Toc75 plays a central role in chloroplast biogenesis in plants as the membrane channel of the protein import translocon at the outer envelope of chloroplasts (TOC). Toc75 is a member of the Omp85 family of bacterial and organellar membrane insertases, characterized by N-terminal POTRA (polypeptide-transport associated) domains and C-terminal membrane-integrated β-barrels. We demonstrate that the Toc75 POTRA domains are essential for protein import and contribute to interactions with TOC receptors, thereby coupling preprotein recognition at the chloroplast surface with membrane translocation. The POTRA domains also interact with preproteins and mediate the recruitment of molecular chaperones in the intermembrane space to facilitate membrane transport. Our studies are consistent with the multi-functional roles of POTRA domains observed in other Omp85 family members and demonstrate that the domains of Toc75 have evolved unique properties specific to the acquisition of protein import during endosymbiotic evolution of the TOC system in plastids.DOI:http://dx.doi.org/10.7554/eLife.12631.001 Chloroplasts are a hallmark feature of plant cells and the sites of photosynthesis – the process in which plants harness the energy in sunlight for their own needs. The first chloroplasts arose when a photosynthetic bacterium was engulfed by another host cell, and most of the original bacterial genes have been transferred to the host cell’s nucleus during the evolution of land plants. As a result, modern chloroplasts need to import the thousands of proteins encoded by these genes from the rest of the cell.The chloroplast protein import system relies on a protein transporter in the chloroplast membrane that evolved from a family of bacterial transporters. However, the bacterial transporters were initially involved in protein export, and it was not known how the activity of these transporters adapted to move proteins in the opposite direction.Arabidopsis thaliana. These experiments revealed that a part of the transporter that is conserved in many other organisms, the “protein transport associated domains”, has been adapted for three key roles in protein import. First, this part of the transporter interacts with the other components of the import system that make the transporter more selective and control which direction the proteins are transported. Second, the domains interact with proteins during transport to help move them across the chloroplast membrane. Finally, the domains recruit other molecules called chaperones, which stop the protein from aggregating or misfolding during the transport process. These activities are similar to those for the bacterial export transporters, but clearly evolved to allow transport in the opposite direction – that is, to import proteins into chloroplasts.Paila et al. set out to better understand the chloroplast protein import system and produced mutated forms of the transporter in the model plant The next challenges are to explain how proteins destined for chloroplasts are recognized and transported through the chloroplast’s membrane.DOI:http://dx.doi.org/10.7554/eLife.12631.002 The biogenesis and function of plastids are reliant on the coordinate expression and selective import of >2500 different nucleus-encoded proteins . The majThe evolution of the TOC and TIC system during endosymbiosis required the adaptation or acquisition of protein conducting channels, receptors and molecular chaperones at both envelope membranes to provide a unidirectional conduit for the transport of nuclear encoded polypeptides into the organelle. Protein transport across the outer envelope membrane is mediated by a β-barrel membrane protein, Toc75, which is proposed to constitute the major component of the TOC import channel . Toc75 aTOC75-III, AT3G46740) and null mutants are embryo lethal  superfamily of β-barrel integral membrane proteins . Omp85 p1α1α2β2β3 secondary structural motif  domains, each characterized by a βal motif . The ~45al motif . While tal motif , the funE. coli outer membrane , two (Toc75ΔP1-2) or all three (Toc75ΔP1-3) predicted POTRA domains (toc75-III-1 null mutant. The TOC75-III genomic fragment retains both the native promoter and introns and was previously shown to complement toc75-III-1 (toc75-III-1 plants were transformed with a wild-type TOC75 gene construct (TOC75), TOC75ΔP1, TOC75ΔP1-2 or TOC75ΔP1-3. Transformants were selected for hygromycin resistance linked to the toc75-III-1 T-DNA insertion and for the DsRed fluorescence marker linked to the TOC75 and POTRA-deletion constructs. The presence of TOC75, TOC75ΔP1, TOC75ΔP1-2 or TOC75ΔP1-3  was confirmed by PCR of genomic DNA in T3 transformants homozygous for the DsRed marker. Plants homozygous for the TOC75, TOC75ΔP1, TOC75ΔP1-2 or TOC75ΔP1-3 transgenes were selected in the heterozygous toc75-III-1 (toc75III-1[+/-]) background, and the progeny of these plants were segregated to test for complementation of the lethal phenotype of toc75-III-1 homozygous plants. At least five independent lines were selected for each construct. The segregation analysis of representative T3 lines is shown in toc75-III-1(+/-) progeny carrying the homozygous TOC75 wild type transgene segregated at a near 3:1 ratio of hygromycin resistant:sensitive plants, consistent with complementation of the toc75-III-1 insertion  could be recovered (toc75-III-1(+/-) lines homozygous for the TOC75ΔP1, TOC75ΔP1-2 or TOC75ΔP1-3 deletion constructs segregated at 2:1 or lower ratios of hygromycin resistant:sensitive plants, indicating that the POTRA deletions were unable to complement toc75-III-1 , several transformed lines  showed progressively increased pale phenotypes and reduced growth rates  and those showing aberrant segregation (TOC75ΔP1#2 and TOC75ΔP1-2#2)  background. Consistent with this observation, the levels of Toc75ΔP1 or Toc75ΔP1-2 mRNA in TOC75ΔP1#2 or TOC75ΔP1-2#2 plants were 3.5–4.5 times higher than those of native Toc75 mRNA, whereas the Toc75ΔP1 or Toc75ΔP1-2 protein levels were significantly lower than native Toc75. This suggests that expression of TOC75ΔP1#2 and TOC75ΔP1-2#2 is tightly controlled post-transcriptionally or by proteolytic degradation of Toc75ΔP1 or Toc75ΔP1-2, most likely in response to the negative impact of the POTRA deletions on chloroplast function.The differences in the segregation  and phenonstruct . We compΔP1-2#2) . TOC75ΔPr plates . Real-tir plates  and immur plates  from seen growth  and germn growth  correlatn growth . Althougn growth . These dtoc75-III-1 (+/-), TOC75, TOC75ΔP1#2 and TOC75ΔP1-2#2 plants. Isolated chloroplasts were incubated with [35S]-labeled chloroplast preprotein, preSSU, in the presence of 3 mM ATP to promote import for 2, 5 or 10 min at 26° (toc75-III-1 (+/-) and TOC75 plants were statistically indistinguishable  and TOC75 plants (35S]preE1α)  . PreE1α ]preE1α) . These d35S]preSSU with the import machinery under two distinct energy conditions. In the absence of ATP, preproteins bind to chloroplasts in a low-affinity, energy-independent interaction involving the Toc159 and Toc33 GTPase receptors (TOC75 and toc75-III-1(+/-) chloroplasts showed 53% and 97% increases in chloroplast-associated preprotein, respectively, in the presence of 0.1 mM ATP compared to chloroplasts depleted of ATP  control chloroplasts and TOC75ΔP1#2, and TOC75ΔP1-2#2 chloroplasts ) of preSSU-FLAG-HIS for TOC translocons in TOC75 and TOC75ΔP1#2 and TOC75ΔP1-2#2 chloroplasts were 158.6 ± 29 nM, 187.7 ± 46 nM and 126.5 ± 24 mM, respectively  similar to TOC75ΔP1#2.To test if the POTRA deletions impacted the topology of Toc75 in the outer membrane, we examined the sensitivity of full-length Toc75 and Toc75ΔP1 to exogenous protease in isolated, intact chloroplasts. We were unable to examine Toc75ΔP1-2 due to relatively low levels of expression of this construct in transgenic plants and the consequent inability to reliably detect proteolytic fragments from the deletion construct. To facilitate our studies, we expressed Toc75 and Toc75ΔP1 containing a myc epitope inserted just upstream of the POTRA domains at the N-terminus of the mature proteins in 1 plants . The TOChenotype . Consist75-III-1 . For submyc TOC75and mycTOC75ΔP1 plants were treated with various concentrations of trypsin or thermolysin, resolved by SDS-PAGE and immunoblotted with anti-myc or antibodies specific to the Toc75 POTRA domains. Previous studies have shown that Toc75 is resistant to low-to-moderate levels of thermolysin, whereas trypsin generates several proteolytic fragments, including peptides corresponding to the N-terminal POTRA deletions  is concomitant with the appearance of a ~55 kDa protease-resistant fragment, which reacts with anti-POTRA and anti-myc  plants  plants . The lev) plants , were un) plants , further2 plants . However2 plants , were inon lines .toc75III-1 (+/-) and TOC75 plants, the levels of total transcripts encoding Toc75 and the POTRA deletions  were increased by four- and six fold in TOC75ΔP1#2 and TOC75ΔP1-2#2 plants, respectively , based on the mobility of TOC complexes on blue-native gel electrophoresis . The oveth Toc33 . OEP80, th Toc33 . These dTOC75 chloroplasts co-migrated in complexes, which peaked at ~1.3 MDa (region a). A second complex, with a peak at ~440 kDa (region b) migrated between 400 and 880 kDa and contained Toc75 and Toc33, but lacked detectable levels of Toc159. The presence of the ~1.3 MDa and ~440 kDa complexes is consistent with the 800–1000 kDa and 300–450 kDa TOC complexes previously reported from pea chloroplasts  were increased significantly in roplasts  is likelroplasts . Taken tTOC75ΔP1#2 and TOC75ΔP1-2#2 plants, raised questions regarding the relationship of these two import proteins. Previous work had demonstrated a potential interaction between the N-terminal POTRA domains of an Omp85 family member in the outer membrane of the cyanobacteria, Anabeana sp. PCC 7120, and a chaperone protein in the periplasmic space, which is structurally related to Tic22 (E. coli and tested for binding to Tic22-III and Tic22-IV. The recombinant POTRA-His was immobilized on nickel-nitrilotriacetic acid (Ni-NTA) matrix and incubated with in vitro–translated [35S]Tic22-III and [35S]Tic22-IV. Binding was measured as the fraction of [35S]Tic22-III and [35S]Tic22-IV that co-sedimented with the immobilized POTRA domains. Transit peptide-dependent binding of preSSU to Toc75 POTRAs has been demonstrated previously   . Similartein 80)  and is ptein 80) . OEP80 itein 80) . Althougtein 80) . The evoArabidopsis thaliana Col-0 were grown on phytoagar plates containing 0.5 × Murashige and Skoog growth medium (MS medium), 1% sucrose under long-day condition for 14-day at 22°C. Genetic complementation was carried out as described earlier . The destination vector contains a fluorescent marker, DsRed, under a seed specific promoter and BASTA (glufosinate ammonium) resistance, allowing for rapid screening of seed and seedlings. Final constructs were confirmed by sequencing and introduced into heterozygous toc75-III-1 plants  as per manufacturer’s instructions. RNA isolated was quantified by absorbance at 260 nm, and equivalent amounts were used to synthesize first-strand cDNA using SuperScript III and an oligo(dT) primer . cDNA was used for PCR reactions (35 cycles) using gene-specific primers. Primers used for PCR amplification of Waltham, MA). Baseline and threshold cycles (Ct) were analyzed by Realplex 2.2 Software. Relative gene expressions were calculated with respect to internal reference of actin using the 2∆Ct method , 0.25 mg/ml soybean trypsin inhibitor and 2 μg/ml aprotinin). Intact chloroplasts were reisolated through 35% Percoll and washed with ice-cold HS buffer containing EDTA or trypsin inhibitor cocktail. 1% (v/v) Triton X-100 was included in reactions to disrupt membrane permeability.Thermolysin and trypsin treatments of intact chloroplasts were performed as described earlier . All proTotal protein was extracted directly in SDS-PAGE sample buffer from Arabidopsis seedlings. Samples corresponding to equivalent amounts of total protein were resolved by SDS-PAGE, transferred to PVDF membranes, and subjected to immunoblotting with antisera to the indicated proteins. Immunoblotting was performed as described previously  using chImmunoaffinity chromatography of TOC core complex proteins under native conditions was performed as described previously . The pro35S]Methionine-labeled Arabidopsis preSSU, preE1α, fully processed (mature) forms of Toc75ΔP1 and Toc75ΔP1-2 were generated in a coupled transcription-translation system containing reticulocyte lysate according to the manufacturer’s instructions . The in vitro translation product containing [35S]preSSU or [35S]preE1α was used directly for chloroplast import assays. Chloroplast early binding and import reactions were performed using [35S]methione-labeled preproteins and equal number of chloroplasts (107 chloroplasts), in a total volume of 50 μl of import buffer  for 5 min with 0.1mM ATP and GTP or 20 min at 26°C in the presence of 3 mM ATP for binding and import, respectively as described previously (35S]preSSU was gel filtered on Sephadex G-25 to remove nucleotides (7) containing bound preprotein or the early import intermediate reactions were generated as above. Chloroplasts recovered after isolation over the 35% Percoll cushion were washed once and resuspended in import buffer. Preprotein translocation was initiated with the addition of 3 mM ATP, and samples were incubated at 26°C for 20 min. All samples were resolved by SDS-PAGE and analyzed by phosphorimaging (Fuji Fla-5000 phosphorimager). Equivalent numbers of chloroplasts based on microscopic counting were loaded in all lanes. ImageQuant TL (v 1.00) software was used for analysis. in 100:3.25 (v/v) to give a ratio of the detergent  to the dye (Serva blue G) as 8:1 (w/w). The sample was loaded onto a 4–12% (w/v) polyacrylamide gradient gel in 50 mM BisTris-HCl, pH 7.0, containing 500 mM 6-amino-n-caproic acid . Electrophoresis was carried out in the cathode buffer  and the anode buffer  at a constant voltage of 30 V at 4°C for 14 hr. Spectra multicolor Broad Range Protein Ladder (Thermo Scientific) and ferritin  were used as size standards. After BN-PAGE, the marker lanes were directly stained with Coomassie Brilliant Blue, and the sample lanes were excised and heated at 37°C for 30 min in 3.3% SDS, 4% 2-mercaptoethanol and 65 mM Tris-HCl, pH 6.8. The gel strip was layered on top of a 1.5 mm-thick stacking gel, and the second dimension 5–12% gradient SDS-PAGE under reducing conditions was performed according to standard procedures.2D BN-PAGE was performed as described earlier  with a fE. coli, purified by Glutathione Sepharose 4B chromatography and cleaved using TEV protease to generate soluble POTRA-His. Solid phase binding assays were performed as described previously  with 10 mM imidazole and 0.1% Triton X-100 (binding buffer). 1–3 μl of [35S]Tic22-III, [35S]Tic22-IV, [35S]preSSU and [35S]SSU were incubated in binding buffer with POTRA-bound resin in a final volume of 100 μl for 30 min at room temperature (23°C). After washing, resin-bound proteins were eluted with SDS-PAGE sample buffer containing 500 mM imidazole. All radiolabeled proteins from in vitro pull-down assays were resolved using SDS-PAGE, and radioactivity was analyzed by phosphorimaging (Fuji Fla-5000 phosphorimager) and quantitated using ImageQuant TL (v 1.00).A glutathione S-transferase-POTRA fusion (GST-POTRA-His) was expressed in eviously  with somSequence data from this article can be found in the EMBL/GenBank data libraries under accession number Arabidopsis AGI locus identifier: AT3G46740 for Toc75-III. In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by Nikolaus Pfanner as the Reviewing Editor and Vivek Malhotra as the Senior Editor.Thank you for submitting your work entitled "Multi-functional roles for the polypeptide transport associated (POTRA) domains of Toc75 in chloroplast protein import" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:TOC75 POTRA domains and there is also uncertainty about the orientation of these domains in the outer membrane. The manuscript describes a first attempt at a comprehensive analysis of the TOC75 POTRA domains and also addresses the membrane orientation. The authors analyzed the outcome of deleting one, two, or all three POTRA domains of Toc75. In addition they characterized the import behavior of organelles coexpressing one of the deletion variants together with a native form. They also analyzed the assembly of the TOC complex in such chloroplasts. An interesting side aspect of this report is the Toc75 topology analysis, which is in agreement with the bacterial and mitochondrial Omp85 homologs. This is important since a previous controversial study  proposed just the opposite orientation for chloroplasts. Finally, the authors show that a recombinant polypeptide corresponding to the three POTRA domains can interact with precursor protein and with Tic22. In general, the data presented in the manuscript is technically sound and represents the first characterization of the Toc75 POTRA domains for protein import into chloroplasts. The elucidation of TOC75 POTRA function will represent a significant step forward in the field, and so this manuscript is potentially of considerable interest.The chloroplast protein Toc75 is a member of the Omp85 family of proteins. Proteins of this family contain a β-barrel membrane-embedded domain in their C-terminal part and one to five POTRA domains in the N-terminal region. However, little is known about the functions of the Essential revisions:TOC75 POTRA deletion constructs were used to try and complement the toc75-III-1 mutant. These were dP1, dP1-2, dP1-3. As none of these was able to complement the mutant, this only reveals a necessity for POTRA 1. Alternatively perhaps the folding of the whole POTRA domain was already disrupted by the dP1 deletion and so further deletions had no further effects.1) Three toc75-III-1 background. Single plant photos show an increasing severity of effect related to deletion of the P1, P1&2 or P1, 2&3 POTRA domains. Transformants with no phenotype are shown to have near WT levels of overexpression, whereas it appears that the negative phenotypes are associated with high overexpression of the deletion constructs. However, as only one WT-like and one dominant-negative line containing each construct is assessed, the asserted dosage dependency is not clear (this is referring to Putative dominant-negative phenotypes were found in some transformed plants containing the POTRA deletion constructs in the heterozygous TOC75ΔP1#2 and Toc75ΔP1-2#2 are much higher than those of TOC75 (2) There is a discrepancy between the results of of TOC75  the protToc75ΔP1-2#2 are at most about 20% of those of native Toc75. It is not clear how such low levels can cause such strong "dominant negative" effects. Can the authors exclude the possibility that part of these variant molecules are clogging the TOC complex as substrate in transit and thus affecting function and assembly of the TOC complex?According to TOC75 and WT TOC75, implying necessity of the POTRA domains (this is supported by data in TOC75 was imported in vitro and has the same in vivo topology as TOC75). However results for a highly overexpressed TOC75 line are not shown. Therefore it is not possible to tell if the deleterious effect is truly due to assembled TOC75 lacking POTRA domains .The phenotypes in TOC75 and toc75-III-1+/-, but not between each other. The only data potentially revealing differing roles of the POTRAs are in TOC75 does not form part of the largest TOC complex.The title asserts multifunctional roles for POTRA domains in TOC75. However many of the analyses  of the d3) TOC75ΔP1#2 and Toc75ΔP1-2#2 chloroplasts while Toc159 remains unchanged. Additionally, TOC75ΔP1#2 and TOC75ΔP1-2#2 in As shown in mycToc75ΔP1 is behaving differently than Toc75 as the former is completely resistant to trypsin. Surprisingly, in the anti-POTRA western is seems as if the vast majority of molecules of both forms are resistant to trypsin. Since the authors have an antibody against Toc75 it will be informative to show a full gel immunodecorated with anti-Toc75 to see also smaller fragments. Currently, I am not convinced that TOC75ΔP1 has the same topology as native Toc75.4) TOC75ΔP1 and TOC75ΔP1-2 this analysis does not allow any conclusion about the interaction between TOC75ΔP1 and TOC75ΔP1-2 and the other Toc and Tic subunits. Alternatively, the authors could perform an anti-myc co-immunoprecipitation using their Toc75myc chloroplasts.5) In Toc75ΔP1-2#2 very similar, whereas in many other experiments  Three TOC75 POTRA deletion constructs were used to try and complement the toc75-III-1 mutant. These were dP1, dP1-2, dP1-3. As none of these was able to complement the mutant, this only reveals a necessity for POTRA 1. Alternatively perhaps the folding of the whole POTRA domain was already disrupted by the dP1 deletion and so further deletions had no further effects.Putative dominant-negative phenotypes were found in some transformed plants containing the POTRA deletion constructs in the heterozygous toc75-III-1 background. Single plant photos show an increasing severity of effect related to deletion of the P1, P1&2 or P1,2&3 POTRA domains. Transformants with no phenotype are shown to have near WT levels of overexpression, whereas it appears that the negative phenotypes are associated with high overexpression of the deletion constructs. However, as only one WT-like and one dominant-negative line containing each construct is assessed, the asserted dosage dependency is not clear  The reviewers asked us to clarify our conclusion that the three POTRA domains are essential for Toc75 function. We agree with the reviewers that we can only conclude from our analyses that POTRA1 is essential for Toc75 function. We have modified the Abstract to avoid implying otherwise. However, the increasing severity of the lines expressing TOC75ΔP1#2 and TOC75ΔP1-2#2 are representative of multiple lines expressing the POTRA deletion constructs. We have included data on the phenotypes and levels of expression of the POTRA deletions from six independent transformed lines each for TOC75ΔP1 and TOC75ΔP1-2 .b) Second, the reviewers raised the issue of whether our analyses of C75ΔP1-2 . These l2) There is a discrepancy between the results of According to The phenotypes in The title asserts multifunctional roles for POTRA domains in TOC75. However many of the analyses  of the dThese comments focus on the expression levels of the Toc75 POTRA deletions and wild type Toc75 in transgenic plants.TOC75ΔP1#2 and TOC75ΔP1-2#2 lines relative to expression of wild type Toc75. The transgenes are all expressed from the native promoter, and the gene constructs retain all corresponding intron and exon organization. However, it is well known that positional effects due to the sites of insertion in the Arabidopsis genome can result in significant differences in the levels of expression of the same gene constructs. With regards to the fact that the protein levels of the POTRA deletions are significantly lower than wild type Toc75 protein, we speculate that this might be due to relative instability of the deletion constructs and their apparent toxicity to the plants. Statements to these effects are added to the text .a) We did not examine the cause of the high levels of mRNA accumulation of the POTRA deletions in the E. coli. We hypothesize that the introduction of defective subunits into these complexes can disrupt the function of the large complexes by interfering with the coordinate activities of Toc33, Toc159 and Toc75. This hypothesis is consistent with other known cases in which mutations that impact a small proportion of subunits of oligomeric complexes have significant impacts on function. We have added a statement to the text to explain this possibility .b) TOC complexes are large multimeric assemblies. Toc75 appears to self-associate when expressed and reconstituted from TOC75ΔP1, assume the same topology as native Toc75 (c) Our data do not support the possibility that the POTRA deletions might be clogging the translocon channel and thereby causing protein import defects. The deletions are targeted, fully processed and membrane integrated as shown in ve Toc75 .TOC75ΔP1#2 without any obvious phenotypes.d) It is very difficult to obtain highly over-expressed lines of wild type Toc75. High level overexpression leads to co-suppression and a lethal phenotype. In unpublished work, we previously expressed Toc75 under strong constitutive promoters (e.g. 35S CaMV), and could obtain up to a 2-fold increase in expression similar to what we observe for wild type Toc75 in TOC75ΔP1 and TOC75ΔP1-2 have distinct effects on TOC assembly (e) As stated above, we agree that our data do not demonstrate that the individual POTRA repeats have entirely distinct functions. However, it is clear that the POTRAs together play multiple roles in TOC function and assembly. Furthermore, the fact that assembly  suggest 3) As shown in The reviewers raised questions regarding the nature of the protein import defect in the POTRA deletion lines.a) We have added import experiments with an additional preprotein, the precursor to the E1 α subunit of pyruvate dehydrogenase . This prd(app). We would expect that significant binding by “free” Toc33 would impact the Kd(app) as this interaction would most certainly have a distinct affinity from intact TOC complexes.b) Although we cannot eliminate the possibility that excess Toc33 contributes to the import defect, we would expect to see increased binding to chloroplasts under no energy conditions . Rather,myc is behaving differently than Toc75 as the former is completely resistant to trypsin. Surprisingly, in the anti-POTRA western is seems as if the vast majority of molecules of both forms are resistant to trypsin. Since the authors have an antibody against Toc75 it will be informative to show a full gel immunodecorated with anti-Toc75 to see also smaller fragments. Currently, I am not convinced that Toc75ΔP1 has the same topology as native Toc75.4) TOC75ΔP1 exhibit indistinguishable protease sensitivity patterns, consistent with our conclusion that they assume the same topology in the outer membrane.We believe that the questions regarding the interpretation of results in myc chloroplasts. 5) In These comments address the assembly of the POTRA deletions with other TOC components.a) The intent of the data in b) We attempted to use anti-myc to immunoprecipitate complexes, but were unsuccessful, perhaps due to the inaccessibility of the epitope on Toc75 within TOC assemblies.c) We also included immunoblots of OEP80, an outer membrane negative control, in Arabidopsis chloroplasts, whereas the published study was analyzing pea chloroplasts. The GTPase receptors are different sizes in the two organisms. We do not think this is significant. To avoid confusion, we now refer to the complexes we observe as 1.3 MDa and 440 kDa complexes and explain the slight differences from the results of Kikuchi et al. in the text .d) The minor size differences in the TOC complexes shown here compared to the previous study is likely due to the fact that we are analyzing TOC75ΔP1 observed in other experiments.e) We have repeated the 2D blue-native gel analysis and included higher quality images in Arabidopsis than in pea chloroplasts in our hands.f) We did not detect an 86 kDa fragment when the blots were probed with an anti-serum to the C-terminal domain of Toc159. We take extra precautions to avoid proteolysis during our experiments. Furthermore, Toc159 is much less susceptible to proteolysis in g) As mentioned above, we repeated the 2D blue-native gel analysis and included higher quality blots in


Treatment with tumour necrosis factor (TNF) antagonists may lead to enhanced susceptibility to certain malignancies. In particular, an association is seen emerging between TNF antagonists and development of squamous cell carcinomas (SCCs) of the skin (in association with psoriasis), the oral cavity, and in the anogenital areas (possibly related to prior human papilloma virus infection). We present here a case of a 53-year old woman with a history of severe rheumatoid arthritis (RA), most recently treated with the TNF antagonist etanercept plus methotrexate, presented to our service after several months of increasing left pelvis and buttock pain. Evaluation with a computerised tomography (CT)-directed biopsy of a pelvic side wall mass revealed a metastatic SCC. On a fluorodeoxyglucose (FDG) positron-emission tomography (PET) an additional area of uptake was identified in the left posterior rectum corresponding to a 1 cm nodule palpable on digital exam. Colonoscopic biopsy revealed a basaloid SCC of the rectum as the likely primary site. Immunosuppression following TNF antagonist therapy may have given arise to this unrestrained neoplastic growth. It thereby underscores the need for an initial baseline study of risk factors and identification of patients who are at higher risk for development of a malignancy, in order to achieve a diagnosis at an early stage. However, some reports suggest there is no obvious link between HPV and squamous cell carcinoma of the rectum [Tumour necrosis factor (TNF) is a pro-inflammatory cytokine that plays an important role in immune response and is implicated in the pathogenesis of many inflammatory conditions –4. Etanee rectum –11. StudA 53-year old woman with severe rheumatoid arthritis (RA) presented to her primary care physician in March 2014 with increasing left pelvis and buttock pain over several months. Treatment for her RA dated back to 1995 and included several regimens including steroids, methotrexate alone, and for the past six years she appeared to have benefitted from the addition of etanercept to methotrexate (50 mg weekly). In addition to the pelvic pain, she was experiencing fatigue but there were no neurologic signs, no weight loss, fevers, or night sweats. A computed tomography (CT) scan of the abdomen and pelvis revealed a lobular, infiltrative 3.1 x 6.1 x 4.4 cm soft tissue mass extending along left obturator space with encasement of the inferior branch of left internal iliac vessels and likely involvement of the sciatic bundle with extension to the left vaginal wall.CT-guided core biopsy was consistent with squamous cell carcinoma (SCC), poorly differentiated. A fluorodeoxyglucose (FDG) positron-emission/computerised tomography (PET/CT) scan demonstrated a markedly FDG-avid left femoral lymph node and intense uptake in the left posterior rectum corresponding to a 1 cm nodule palpable on digital exam that was concerning for malignancy  in additRadiation consisted of treatment to the pelvis with a 3D-conformal four-field set-up with a dose of 4320 cGy in 24 fractions using a mixed 16/6 MV photon beam. Treatment to the pelvis was sequentially followed by a boost to the pelvic mass to a dose of 1000 cGy in five fractions using a 3D-conformal anterior-posterior field set-up with a 16 MV photon beam. The patient experienced a gradual response with eventual full relief of symptoms. At completion, she relocated her care to Florida where she was consolidated with seven cycles of carboplatin and paclitaxel. She was followed until September 2015 but details of her disease status are unknown.The association of TNF antagonists and development of SCCs has come to light in several reports –21. Patiet al of nine randomised placebo-controlled trials of TNF antagonists in RA, found a malignancy of 0.2% in controls who received only placebo versus 0.8% in patients receiving anti-TNF medications for their RA. The number needed to harm was 154  for one additional malignancy within a treatment period of 6–12 months [In fact, one such study of the Swedish inpatient registry compared 53,067 RA patients admitted to hospital between 1990–2003 to patients treated with TNF antagonists for RA during 1999–2003 (n = 4160). Results demonstrated an increased occurrence of non-melanoma skin cancers (standardised incidence ratio = 3.6) but no rise in solid tumours frequency in the TNF antagonist-treated group. In fact, patients treated with TNF antagonists actually had a 20% decreased risk for breast cancer that echoed those of the inpatient registry as well as historical RA cohorts. However, the comparator RA cohort did have a 25% reduction in colorectal cancer relative to other admissions that was not seen in the TNF antagonist treated patients . Patient2 months . These lA case report by Smith and Skelton (2001) described seven patients with RA who experienced one or more rapidly growing cSCC that developed shortly after the initiation of etanercept therapy . Similaret al (2014) described a case of a 45-year-old man with RA who was started on etanercept and within two months developed oropharyngeal pain and ulcerations and was diagnosed with tonsillar carcinoma [SCCs of the oral cavity have also been reported with TNF antagonist therapy , 18, 21.et al 20 describeIn conclusion, our report of the rare occurrence of a basaloid SCC of the rectum with positive p16 expression occurring after treatment of RA arthritis with etanercept and methotrexate also raises questions about enhanced susceptibility to human papilloma virus (HPV) as a risk factor for secondary malignancies following TNF inhibitor use. Of possible relevance, TNF antagonists have been noted to exacerbate genital HPV recurrences . TherefoThe authors have no conflicts of interest to declare.


Biomarkers are widely used and studied in heart failure. Most studies have described the utility and performance of biomarkers in sub-studies of randomised clinical trials, where the vast majority of the patients suffered from heart failure with reduced ejection fraction (HFrEF), and not with preserved ejection fraction (HFpEF). As a result, there is a scarcity of data describing the levels, dynamics, clinical and biochemical correlates, and biology of biomarkers in patients suffering from HFpEF, whereas HFpEF is in fact a very frequent clinical entity. This article discusses the value of different biomarkers in HFpEF. We describe various aspects of natriuretic peptide measurements in HFpEF patients, with a focus on diagnosis, prognosis and the risk prediction of developing heart failure. Further, we will discuss several emerging biomarkers such as galectin-3 and suppression of tumorigenicity 2, and recently discovered ones such as growth differentiation factor-15 and syndecan-1. In the last decades we have seen an explosion in numbers of new and emerging biomarkers, and the number of articles on this topic has steadily risen –3. ThereThe aim of this overview is to describe several generally accepted and several novel biomarkers in HFpEF, focusing on the value these biomarkers have for diagnostic and prognostic performance, and potential clinical utility.The natriuretic peptides are by far the best studied and most widely accepted and employed biomarkers in heart failure, both in HFrEF and in HFpEF. The best studied natriuretic peptides are atrial natriuretic peptide and B-type natriuretic peptide (BNP). Mechanistically, transcription and release of both natriuretic peptides occurs in response to myocardial stretch , 8. BesiNowadays, natriuretic peptides can easily be measured by fully automated and commercially available assays, which have proven excellent precision and reproducibility, also between laboratories. BNP and NT-proBNP correlate very well, but the clearance of NT-proBNP is exclusively via the kidney whereas BNP is also cleared via the liver, resulting in different half-lives of the hormones.Natriuretic peptides are moderately elevated in HFpEF patients –14, and There are no established cut-off points for HFpEF versus HFrEF, or versus heart failure as an entire group.According to the heart failure guidelines  natriureTschöpe et al. conducted a study that provided evidence that natriuretic peptides may be used to diagnose HFpEF. Herein, the authors demonstrated that NT-proBNP levels were strongly related to increased LV diastolic filling pressures, as determined by invasive measurements and end-diastolic wall stress in HFpEF patients . The latClearly, acute heart failure patients present completely differently than chronic stable heart failure patients. A crucial difference is that patients present acutely, usually to the emergency department, with symptoms of dyspnoea. During such episodes, the cardiac muscle endures high wall stress, which results in high values of natriuretic peptides in those with acute heart failure, regardless of a preserved or reduced ejection fraction. Maisel et al. showed that HFpEF patients who present with acute decompensated heart failure typically had BNP values of 600–1000 pg/ml . Less seBesides their diagnostic utility, natriuretic peptides might be used to identify subjects at risk of developing HFpEF over the course of many years, which is different from the diagnostic utility of natriuretic peptides. A possible utility for ‘signalling’ heart failure over a very long course has been shown for natriuretic peptides, and high-sensitivity troponin, for new-onset HFpEF in high-risk subjects from the general population , and theEpidemiological studies suggest that the overall prognosis is comparable for patients with HFpEF compared with HFrEF. However, in randomised controlled trials (RCTs), a certain bias exists because a ‘typical’ HFpEF patient usually has multiple comorbidities and often these comorbidities are exclusion criteria for trials, and as a result patients suffering from them are excluded from such trials. Therefore, the patients with a high likelihood for an adverse event are paradoxically often excluded from HFpEF trials, and this results in a substantially lower cardiovascular risk in HFpEF patients enrolled in RCTs than in HFrEF patients enrolled in RCTs.Nevertheless, the i-PRESERVE and the PEP-CHF studies demonstrated that both baseline values of NT-proBNP and change in NT-proBNP from baseline have prognostic value in patients with HFpEF, improving the prediction of mortality and heart failure rehospitalisation , 23. WheAtrial fibrillation is very common in HFpEF, and the presence of atrial fibrillation has a strong impact on circulating levels of natriuretic peptides. In patient cohorts with paroxysmal and chronic atrial fibrillation, natriuretic peptide levels are strongly elevated, often exceeding the values reported in HFpEF . McKelvi2. Finally, obesity is associated with lower BNP levels [2. Therefore clinicians who treat HFpEF patients and measure natriuretic peptide levels are advised to take the above-mentioned comorbidities into consideration when assessing the natriuretic peptide values.Besides atrial fibrillation, female sex and advanced age, both very common in HFpEF, are associated with elevated NT-proBNP . In a stP levels , 29, andGalectin-3 and suppression of tumorigenicity 2 (ST2) are emerging biomarkers that are not only predictive for hospitalisation and death in patients with heart failure, but also add additional prognostic value over natriuretic peptides. As such, they received a class IIB recommendation by the 2013 American College of Cardiology/American Heart Association guideline for the management of heart failure for risk stratification .Evidence has been generated specifically regarding the prognostic value of galectin-3 and ST2 in HFpEF patients, and patients with preclinical diastolic dysfunction.One of the first studies that compared galectin-3 levels between HFpEF and HFrEF patients was conducted by de Boer et al. . The CooThree additional HFpEF cohorts have since demonstrated the relation between galectin-3 and HFpEF.First, Carrasco-Sánchez and colleagues demonstrated the predictive value of galectin-3 in 419 HFpEF patients (LVEF > 45 %) who were admitted with acute heart failure. Galectin-3 independently predicted all-cause mortality and heart failure rehospitalisation, and yielded significant reclassification indices . CurrentSecondly, the ALDO-HF trial randomised HFpEF (LVEF ≥ 50 %) patients to either a mineralocorticoid receptor antagonist  or placebo. Spironolactone improved LV diastolic function, but did not affect maximal exercise capacity, patient symptoms, or quality of life . GalectiThe RELAX trial  was a muThe first evidence that ST2 levels could be of interest in HFpEF patients was provided by a post-hoc analysis of 200 HFpEF patients . AlthougMore clinical data on ST2 in HFpEF was provided by Friões et al. . The autHypertension, a comorbidity that is most frequently present in HFpEF patients, might lead to different activated pathophysiological mechanisms in HFpEF. This was recently investigated by Zile et al. . The autBesides natriuretic peptides, galectin-3 and ST2 several other biomarkers have been identified as potential markers for HFpEF. With respect to the biomarker levels, tissue inhibitor of metalloproteinases (TIMPs), aminoterminal propeptide of type III procollagen (PIIINP), homocysteine and resistin are particularly upregulated in HFpEF compared with HFrEF. Whereas biomarkers of inflammation, such as pentraxin-3, C-reactive protein, tumour necrosis factor alpha (TNF-α), interleukin (IL)-6 and IL-10, show higher levels in HFrEF compared with HFpEF  2 and 9, carboxy-terminal telopeptide of collagen type I (CITP) and PIIINP. All these biomarkers are associated with outcome in HFpEF, although the independent predictive value of each marker was limited . SyndecaInflammatory markers play an important role in the development of heart failure. TNF-α and IL-6 were both associated with new-onset HFpEF, but less associated with HFrEF .Next to markers of myocyte stress, inflammation and extracellular matrix remodelling, also other biomarkers are studied for their value in HFpEF. These biomarkers include growth differentiation factor-15 (GDF-15), cystatin C, resistin, cancer antigen-125 (CA-125) and von Willebrand factor (vWF).GDF-15 is a member of the transforming growth factor (TGF)-beta family and correlates with cardiac hypertrophy and fibrosis. In 149 patients with LV diastolic dysfunction and normal ejection fraction, GDF-15 was found to be a potentially useful prognostic biomarker in patients with HFpEF, although the independent value was not assessed . HoweverCystatin C is a marker of renal function that also predicts cardiovascular outcome. The serum cystatin C level in patients with HFpEF is an independent predictor for all-cause mortality and/or readmission in patients with acute heart failure, regardless of renal function . HoweverResistin is derived from adipose tissue and associated with inflammation. Resistin was measured in 2902 subjects in the Health ABC study and was associated with incident HFpEF and to a lesser extent with HFrEF. The prognostic value is yet to be examined .CA-125 is a tumour marker for ovarian cancer, but is also elevated in heart failure and is related to the severity of heart failure . CA-125 vWF is an independent predictor of long-term outcome in patients with HFpEF, which may be explained by its role in endothelial dysfunction . vWF wasA large proportion of patients with heart failure suffer from HFpEF. In contrast to HFrEF, the role for biomarker assessment in HFpEF has not been validated in full extent. No biomarker is specific for HFpEF; however, several markers have been shown to provide powerful and clinically meaningful information for the setting of the diagnosis and prognosis of HFpEF. As HFpEF is a very heterogeneous syndrome, likely not one but rather a panel of various biomarkers, representing different aspects of the pathophysiology, will provide the most powerful and sophisticated information for clinicians caring for patients with HFpEF. In the coming decades, such panels need to be identified, validated, and prospectively tested in the outpatient and inpatient setting.


TNF‐α) in peripheral blood cells is increased by various psychological stresses, the effect of TNF‐α administration to pregnant mice on telomere length in adulthood was examined in the present study. In utero TNF‐α treatment‐induced telomere shortening in adult mice. Telomere shortening was observed in certain tissues such as the bone marrow, spleen, and lung, and was detected at specific age ranges during adulthood. Telomere shortening was not observed in mice lacking the stress‐responsive transcription factor ATF7, which contributes to heterochromatin formation in the absence of stress. The present study identified the conditions under which in utero TNF‐α treatment induces telomere shortening in adulthood.Epidemiological studies indicate that exposure to stress during intrauterine life is associated with shorter telomeres in young adulthood, and a correlation between telomere length in early life and lifespan has been suggested. However, empirical studies evaluating these phenomena have not been performed, and the mechanism of stress‐induced telomere shortening remains unknown. Since the level of tumour necrosis factor α ( ATFactivating transcription factorChIPchromatin immunoprecipitationdATF2Drosophila ATF2ROSreactive oxygen speciesTNF‐αtumour necrosis factor‐αWTwild‐typeTelomeres consist of tandem TTAGGG repeats and maintain the integrity of chromosome ends during cell division Drosophila ATF2 (dATF2) and yeast S. pombe Atf1, which are orthologs of ATF7, contribute to heterochromatin formation Activating transcription factor‐7 (ATF7) is a vertebrate member of the ATF2 subfamily of transcription factors, which belong to the ATF/CREB superfamily of proteins. The ATF2 subfamily of transcription factors is characterized by the presence of phosphorylation sites for stress‐activated protein kinase p38 and b‐ZIP DNA‐binding domains In this study, the association between maternal psychological stress exposure during pregnancy and telomere shortening in adult life was assessed using TNF‐α, which is induced in peripheral blood cells by various psychological stresses Atf7−/−C57BL/6 mice, which were described previously Congenic wild‐type (WT) and −1 weight) was intraperitoneally administered to pregnant mice daily between E2.5 and E18.5. DNA was prepared from blood cells or various tissues of 1–6‐week‐old male mice, and used for Q‐PCR to measure telomere length.TNF‐α  was used to remove red blood cells.Different tissues were isolated from mice at different times, and DNA was isolated using a DNeasy Blood & Tissue Kit . For blood samples, a red blood cell lysis buffer . The DNA was sheared into approximately 500 bp fragments by sonication and diluted 10‐fold with dilution buffer . Immunoprecipitation was performed overnight at 4 °C with anti‐ATF7 (2F10), anti‐H3  and anti‐H3K9me3  antibodies. Normal anti‐mouse IgG antibodies were used as controls. Immunocomplexes were recovered using Dynabeads® Protein G beads , and then incubated at 65 °C in 130 μL of IP elution buffer  to release proteins. The DNA was further purified using a QIAquick PCR Purification Kit (Qiagen) and eluted in 100 μL of elution buffer. Eluted DNA samples were blotted onto Biodyne B membranes. Membranes were hybridized overnight in hybridization buffer containing 7% sodium dodecyl sulphate, 1% BSA, 0.5 m phosphate buffer (pH7.2), and 1 mm EDTA at 42 °C with the following 32P‐labelled oligonucleotide telomeric probes: G24 probe, [TTAGGG]4; C24 probe, [CCCTAA]4. Signals were visualized and quantified using Image Analyzer 2500 .Chromatin immunoprecipitation (ChIP) assays were carried out as described previously t‐test.Results are presented as means ± standard deviation (SD). The statistical significance of differences between groups was determined with the Student's −1, which induces inflammatory process but not developmental defect, because we used TNF‐α treatment as the method which mimics psychological stress. Injection of TNF‐α into mice was previously shown to induce the expression of multiple genes Cxcl10, Cxcl9, NF‐κB p65 and Cd14 were significantly induced in the liver in response to administration of 20 μg·kg−1 of TNF‐α to pregnant mice (data not shown). Pregnant mice received daily intraperitoneal injections of 10 or 20 μg·kg−1 of TNF‐α or saline from E2.5 to E18.5, and DNA was prepared from the blood cells of newborn mice at 1 week after birth. Assessment of telomere length by Q‐PCR showed telomere shortening in the blood cells of 1‐week‐old mice exposed to 10 or 20 μg·kg−1 of TNF‐α from E2.5 to E18.5  male and female mice were mated, and pregnant mice received daily intraperitoneal injections of 20 μg·kg−1 of TNF‐α from E2.5 to E18.5. Telomere shortening was not observed in any of the tissues examined in 1‐week‐old Atf7−/− offspring . Therefore, we examined whether TNF‐α treatment during E2.5–E18.5 induced the release of ATF7 from telomeres in splenocytes of 1‐week‐old mice. Splenocytes were prepared from 1‐week‐old mice exposed to 20 μg·kgice Fig. A, suggesin utero TNF‐α treatment reduced the level of H3K9me3 on the telomeres of splenocytes from 3‐week‐old mice  in the splenocytes of 3‐week‐old mice. The results of similar ChIP/Slot‐blot hybridization experiments using anti‐H3K9me3 indicated that ice Fig. B.In utero TNF‐α treatment‐induced telomere shortening was observed only in blood cells in 3‐week‐old mice, indicating that telomere shortening was retained for approximately 1 month. The TNF receptor, p38, and ATF7, which are involved in TNF‐α‐induced and ATF7‐dependent telomere shortening, are not only expressed in blood cells, but also in other tissues, indicating that this specificity is not due to tissue‐specific expression of these factors. The mechanisms underlying the tissue specificity of telomere shortening remain unclear; however, the fact that the lifespan of blood cells is approximately 1 month after differentiation from hematopoietic stem cells could be important.An epidemiological study indicated that exposure to stress during intrauterine life is associated with shorter telomere length in young adulthood In utero TNF‐α treatment‐induced telomere shortening detected at 3 weeks after birth was lost at 6 weeks after birth in tissues and blood cells. TNF‐α induces ATF7 phosphorylation by p38, which may cause telomere shortening. On the other hand, TNF‐α induces NF‐κB p65 nuclear translocation, which promotes the translocation of telomerase from the cytosol to the nucleus, leading to telomere lengthening in utero TNF‐α treatment reduced the level of H3K9me3 on the telomeres of splenocytes from 3‐week‐old mice. Given that Suv39 h1/2 mutations induce telomere lengthening in utero TNF‐α‐induced telomere shortening, as observed at 6 weeks after birth in this study.The results of an epidemiological study indicating that stress exposure in intrauterine life is associated with shorter telomere length in young adulthood in utero TNF‐α treatment‐induced telomere shortening in specific tissues of infant and young adult mice in an ATF7‐dependent manner. The present findings may improve our understanding of the mechanism of stress‐induced telomere‐related human diseases.In summary, we found that BL, TM and SI conceived and designed the experiments. BL and TM performed the experiments. BL, TM and SI analysed the data, and wrote the manuscript.Table S1. Sequences of oligonucleotides and primers used.Click here for additional data file.


Tumor necrosis factor (TNF) is a key signaling molecule orchestrating immune and inflammatory responses and possesses the capacity to trigger apoptotic as well as necroptotic cell death. Apoptotic cell death elicited by TNF has been demonstrated to engage pro-apoptotic Bcl-2 family proteins, most prominently the BH3-only protein Bid, a key substrate of caspase-8, the key effector protease downstream of TNF receptor I. Most recently, the BH3 domain-containing protein Bad (Bcl-2-antagonist of cell death) has been shown to be rate limiting for TNF-mediated cell death, suggesting possible synergy with Bid, but genetic analyses presented here demonstrate that it is dispensable for this process. TNF can also trigger cell death by engaging TNF receptor I (TNFRI) along apoptotic or necroptotic pathways, which is believed to limit or exacerbate inflammatory responses, respectively.1 Excessive TNF release during fulminant bacterial infection can lead to a cytokine storm, vascular leakage driven by TNF-mediated endothelial damage, septic shock and death.2 The pro-death and inflammatory potential of TNF therefore need to be tightly regulated and cells have developed highly specialized means to control the biological outcome of TNFRI activation.Tumor necrosis factor (TNF) is a pleiotropic cytokine critical for inflammation during innate and adaptive immune responses, orchestrating cytokine expression via activation of the canonical NF-4 Its pro-death function is thereby constrained by different means but most prominently by NF-κB-dependent transcription of survival genes, such as Bcl-x, XIAP (X-linked inhibitor of apoptosis protein), cIAPs (inhibitor of apoptosis proteins) or c-FLIP (cellular FLICE inhibitory protein), a dimerization and activation partner of procaspase-8 that is rate limiting for TNF-driven apoptosis and also prevents RIPK1/RIPK3-dependent necroptosis that can ensue when caspase-8 is inhibited.5Recent studies define a delicate network of linear and branched ubiquitylation/deubiquitylation-controlled signaling events downstream of TNFRI as critical for the decision between life and death upon TNF stimulation.6 TNF has also been shown to trigger NF-κB-dependent transcriptional activation of Puma and deficiency of this BH3-only protein can also provide protection from hepatocyte killing upon systemic TNF administration.7 Together these findings show that mitochondrial apoptosis, controlled by the Bcl-2 family, can be a critical event in pathological hepatocyte cell death in response to TNF.To promote death, TNFRI signaling can also engage pro-apoptotic members of the Bcl-2 family, for example, the BH3-only proteins Bim, Bid, Puma and Bad (Bcl-2-antagonist of cell death). Bid is processed and thereby activated by caspase-8 in many cell types and systems. Bim activity, on the other hand, appears to be controlled by TNF-driven JNK-mediated phosphorylation and both, Bid and Bim, contribute to T-cell-driven TNF-dependent liver toxicity.8). More recently, Yan et al.9 showed that the activity of the BH3 domain-containing protein Bad is repressed downstream of TNFRI activation by direct IKK-mediated phosphorylation on Ser-26. This study showed that mouse embryonic fibroblasts (MEFs), derived from Ikbkb−/− mice, that are deficient in IKKβ/IKK2, where Bad expression was also repressed by RNA interference, or MEFs from Bad−/− mice that were pre-treated with the IKK inhibitor PS-1145, showed reduced caspase-3/7 activity, delayed PARP cleavage and strongly reduced cell death upon TNF treatment. Similar observations were made in primary thymocytes and hepatocytes, pretreated with IKK-inhibitor prior to TNF stimulation. Most strikingly, Bad-deficient animals were protected from the lethal effects of hepatitis elicited by D-GalN priming and subsequent TNF injection. Together, these data provided a strong evidence for an NF-κB-independent anti-apoptotic function of the IKK complex, explaining, at least in part, the increased cell death susceptibility of IKKβ-deficient MEF over those lacking the NF-κB family members RelA plus cRel9 and supported a rate-limiting role of Bad in TNF-induced cell death signaling.Bad phosphorylation on residues Ser-112, -136, -155, has previously been shown to prevent mitochondrial translocation of Bad and restrain its pro-death function  was used as an unrelated cell death inducer. Cell death was assessed by Annexin V and 7-AAD staining and flow cytometric analysis. In contrast to published findings,9 we were unable to detect a survival difference between wt and Bad−/− thymocytes. In fact, we did not see a sensitization of thymocytes toward TNF killing by PS-1145 pretreatment. If anything, this inhibitor delayed spontaneous cell death of thymocytes in culture at later time points, while IKK-VII treatment sensitized thymocytes to spontaneous cell death in culture as well as TNF killing. However, wt and Bad−/− thymocytes responded at similar rates .First, we isolated thymocytes from wt or ar rates . Both inreatment . We concBad−/− cells, were equally sensitive to STS or IKK inhibition in the absence or presence of TNF. In contrast to the situation in thymocytes, PS-1145 sensitized MEF to TNF, in line with a prosurvival effect of NF-κB-regulated genes, and/or, modification of alternative direct targets by IKK in these cells. However, absence of Bad failed to protect from or delay cell death under conditions of IKK inhibition  liver enzyme, histological assessment or TUNEL staining of liver sections of these mice . A comparison between C57BL/6N and C57BL/6J mice did not reveal significant differences in survival upon GalN/TNF treatment (not shown). Genetic background differences, however, have been shown to impact on the activity of cell death related genes . Differences in MEF sensitivity might be explained, next to genetic background issues, by different modes of immortalization. Although we used six independently generated batches of SV40 large T antigen-immortalized MEF per genotype . Genotype-blinded animals, 8–12 weeks of age, were sensitized by administration of 700 mg/kg body weight of D-GalN  i.p. and 30 min later treated with 15 mg/kg body weight of hTNF  along the same route. Mice were monitored up to 12 h post TNF challenge, liver specimens were collected at the time of death and fixed for the preparation of histological sections. Blood was collected from the submandibular vein.The generation of μM L-glutamine , 50 μM 2-mercaptoethanol, penicillin/streptomycin (Sigma) and 10% fetal calf serum (PAA Laboratories). Staurosporine (Sigma) was used at 100 nM, IKK inhibitors IKK-VII and PS-1145 were purchased from Sigma or Calbiochem , for reference purposes. Cells were pretreated with 10 or 25 μM of inhibitors for 2 h prior addition of 5 ng/ml or 25 ng/ml hTNF (Peprotech). Antibodies for western analysis were purchased from Cell Signaling .Thymocytes or MEF, derived from E13.5 embryos, were cultured in DMEM , 250 Liver lobes were fixed in 4% paraformaldehyde in PBS. Tissues were processed according to standard procedures, cut in 3 μm sections and stained in hematoxylin and eosin or were processed for TUNEL staining by immunofluorescence.μm, deparaffinized and rehydrated. For visualization of apoptotic cells via the in situ terminal transferase-mediated dUTP nick end-labeling method , rehydrated slides of tissue sections on cover slips were treated with 0.1% Triton X-100/0.1% sodium citrate buffer and dehydrated by graded series of alcohol and chloroform. The TUNEL reaction was carried out in a humidified chamber for 1 h at 37°C using recombinant terminal transferase and FITC-dUTP (Roche) as described.20 Counterstaining of nuclei was done with DAPI (Sigma).Liver tissue embedded in paraffin was cut at 3 P-value <0.05 was considered to be statistically significant.Two-way ANOVA with Bonferroni post-test was used for statistical analysis. A


Healthy People 2020 objective (IID-18) is to increase to 95% the proportion of children aged <6 years whose immunization records are in fully operational, population-based IIS (Immunization information systems (IIS) are confidential, computerized, population-based systems that collect and consolidate vaccination data from vaccination providers and provide important tools for designing and sustaining effective immunization strategies ,2. A Heaased IIS . The Natased IIS ,5. Two otive IID-8 is to iOf the 56 immunization program grantees  participated in an IIS in 2011. Child participation in IIS has increased steadily, from 63% in 2006 .Each year, CDC collects IISAR information on the percentage of grantees meeting 12 NVAC functional standards for IIS ,5. The iAmong the 54 grantees reporting in 2011, 32 (59%) included a field for each of the 18 data elements in their IIS. The most common data elements not included in IIS were birth order for multiple births only (nine grantees), mother’s middle name (eight grantees), and birth country (six grantees). Average completeness of NVAC core data elements for children aged <6 years ranged from 38% for mother’s middle name to ≥98% for six fields  .Vaccine manufacturer and vaccine lot number are two data elements recognized for their importance in IIS for vaccine inventory management and their potential to increase patient safety through identification of persons who were administered recalled vaccine and reporting of vaccine-associated adverse events. The proportion of IIS including fields for these data elements in IIS has been high. In 2006, 89% of IIS contained a field for recording vaccine manufacturer and 88% contained a field for lot number; these increased in 2011 to 98% and 100%, respectively. The completeness of data in these fields has increased from 37% for both in 2006 to 63% for vaccine manufacturer and 60% for lot number in 2011 .Healthy People 2020 objective for child participation to increase to 95% the proportion of children aged <6 years whose immunization records are in fully operational, population-based IIS, and completeness of the vaccine fields remains suboptimal. Maximal child participation and complete records are needed to fully realize the benefits of IIS, which include clinical decision support, vaccination coverage reports, support for vaccine-preventable disease outbreak response, vaccine inventory management, and the ability to generate reminder and recall messages. Developing and promoting these beneficial tools that IIS offer to providers can encourage provider participation in IIS. If provider participation in IIS increases, child participation increases.Child participation in IIS and completeness of data for vaccine manufacturer and lot number in IIS increased steadily from 2006 to 2011. Despite this progress, challenges remain to meeting the Several ongoing initiatives are expected to increase participation in IIS and improve the completeness and accuracy of data contained in IIS, including enhanced interoperability between IIS and electronic health records (EHRs), the use of IIS to interface with VTrckS, CDC’s vaccine tracking system for publicly purchased vaccine, and the use of 2D barcodes to facilitate recording vaccine information in EHRs and IIS. VTrcks and interoperability initiatives have been reported on previously ,7. The 2In September 2011, CDC initiated a 2D vaccine barcode pilot projectWhat is already known on this topic?In 2010, 82% of the 18.8 million U.S. children aged <6 years participated in immunization information systems (IIS).What is added by this report?In 2011, 84% (19.2 million) of U.S. children aged <6 years participated in IIS. Among IIS grantees, completeness of data for vaccine manufacturer and vaccine lot number has increased since 2006, but remained suboptimal at 63% and 60%, respectively, in 2011. In 2011, CDC initiated a vaccine barcoding pilot project to determine best practices for labeling and tracking vaccines using 2D barcodes that include vaccine product information, lot number, and expiration date.What are the implications for public health practice?Widespread adoption of 2D vaccine barcoding among manufacturers and providers has the potential to increase completeness and accuracy of IIS data and improve patient safety. More complete and accurate data elements might provide an additional incentive for providers to participate in IIS, which in turn can increase child participation in IIS.Documenting vaccine product information and lot number is required by the National Childhood Vaccine Injury Act of 1986, and the American Academy of Pediatrics recommends documenting vaccine expiration date to improve patient safety. To report vaccine product data to an IIS, providers must either type them into an EHR, which transmits data to the IIS, or type the information into the IIS directly. In addition to recording data electronically in an EHR or IIS, some providers also record these data by hand in paper records. Recording information by hand and duplication of entries takes time and resources and increases the likelihood of data entry errors.Use of a 2D barcode on vaccines could allow for rapid, complete, and accurate capture of these vaccine product data by a barcode scanner that could transfer the information to EHRs and IIS. A CDC assessment of the impact of 2D barcode for vaccine production, clinical documentation, and public health reporting and tracking analyzed the expected costs and benefits of barcode use by immunization providers. The assessment found that for every $1 expended, $2.70–$2.80 in benefits were expected to accrue from 2011 through 2023 (The findings in this report are subject to at least two limitations. First, data from the IISAR were self-reported and self-validated. Second, because two of the 56 grantees did not report data during the period of data collection, the nationwide IIS participation rates for children aged <6 years and completeness of core data elements might be underestimated or overestimated.Healthy People 2020 objective and NVAC goals.As with other technological advances, adoption of 2D barcode technology for recording vaccination information has the long-term potential to improve vaccine safety monitoring and inventory management, reduce staff time spent manually capturing vaccine data, decrease costs for vaccine stakeholders, and enhance the completeness and accuracy of vaccination information in electronic medical records and IIS. The barcode effort is one of many recent advances in health-care technology that should have the added benefit of reducing the reporting burden on providers while improving the quality of data in IIS. These advances and the resulting increase in data quality might lead to increased vaccine provider, and therefore child, participation in IIS, and achievement of the