one-way repeated measure ANOVA. A p value <0.05 was considered to indicate a significant difference.Data are expressed as means ± standard error (SEM). Statistical differences were estimated using p < 0.001) than the one- and two peptide/lipid mixtures and Infasurf. Therefore, the relative order for the surfactant activities determined with captive bubble surfactometry for the various preparations were as follows: S-MB surfactant ∼ SP-C33 UCLA surfactant ∼ S-MB + SP-C33 UCLA surfactant ∼ Infasurf ≫ Lipids alone.S-MB, SP-C33 UCLA and S-MB + SP-C33 UCLA surfactant and Infasurf (positive control) all had very high surface activity in captive bubble experiments and reached minimum surface tension values ≤ 1 mN/m during each of ten consecutive cycles of dynamic cycling   .p < 0.01)  .n = 39) increased almost 10-fold from 286 ± 10 µg/ml in the first 30 ml lavage to induce surfactant-deficiency to 2,776 ± 132 µg/ml in the first postmortem lavage. This increase was least for S-MB+SP-C33 UCLA and S-MB surfactant and highest for synthetic lipids alone. Protein content of BALF in the first postmortem lavage of the 40 HCl-instilled rabbits was higher than in the 39 lavaged rabbits . Rabbits from the HCl group treated with S-MB + SP-C33 UCLA had the lowest and rabbits treated with lipids only had the highest protein values in the postmortem BALF samples among the 5 treatment groups (Average (±SEM) protein content of BALF  in the tt groups . Phospho®, Discovery Laboratories, Warrington, PA) with continuous positive airway pressure (CPAP) only in newborn pigs with acute lung injury after HCl instillation. Both KL4 preparations improved survival, but arterial oxygenation did not increase very much over the surfactant pretreatment values.The success of rescue therapy for chemical-induced ALI using synthetic surfactant with advanced and robust SP-B and SP-C mimics represents a significant breakthrough, as previous experiments have indicated only minimal or no improvement in lung function with animal-derived and first generation synthetic surfactant preparations . Lamm & in vivo activities of Mini-B, i.e., a ‘truncated’ 34-residue predecessor of S-MB without the 7 amino-acid insertion sequence at the N-terminal  after HCl instillation because values <80 torr were associated with a quick demise. In fact, the average PaO2 values after lung lavage were 53 torr  and 117 torr  after HCl treatment. Lung compliance corresponded with oxygenation, lung lavage decreased lung compliance by 51% versus 39% after HCl treatment. These differences can be explained by a lesser degree of capillary-alveolar protein leakage after lung lavage than acid treatment that not only results in loss of active surfactant, but also leads to epithelial and endothelial damage, lung hemorrhages and copious edema formation resulting in severe surfactant dysfunction.Animal models have their advantages and limitations. In this study animals were studied for 2 h after surfactant treatment, but longer duration of the experiments (at least 6 h) might better correlate with clinical outcome in ALI/ARDS patients. Lung lavage and HCl instillation each result in significant hypoxemia without hemodynamic effects that are fairly stable over at least the first 6–9 h . In our As opposed to animal-derived (Infasurf) or first generation synthetic (Surfaxin or Venticute) surfactant preparations, rescue therapy with a second generation synthetic (S-MB and SP-C33 UCLA in a synthetic three-lipid mixture) was highly effective in stabilizing and improving oxygenation and lung compliance in rabbits with chemical-induced lung injury.