The recovery of motor function in rats is inhibited following contusion spinal cord injury (cSCI). However, the mechanism of tumour necrosis factor α (TNF-α) in motor function after cSCI associated with peroxiredoxin 6 (PRDX6) remains unknown. We randomly divided rats into four groups: sham, cSCI, vector and lentivirus mediating TNF-α RNA interference (TNF-α-RNAi-LV) group. The Basso, Beattie, Bresnahan (BBB) scale was used to evaluate motor function. Real-time quantitative PCR (qRT-PCR) and western blotting were used to detect the expression of TNF-α and PRDX6, which were located in neurons using immunohistochemistry (IHC) and immunofluorescence. Subsequently, lentiviral-mediated TNF-α was used to determine the role of TNF-αand the relationship of PRDX6 and TNF-α in cSCI. After cSCI, the motor capability of hind limbs disappeared and was followed by recovery of function. IHC analysis indicated that TNF-α and PRDX6 were primarily located in spinal cord neurons. TNF-α interference significantly improved neural behaviour and increased expression of PRDX6. Our study suggests that inhibition of TNF-α can promote the recovery of motor function. The underlying mechanism of TNF-α-promoted motor function may be connected with the up-regulation of PRDX6. This provides a new strategy or target for the clinical treatment of SCI in future. Spinal cord injury (SCI), a type of serious trauma of the central nervous system, can eventually lead to motor and sensory dysfunction below the level of injury134672+-independent phospholipase A2 (iPLA2) activity to protect against oxidative stress and to prevent cells from peroxidation leading to membrane injury18192+ homeostasis2O2TNF-α, an inflammatory factor produced by astrocytes and microglia after SCI910111216In this study, we used the lentivirus-mediated RNA interference to suppress the expression of TNF-α (TNF-α-RNAi-LV) and attempted to find the possible association of TNF-α and PRDX6 and explore its mechanism in SCI. We aimed at providing new targets for the clinical treatment of SCI.The experiments were conducted with a total of 282 adult female Sprague-Dawley (SD) rats , aged 12 weeks, with a body weight of 210 ± 10 g. They were randomly divided into groups of varying number for the following procedures.−3 N (0.10 kg × 0.03 m × 9.8 N/kg). The sham group and vector group animals underwent identical surgical dissection and exposure, but did not undergo injury to the spinal cord.Contusion spinal cord injury (cSCI) models were produced using a modified weight-dropping device. Briefly, after the animals were deeply anaesthetised with sodium pentobarbital , they were fixed in a supine position and underwent a partial laminectomy of T9. A contusion cSCI was produced using a patented effect cSCI device (Patent number: CN200820141443) with an impact force of 3 × 10After the surgery, the rats were maintained in an isothermic cage until their recovery. Then, they were transferred to a bacteria-free biologically clean room set on a 12-hour light/dark cycle and provided with food and water. The bladders of the injured animals were manually emptied twice daily until normal function returned.The usage of animals and experimental protocol in the ethics has been confirmed by a named institutional and licensing committee of ethics in Sichuan University. All experimental procedures were conducted in accordance with the guidelines outlined in the National Institute of Health (NIH) Guide for the Care and Use of Laboratory Animals (1996) and were approved by Institute of Neurological Disease, Translational Neuroscience Center, West China Hospital, Sichuan University.The shRNA fragment with the highest interference efficiency was provided by GeneCopoeia  and the TNF-α lentiviral expression vector used to inhibit TNF-α was constructed. After this, TNF-α expression vector (5 μg) and viral packaging vectors  were co-transfected into 293 cells to produce lentiviral particles, TNF-α-RNAi-LV. The viral supernatant was harvested at 48 hours post-transfection and filtered through a 0.45 μm cellulose acetate filter. Then, the 5 ml cell supernatant containing lentivirus was centrifuged . The precipitate was re-dissolved in 500 μl PBS. Finally, the lentivirus was frozen at −80°C.Injection of TNF-α-RNAi-LV and vector into the spinal cord in vivo was performed within 48 hours before the cSCI. Briefly, the rats were deeply anesthetised with sodium pentobarbital (35 mg/kg), fixed in a supine position, and then underwent a partial laminectomy to expose T9 of the spinal cord. An equal volume of lentiviruses (5 μl per rat) was injected into two points on a coronal plane of the T9 segment of the spinal cord. The animals were maintained at the optimum temperature and housed in comfortable cages.Hind limbs motor function recovery was assessed using the Basso, Beattie, and Bresnahan (BBB) motor rating scaleThe animals were placed in a 1 m × 1 m field and allowed to move freely. The ipsilateral hind limb motor functions were rated with a 21 point scale. The measures were performed prior to surgery and at daily intervals thereafter.According to the BBB scoring system, neurobehavioral assessments were performed by five people mastering the experimental criteria but did not know the groupings. Four colleagues acquired points, one acted as a quality control (remove one score of four colleagues) and data recorder. Every rat was assessed over 4 minutes and scores were obtained from the three of four evaluators. The mean score was recorded as the final result.Once deep anaesthesia was achieved, transcardial perfusion for the models was performed at the corresponding survival time point. According to the requirements of the different experimental techniques, models were also divided into morphological staining groups, sham group (n = 6), cSCI group (n = 6), vector group (n = 6), and TNF-α-RNAi-LV group (n = 6).The former was firstly rinsed quickly with 0.9% normal saline (4°C), then slowly pre-fixed with 4% paraformaldehyde . After 48 h, post-fixed specimens (T9s were removed) were dehydrated, then embedded in paraffin and sectioned (5 μm); the latter was directly rinsed quickly with 0.9% saline (4°C) for samples (1 cm long injury segment) and then preserved at −80°C.Immunohistochemistry was performed using the SP Kit (Zhongshan Jinqiao Biotechnology Co. Ltd.) method. After sections were dried, dewaxed, hydrated and underwent high-pressure antigen repairing. Sections were incubated in 3% hydrogen peroxide (filtered 0.01 M PBS preparation) at 37°C for approximately 10 min and then incubated in 5% sheep serum (filter 0.01 M PBS preparation) at 37°C for 25 min. Subsequently, anti-TNF-α  and anti-PRDX6  antibodies were incubated overnight at 4°C. Sections were then washed with 0.01 M PBS according to SP kit instructions, then sequentially incubated with reagent B (37°C incubating for 15 min) and reagent C (37°C incubating for 15 min) at intervals with 0.01 M PBS. Then, diaminobenzidine (DAB) was used to develop the stain and 0.01 M PBS was used to terminate the reaction. The sections were then subjected to haematoxylin staining, separation, transblue, and dehydration. Sections were made transparent, and mounted. They were observed with a light microscope (Motic inverted microscope) with a computer-assisted video camera.After tissue was dewaxed, hydrated, high pressure antigen repaired and 5% sheep serum (filter 0.01 M PBS preparation) blocked 37°C for 25 min, Anti-TNF-α  and anti-PRDX6  were added overnight at 4°C. After they were washed, sections were incubated with fluorescence secondary antibody IgG /IgG  for 2 hours at 37°C. After DAPI mounting, the slides were observed using fluorescence microscopy. For double labelling, anti-GTAP/anti-NEUN was chosen to adapt to species of anti-TNF-α/anti-PRDX6.The spinal cord segments at rostral thoracic vertebrae 9 were obtained from the model group, specifically including sham group, 12 hour post operation (h), 1 day post operation (dpo), 3 dpo and 7 dpo.After carefully removing the spinal cord meninges, spinal cord segments were homogenised on ice in a lysis buffer containing 0.05 M Tris-HCL , 0.5 M EDTA (Amresco), 30% TritonX-100 (Amresco), NaCl (Amresco), 10% SDS , and 1 mM PMSF (Amresco) and then centrifuged at 12000 r/min for 30 min. The supernatant was obtained and stored at −80°C for later use. A 20 μl sample was loaded onto each lane and electrophoresed on 12% SDS-polyacrylamide gel (SDS-PAGE) for 2.5 h at a constant voltage of 120 V. The proteins were transferred from the gel to a nitrocellulose membrane blocked with PBS containing 0.05% Tween-20 (PBST) with 10% non-fat dry milk overnight at 4°C. The membrane was rinsed with PBST and incubated with the primary antibodies for anti-TNF-α (1:1000) and anti-PRDX6 at 4°C. The membrane was incubated with a HRP-conjugated goat anti-rabbit IgG  for 2 h at 37°C and was developed with an ECM kit and exposed against to X-ray film in a darkroom. Densitometry analysis for the levels of TNF-α protein was performed using the Bio-Gel Imaging system equipped with Genius synaptic gene tool software. β-actin  was used as an internal control.−ΔΔCt method.The T9 segments of the spinal cords were obtained from the model group. Total RNA was extracted from spinal cord tissue of rats using Trizol reagent (Invitrogen) according to the manufacturer's protocol and reversed transcription to cDNA with the First Strand cDNA Synthesis kit . qRT-PCR was then performed to determine the expression of genes. For qRT-PCR, the primer sequences were as shown in All data are expressed as the means ± SEM. The numbers of positive cells were compared using one-way analysis of variance (ANOVA). The significance of the difference between the groups was calculated followed by Fisher's least significant difference post hoc test. Probability values (P) of less than 0.05 were considered to represent significant differences.The recovery of hind limb function following spinal cord contusion in rats was recorded using the BBB scale. Rats exhibited immediately paraplegia with no hind limb movement after surgery compared to the sham rats, obtaining a BBB score of 21. After spinal cord contusion, the rats exhibited significant motor dysfunction in hind limbs on 1 dpo and 3 dpo compared with the sham rats (P < 0.05). BBB score increased significantly on 7 dpo and 14 dpo compared to 3 dpo (P < 0.05). However, no further increase in recovery of motor function was observed 28 dpo. Molecular network prediction methods were used to detect whether there is a relationship between TNF-α and PRDX6. The results showed that TNF- α and ATF4 are co-expressed, PRDX6 and ATF4 are co-expressed, TNF- α and ATF3 are co-expressed, and ATF4 and ATF3 have physical interactionsThe expression of TNF-α and PRDX6 mRNA was detected using real time quantitative PCR, Compared to the sham group, the level of TNF-α mRNA was significantly increased 6 h, 24 h and 14d after cSCI in the rats (P < 0.05) . ComparaImmunofluorescence was used to observe the location of TNF-α and PRDX6 in the rats with spinal cord injury. After cSCI, TNF-α was observed in spinal cord grey matter neurons and in neuroglial cells. TNF-α and PRDX6 was evidently co-localised in spinal cord neurons and neuroglial cells in the anterior horn of spinal cord .The TNF-α-positive cells were located in the cytoplasm of neurons of the anterior horn in the spinal cord . The numUsing western blotting, we measured whether there was a significant change in protein expression of TNF-α and PRDX6 in spinal cord grey matter after cSCI. The results indicated that protein expression of TNF-α was significantly increased on 1 dpo and then decreased on 3 dpo, but the value was still higher than in the sham group , which suggested the TNF-α-RNAi-LV had successfully transfected host cells compared to those of the control group .The BBB score after TNF-α-RNAi-LV administration was significantly higher than the vector group at 5 dpo, 7 dpo and 14 dpo . The difference existed until 28 dpo (p < 0.05) after cSCI. However, when comparing the number of PRDX6-positive cells between the TNF-α-RNAi-LV group and the vector group, the TNF-α-RNAi-LV group had higher counts than the vector group at 3 dpo, 7 dpo and 28 dpo, and the number of PRDX6-positive cells were significantly different . Therefore, it is assumed that TNF-α and PRDX6 were likely involved in certain cellular processes. To further verify this hypothesis, a series of experiments were conducted. The results of Q-PCR indicated that expression of TNF-α significantly increased and the expression of PRDX6 significantly decreased after the cSCI. However, the expression of PRDX6 was increased when lentiviral-mediated TNF-α RNAi vector was transduced into the spinal cord. These results suggested that inhibition of TNF-α can serve not only as a possible strategy for the treatment of SCI but also as an underlying mechanism related to the expression of both TNF-α and PRDX6.In the present study, the effect of TNF-α on motor function recovery of SCI rats was observed. We evaluated the motor function of the rats via BBB scores and detected the expression of TNF-α using Q-PCR, IHC and WB. The results indicated an increase in TNF-α and a decrease of motor function after SCI, whereas lentivirus-mediated inhibition of TNF-α promoted motor function recovery in rats after SCI. This suggested that TNF-α is a predominant mediator in the spinal cord after injury. The results of TNF-α interference provided further indication of the role of TNF-α in the injured spinal cord, indicating its potential usage in future clinic trials.TNF-α, as an anti-inflammatory factor, was associated with secondary injury after cSCI. TNF-α may aggravate the inflammatory response after cSCI and inhibit the recovery of spinal motor function as reported by numerous studies. TNF-α was increased rapidly after 1 hour in a model of traumatic spinal cord injuryIn the present work, we observed a decrease in PRDX6, which accompanied the increase of TNF-α after cSCI. As a result, it is assumed that a relationship between TNF-α and PRDX6 is probable. To illustrate the relationship between TNF-α and PRDX6, bioinformatics analysis was performed. The results of GeneMANIA analysis show that TNF-α and ATF4 are co-expressed, that PRDX6 and ATF4 are co-expressed, and that TNF-α and ATF3 are co-expressed. At the same time, ATF4 and ATF3 have physical interactions. Therefore, PRDX6 and TNF-α may have a direct relationship. M'DahomaCurrently, there are a number of reports on the signalling pathway of TNF-α. Binding to the homotrimeric TNF-α receptor can activate three major signalling cascades, including caspase-8, JNK, and NF-κB3637T.W. and X.Z. designed the experiments. X.Z. and L.S. wrote the main manuscript text. X.G. and D.J. prepared all figures. Z.Z. and T.L. conducted the western blot experiments. X.Z. and Y.R. worked the animal model. X.L., L.L. and X.H. conducted the behaviour tests. Q.X. and Z.Z. conducted the Q-PCR. X.H. and J.C. conducted the immunohistochemistry and immunofluorescence. All authors reviewed the manuscript.


Diagnostics is now indexed and searchable in PubMed. Further, the full-text versions of the papers are archived in PubMed Central. Since the launch of our journal in 2011, this has been a major goal for the editors and the publisher. With coverage in both the index and full text repository of the National Library of Medicine (NLM) articles published in Diagnostics will be more visible and hopefully reach more readers. The coverage will be from the first issue, so, additionally, papers published prior to coverage will become accessible via the databases.It is with great pleasure and proud that we can announce that The journal is currently developing in a most positive way with an increasing number of submissions, a total of 60 in 2015. A total of 37 papers were published in 2015 of which 43% were full-length research articles, 35% reviews and the remaining case reports or interesting images. Interesting images was introduced in 2015 [Diagnostics papers from our website was 37,377 in 2015, an increase of 139% compared to 2014. Citations are also increasing steadily with many papers being highly cited. Information on most cited papers may be found on the webpage of Diagnostics.The total number of downloads of With the continued support from the scientific community through submissions of high quality scientific papers and the extraordinary work performed by the editorial board, office staff and the publisher, I have no doubt that 2016 will be another successful year for our journal.


Cdc20) in anaphase, APCCdc20 is neither necessary nor sufficient for complete Acm1 degradation at the end of mitosis. An APC-independent, but 26S proteasome-dependent, mechanism is sufficient for complete Acm1 clearance from late mitotic and G1 cells. Surprisingly, this mechanism appears distinct from the canonical ubiquitin targeting pathway, exhibiting several features of ubiquitin-independent proteasomal degradation. For example, Acm1 degradation in G1 requires neither lysine residues in Acm1 nor assembly of polyubiquitin chains. Acm1 was stabilized though by conditional inactivation of the ubiquitin activating enzyme Uba1, implying some requirement for the ubiquitin pathway, either direct or indirect. We identified an amino terminal predicted disordered region in Acm1 that contributes to its proteolysis in G1. Although ubiquitin-independent proteasome substrates have been described, Acm1 appears unique in that its sensitivity to this mechanism is strictly cell cycle-regulated via cyclin-dependent kinase (Cdk) phosphorylation. As a result, Acm1 expression is limited to the cell cycle window in which Cdk is active. We provide evidence that failure to eliminate Acm1 impairs activation of APCCdh1 at mitotic exit, justifying its strict regulation by cell cycle-dependent transcription and proteolytic mechanisms. Importantly, our results reveal that strict cell-cycle expression profiles can be established independent of proteolysis mediated by the APC and SCF enzymes.Regulated proteolysis mediated by the ubiquitin proteasome system is a fundamental and essential feature of the eukaryotic cell division cycle. Most proteins with cell cycle-regulated stability are targeted for degradation by one of two related ubiquitin ligases, the Skp1-cullin-F box protein (SCF) complex or the anaphase-promoting complex (APC). Here we describe an unconventional cell cycle-regulated proteolytic mechanism that acts on the Acm1 protein, an inhibitor of the APC activator Cdh1 in budding yeast. Although Acm1 can be recognized as a substrate by the Cdc20-activated APC (APC Skp1/cullin/F-box protein complex (SCF) is well known for promoting the degradation of G1 cyclins, cyclin-dependent kinase (Cdk) inhibitors, and numerous other substrates, and is thought to be constitutively active. However, recognition of most SCF substrates requires their cell cycle-dependent phosphorylation anaphase-promoting complex (APC), or cyclosome, targets the chromosome segregation inhibitor securin, S and M phase cyclins, and many other proteins for degradation during mitosis and G1 Proper execution of the eukaryotic cell division cycle depends heavily on ubiquitin-mediated proteolysis, involving the conjugation of polyubiquitin chains to substrate proteins by E3 ubiquitin ligases and their subsequent recognition and degradation by the 26S proteasome In this report, we describe an unconventional proteolytic mechanism, independent of SCF and APC, that helps establish the strict cell cycle expression profile of the APC inhibitor Acm1 in budding yeast. Acm1 was identified several years ago by our lab as a tight binding partner and inhibitor of the APC activator Cdh1 ACM1 promoter is also cell cycle regulated as part of a large collection of genes turned on at the beginning of S phase Cdc20 during anaphase Acm1 expression is very tightly cell cycle-regulated. Acm1 protein is absent from G1 cells, appears around the onset of S phase, and rapidly disappears in late mitosis, after anaphase onset Cdc20 prior to mitotic exit cdc15-2 strain Cdc20 might not be sufficient for complete elimination of Acm1. To test this rigorously we had a polyclonal antibody raised against recombinant Acm1 so we could monitor endogenous Acm1 protein without addition of an epitope tag. Synchronized dbf2-2 cultures were released from α-factor-induced G1 arrest into fresh medium at 37°C so they would arrest in late anaphase, a point where APCCdc20 has been activated and has targeted its substrates for degradation (Acm1 was reported to be effectively eliminated via APCradation . As expe10xapc2Δ apc11Δ cdc20Δ cdh1Δ pds1Δ clb5Δ SIC1) engineered to survive in the complete absence of APC activity by deletion of the essential APC substrate genes CLB5 and PDS1, and 10-fold overexpression of the Cdk inhibitor Sic1 Cdc20 is not sufficient for complete elimination of Acm1 in late mitosis and that an APC-independent mechanism is also required. In the absence of APC activity this APC-independent mechanism is sufficient for complete Acm1 elimination in late mitosis and G1. We therefore set out to characterize the APC-independent proteolytic mechanism responsible for Acm1 degradation.Next, to definitively test for the presence of an APC-independent proteolytic mechanism acting on endogenous Acm1 protein in late mitosis and G1, we probed for Acm1 in extracts from synchronized cultures of a yeast strain (5A), which is unstable throughout the cell cycle 5A was strongly stabilized after MG-132 treatment in a GAL1 promoter shutoff/cycloheximide chase assay .To identify the proteins directly responsible for Acm1 ubiquitination and proteolysis, we systematically screened a collection of yeast strains individually lacking non-essential yeast E2 ubiquitin conjugating enzymes and E3 ubiquitin ligases  for effemponents . Howevermponents . We suspACM1 promoter we re-screened the entire collection of E2 and E3 deletion strains using the GAL1 promoter stability assay and directly monitored stability of untagged Acm15A with our Acm1 antibody. We also added strains lacking genes encoding predicted RING domains without known E3 activity and other recently verified E3 ligases . We conclude that Acm1 proteolysis by the APC-independent mechanism does not require any single E2–E3 modules, although we cannot rule out the possibility that multiple redundant E3 enzymes are capable of promoting Acm1 degradation or that an unknown E3 exists that was not tested.To avoid problems with expression from the highly regulated  ligases . Since iuba1-204 experiment described above, we analyzed Acm1 stability after overexpressing a mutant ubiquitin in which all lysines have been replaced with arginine . This mutant blocks poly-ubiquitin chain extension when conjugated to a protein doa4Δ strain, which has a defect in processing of ubiquitin precursors and therefore a lower level of endogenous ubiquitin for Ub-K7R to compete with  to the Acm1 C-terminus . Full length Acm1-ProtA was highly unstable. Removal of 42 amino acids or less had little effect on the rate of Acm1-ProtA degradation . StrikinNΔ52 protein was unstable in the absence of the C-terminal Protein A fusion . Thus, although Acm1NΔ52-ProtA is highly stabilized in G1-arrested cells, it is still effectively cleared by cells during mitotic exit. Possible explanations for this are discussed below.We next used the stabilized Acm1blotting . SurprisACM1 promoter is cell cycle-regulated, loss of Acm1NΔ52 as cells exit mitosis could be in part due to termination of ACM1 transcription. To test for defects in APCCdh1 function in cells expressing stabilized Acm1NΔ52 we expressed ACM1 and NΔ52acm1 alleles from the constitutive ADH promoter in a sic1Δ background. Either Sic1 or Cdh1 is sufficient for Cdk inactivation and mitotic exit in budding yeast, however loss of both is lethal. Therefore, activation of Cdh1 becomes critically important in sic1Δ cells. We observed a significant growth delay in sic1Δ cells constitutively expressing Acm1NΔ52 compared to wild-type Acm1, both in liquid culture and on agar plates , which catalyzes the rate-limiting first step in polyamine biosynthesis Several features of Acm1’s proteolytic mechanism reported here are reminiscent of ubiquitin-independent proteolysis. First, Acm1 degradation is insensitive to inhibition of ubiquitin chain assembly . Second,Additional work will be required to illuminate the biochemical mechanism by which Acm1 is specifically targeted to the proteasome in its dephosphorylated state. Many ubiquitin-independent proteasomal targets are thought to possess unstructured regions that mediate their direct recognition by the proteasome, much like a mis-folded protein NΔ52-ProtA in our GAL1 promoter stability assay . Finally, there may be some difference in conditions present in an α-factor arrest compared to mitotic exit that affect the turnover rate of Acm1 by the APC-independent mechanism.Despite the strong stabilization of Acm1Cdc20 in anaphase as described previously Cdc20 is not sufficient to completely eliminate Acm1 and is not actually required for Acm1 levels to oscillate during the cell cycle . The K0acm1 mutant allele was created in pBluescript by site-directed mutagenesis using the QuikChange Multi kit (Stratagene) and, amplified by PCR, and subcloned into the XhoI sites of pHPL117 SacII and XhoI sites of p415GAL1 to create pHLP329. The one lysine codon in the 3HA sequence of pHLP329 was altered to an arginine codon to create pHLP330. The K03HA-acm1 alleles in pHLP328 and pHLP330 were confirmed by DNA sequencing. pHLP298 was constructed by sub-cloning the 5A3HA-ACM1 sequence from pHLP209 into p415GAL1. Plasmids for Acm1 stability experiments were constructed by first ligating the PCR-amplified ZZ domain of Protein-A into the HindIII and XhoI sites of p415GAL1 and then ligating the appropriate ACM1 PCR fragments into the PstI and HindIII sites. All plasmid constructs were confirmed by DNA sequencing.All yeast strains used in this study, except those for screening ubiquitin system mutants, are listed in BAR1 or 50 µg/l for bar1Δ strains. For S phase arrest, solid hydroxyurea (Sigma Aldrich) was added directly to cultures at 10 mg/ml. For G2/M arrest, nocodazole (Sigma Aldrich) was added at a final concentration of 15 µg/ml from a 1.5 mg/ml stock in DMSO. For telophase arrest, strains harboring temperature-sensitive MEN mutants cdc15-2 or dbf2-2 were grown initially at 23°C and then shifted to 37°C. For all temperature sensitive strains, 23°C was used as permissive and 37°C as restrictive temperatures. For synchronous growth from G1 to telophase, α-factor treated cdc15-2 or dbf2-2 cells were released from arrest by extensive washing using a vacuum filtration device and resuspension in fresh medium pre-warmed to 37°C. Growth was continued at 37°C and samples were removed at the indicated times for analysis. Extracts of synchronized yBR135 and yBR159 cultures for analyzing Acm1 levels in the absence of APC activity Standard yeast growth conditions and media were used. For G1 arrest, α-factor peptide (GenScript) was added to cultures at a final concentration of 5 µg/ml for The following antibodies were used. Monoclonal anti-HA 12CA5 and anti-Myc 9E10 were from Roche Applied Science  and were used at concentrations of 0.5 µg/ml  and 1 µg/ml (1∶5000 dilution), respectively. Rabbit anti-glucose-6-phosphate-dehydrogenase (G6PD) and rabbit anti-Protein A were from Sigma  and were used at concentrations of 3 ng/ml  and 0.6 ng/ml , respectively. Rabbit anti-Clb2, used at 40 ng/ml  was from Santa Cruz Biotechnology . Horseradish peroxidase-conjugated donkey anti-rabbit and anti-mouse were from Jackson Immuno Research  and were used at concentrations of 80 ng/ml each. Immunoblots were developed using ECL plus  or Luminata (Millipore) detection reagents.ACM1 open reading frame was cloned into pGEX6P-1  for overexpression in E. coli as an N-terminal GST-fusion protein. The majority of GST-Acm1 is found in the insoluble fraction of bacterial cell extracts. Insoluble proteins were pelleted by centrifugation and resolubilized in 8 M urea, separated by SDS-PAGE and stained with Coomassie blue. The predominant band was GST-Acm1 and was excised and submitted to Pacific Immunology for polyclonal antibody production. Total Rabbit IgG was purified from serum using Protein A-agarose resin (Sigma Aldrich). Subsequently, anti-Acm1 antibodies were affinity purified using a GST-Acm1 affinity column generated by crosslinking recombinant GST-Acm1 to glutathione-agarose with disuccinimidyl suberate (Thermo Scientific). Acm1 antibody was eluted from the affinity column with 50 mM glycine pH 1.9 and immediately neutralized by addition of Tris-HCl pH 8.0 to 100 mM. Antibody specificity was tested by immunoblotting of yeast whole cell extracts using acm1Δ cells as a control. 1∶5,000 dilutions of the affinity purified antibody were used for all immunoblots.For Acm1 polyclonal antibody production and purification, the complete Quantification of chemiluminescent immunoblots was performed using a Bio-Rad Laboratories ChemiDoc XRS+ digital imager and ImageLab software. Images obtained following incubation of blots with chemiluminescent reagent were analyzed using the Image Lab software. Signals were normalized to the G6PD load control signal and then to the 0 min timpoint.GAL1 promoter shutoff/cycloheximide chase assays as described cim3-1 and pre1-1 pre2-2), cells were first arrested with α-factor at 23°C and then protein expression induced for 30 min prior to shift to 37°C. For ubiquitin mutant overexpression experiments, actively growing cells were arrested with α-factor in early exponential phase (OD600 = ∼0.4) and then CuSO4 was added to 100 µM to induce overexpression of mutant or wild-type ubiquitin from the CUP1 promoter. After 30 min 2% galactose was added to induce expression of the Acm1 or control proteins. To measure stability of endogenous Acm1, mid-exponential phase cells were treated only with 0.5 mg/ml cycloheximide to terminate expression. For MG-132 treatment, a pdr5Δ strain was used to maximize efficacy Measurements of protein stability were performed by NΔ52-ProtA were expressed from the natural Acm1 promoter on CEN plasmids  in a MET3-CDC20P background. Cultures were arrested in mitosis by Cdc20 depletion as described above and protein levels monitored over time by quantitative immunoblotting.For synchronized mitotic exit experiments, Acm1 or Acm15A mutant 5A from the ACM1 promoter were grown until mid-exponential phase and then treated with 2 µg/ml doxycycline for 2 hrs before processing for immunoblotting.Yeast strains individually lacking all known non-essential E2 ubiquitin conjugating enzymes, E3 ubiquitin ligases, and proteasome components were obtained from the Open Biosystems yeast deletion library . RelativCdh1in vivo was measured exactly as described previously Inhibition of APCsic1Δ cells transformed with either p415ADH, pHLP361 (ADHP-Acm1-ProtA) or pHLP363 (ADHP-NΔ52-ProtAacm1) as well as isogenic wild-type and cdh1Δ strains transformed with p415ADH were grown to mid-exponential phase selective media, then washed and resuspended in sterile TE to OD600 of 1.0. Serial 10-fold dilutions were spotted onto SD-Leu agar plates and grown at 37°C for 2 days.Cultures of 600 of 0.05 in selective media and growing in 96-well plates at 30°C with shaking in a BioTek Synergy 2 plate reader. Absorbance measurements at 600 nm were taken every 60 minutes.Growth rates were measured by diluting exponentially growing cultures to an ODRPN11-TAP fusion was used and for 20S proteasomes a strain expressing a PRE1-TEVProA fusion was used. To assay general activity of proteasome preparations, the fluorogenic substrate Suc-LLVY-AMC  was used as described 2, 10% glycerol, and 1 mM ATP). Fluorescence of the hydrolyzed AMC moiety was measured at excitation and emission wavelengths of 380 and 440 nm, respectively. Specific activities of purified 26S proteasome and 20S proteasomes were measured as described Proteasomes were purified from yeast soluble whole cell extracts by IgG-agarose (Sigma-Aldrich) affinity chromatography using a protocol described previously E. coli as described previously GST-Acm1 and 6His-Acm1 were purified from Figure S15A expression level.Screening of non-essential E2 conjugases and E3 ligases for effects on 3HA-Acm1(PDF)Click here for additional data file.Figure S25A stability in selected strains lacking non-essential E2 conjugases and E3 ligases.Measurements of HA-Acm1(PDF)Click here for additional data file.Figure S35A stability.Screening of non-essential E2 conjugases and known and putative E3 ligases for effects on Acm1(PDF)Click here for additional data file.Figure S4Acm1 is not degraded by purified 20S or 26S proteasomes.(PDF)Click here for additional data file.Table S1Yeast deletion strains screened for effects on Acm1 stabilization.(PDF)Click here for additional data file.Table S2Yeast strains used in this study.(PDF)Click here for additional data file.Table S3Yeast plasmids used in this study.(PDF)Click here for additional data file.


Results showed that sequence variation within CTL epitopes at the first time point (within six months of the estimated date of seroconversion) was consistent with most mutations being transmitted in the infecting viral strain rather than with escape arising within the first few weeks of infection. Escape arose throughout the first three years of infection, but slowly and steadily. Approximately one third of patients did not drive any new escape in an HLA-restricted epitope in just under two years. Patients driving several escape mutations during these two years were rare and the median and modal numbers of new escape events in each patient were one and zero respectively. Survival analysis of time to escape found that possession of a protective HLA type significantly reduced time to first escape in a patient (p = 0.01), and epitopes escaped faster in the face of a measurable CD8+ ELISpot response (p = 0.001). However, even in an HLA matched host who mounted a measurable, specific, CD8+ response the average time before the targeted epitope evolved an escape mutation was longer than two years.The existence of viral variants that escape from the selection pressures imposed by cytotoxic T-lymphocytes (CTLs) in HIV-1 infection is well documented, but it is unclear when they arise, with reported measures of the time to escape in individuals ranging from days to years. A study of participants enrolled in the SPARTAC  clinical trial allowed direct observation of the evolution of CTL escape variants in 125 adults with primary HIV-1 infection observed for up to three years. Patient HLA-type, longitudinal CD8+ T-cell responses measured by IFN-γ ELISpot and longitudinal HIV-1  The cytotoxic T-lymphocyte (CTL) arm of the immune response is thought to play a significant role in the control of HIV-1 infection. Mutations within the HIV-1 genome allow the virus to escape recognition by CTLs and so evade the immune response. These escape mutations have been well documented but observed waiting times to escape within an individual have ranged from days to years. Many studies describing CTL escape have taken a detailed look at a few patients. Our analysis is based on a cohort of 125 clinical trial participants with immunologic and viral sequence data taken at regular longitudinal time points within the first few years of infection. Results suggested that the majority of CTL-related mutations present early in infection had been transmitted in the infecting viral strain as opposed to arising in the new host due to selection pressure imposed by CTLs. Whilst the prevalence of CTL escape mutations in the dataset was high, the incidence of new escape was relatively low; around one third of patients did not drive an escape within the first two years. Patients possessing a ‘protective’ HLA genotype had a significantly shorter waiting time to first escape than those without. In ad of AIDS . However of AIDS , 9.+ T-cells or prevent antigen presentation by HLA class I molecules and so evade the immune response [env mutations that rise to fixation in the first few years represent viral adaptation to CD8+ T-cell responses [Many studies have demonstrated the capability of the HIV-1-specific CTL response to select for viral variants that escape recognition by CD8response –14. Indeesponses  whilst aesponses .+ T-cell response as well as pre-existence of compensatory mutations in sequences surrounding epitopes [CTL escape has been observed to occur throughout the different stages of HIV-1 infection, from soon after seroconversion , 17–20 tepitopes , 21, 27.epitopes –30. In tepitopes .gag, pol, nef and env genes. We used longitudinal viral sequence data covering the first three years of infection to count escape events in each of 46 epitopes in all available patient samples, and only considered periods when patients were off therapy. Approximately one third of patients drove escape in an epitope for which they were HLA-matched within their first year of infection and escape continued to be seen into the second year. However an estimated 33% of patients had still not driven any escape in their HLA-restricted epitopes across gag, pol or nef by the end of their second year off therapy. In looking at whether escape was clustered in patients, we found little evidence to suggest that this was the case. We also observed that time to escape for individual epitopes was faster in HLA-matched patients and in patients who were able to mount a measurable CD8+ T-cell response, and that patients with protective HLA alleles were more likely to have their first escape sooner.In this study we analysed the timing and extent of CTL escape in 125 patients across the HIV-1 gag, pol, env and nef), and IFN-γ CD8+ T-cell ELISpot data were collected longitudinally. Participants were followed for up to three years whilst off ART ; some participants initiated long-term ART before the end of the three years, upon reaching the trial clinical endpoint. The amount of data available at each time point is summarised in The cohort consisted of 125 treatment-naive, HIV-1 subtype B infected adults who were recruited soon after the estimated date of seroconversion  as part of the SPARTAC clinical trial and followed for up to three years see . Particiany amino acid variation in previously identified  escape sites within optimal defined epitopes in gag, pol, env or nef  the data comprised a total of 3831 epitope sequences from 122 patients in which we were able to look for variation within known escape sites. The range of times from seroconversion amongst these patients matched that of the whole cohort. Of these, 1235 contained escape mutations. However, this escape at baseline was, for almost all epitopes, equally prevalent in HLA-matched and HLA-mismatched hosts . This paed hosts . Furtherenv gene, the vast majority of epitopes in env  were already escaped at baseline, and hence whilst these were included in our analyses we were not able to comprehensively study incident escape in env. In the 2297 candidate epitopes, just 108 ‘escape’ events were observed in patients studied while off ART and only 37 of those were in epitopes in an HLA-matched patient. To see if there was evidence for a separate group of particularly fast escaping patients we compared the distribution of the number of escape events per host (in their HLA-restricted epitopes) with a random distribution, calculated using a Poisson process, and found no significant difference between the distributions . Due to the high variability of the ibutions . In factibutions  we used ibutions . Even afThis analysis is meaningful if patients with missing data would have had similar rates of escape to those observed for a full two years whilst untreated. We believe this to be a valid assumption and assess it in further detail later.Time to escape can be described either for each patient in a population  or for each epitope within each patient . By considering the first such escape event (in patients or in epitopes) the well-developed statistical tools of survival analysis become applicable. Since only 9% of patients have more than one incident escape event in an epitope for which they are HLA-matched  very litIn assessing the suitability of these analyses we also needed to address the question of whether the time at which patients are censored from the survival analysis (which occurs when they have no further sequence data) is independent of the rate at which they drive escape mutations. For some patients the fact that we were unable to obtain sequence data at later time points was a direct consequence of the fact that they had reached the trial primary endpoint (a CD4 cell count < 350 cells /μ l or commencing long-term ART) within the time frame of our observation period. Hence if, as some might hypothesise, escape events can drive clinical failure, it is possible that censored patients would have high rates of escape that we’d fail to observe.We looked in more detail at those patients who reached the trial endpoint  within our three-year observation period. An incident escape was observed in 6/20 patients for whom we had no sequence data after clinical failure compared to 7/24 patients who did have sequence data after clinical failure. In the 7 patients in the latter group who drove at least one escape event, there were just two cases where an escape mutation was first seen at the time point after clinical failure (see nef and gag (the genes for which the most data were available) were used to avoid underestimating the occurrence of escape due to missing data. This left 65 patients for this analysis. Note that this number includes both treated and untreated patients (range of previous ART duration 2–12 months). We also considered these patient groups separately  whilst an estimated 33% of patients did not drive any new escape in their HLA-restricted epitopes in 92 weeks off ART %). Note also that the slope of the curve is close to constant; there is no indication of a propensity for many incident escape events to arise early during the observation period and fewer later.Although there was no evidence for a separate group of patients with a high frequency of escape , there w+ T-cell ELISpot response to that epitope in that host . Even here the incidence of escape was low , and by extending the list of epitopes beyond the set of optimal defined epitopes see . Repeate+ T-cells or the antibody response. But understanding the impact of these alternative selective pressures is not necessary for assessing the concern that our methods are blind to CTL escape occurring in previously unidentified epitopes. We have previously shown that HLA-association studies favour the detection of rapid escape mutations. Hence any escape in unknown or poorly characterised epitopes that we have not looked in is likely to be slower than the time frame of this study and consequently not affect our conclusions [We acknowledge that even this extended epitope list is unlikely to be complete, and that the Los Alamos ‘A-list’ will evolve to include new peptides and HLA-restrictions , 41. Indclusions .Our definition of escape was also based on consensus viral sequence data; hence we were unable to detect the presence of any low frequency variants unlike with deep sequencing methods , 17. HowThe final aspect of our definition of escape to address is the use of HXB2 as our comparison sequence. Although there is no ‘gold standard’ comparator for this analysis, there were only four epitopes in which HXB2 differed from the ‘A list’ optimal at a documented escape site. We therefore re-ran the analysis re-classifying these four epitopes and found no significant difference in the distribution of baseline escape prevalence between HLA-matched and-mismatched hosts. We did identify 4 new incident escapes . Although the EVK escape variant is only poorly defined, its inclusion would mean that an estimated 25% of patients had not driven an escape by the end of 2 years off-therapy (as opposed to 33%).In conclusion, this study of a cohort from a randomised controlled trial, the proper design for assessing rates of escape across a population, reveals that escape is on average both less frequent and less concentrated in the first few months of infection than would be expected from recent intense studies of much smaller numbers of acutely infected individuals.The SPARTAC trial was approved by the following authorities: Medicines and Healthcare products Regulatory Agency (UK), Ministry of Health (Brazil), Irish Medicines Board (Ireland), Medicines Control Council (South Africa), and The Uganda National Council for Science and Technology (Uganda). It was also approved by the following ethics committees in the participating countries: Central London Research Ethics Committee (UK), Hospital Universitrio Clementino Fraga Filho Ethics in Research Committee (Brazil), Clinical Research and Ethics Committee of Hospital Clinic in the province of Barcelona, Spain, The Adelaide and Meath Hospital Research Ethics Committee (Ireland), University of Witwatersrand Human Research Ethics Committee, University of Kwazulu-Natal Research Ethics Committee and University of Cape Town Research Ethics Committee (South Africa), Uganda Virus Research Institute Science and ethics committee (Uganda), The Prince Charles Hospital Human Research Ethics Committee and St Vincent’s Hospital Human Research Ethics Committee , and the National Institute for Infectious Diseases Lazzaro Spallanzani, Institute Hospital and the Medical Research Ethics Committee, and the ethical committee Of the Central Foundation of San Raffaele, MonteTabor . All participants signed a written informed consent.3 (> 3 months after randomisation and confirmed within 4 weeks) or initiated long-term ART. This provided an immunological surrogate of clinical progression, but also allowed inclusion of those participants who commenced ART at CD4 cell counts greater than cells/mm3.The design of the SPARTAC trial is reported elsewhere . In brieThe participants studied here consisted of a sub-group 125 HIV-1 subtype B positive individuals of whom 42, 41 and 42 were randomised to ART-12, ART-48 and SOC, respectively. Peripheral blood mononuclear cells (PBMCs) were collected at regular intervals during the treatment period and follow-up. The sub-group of 125 participants was based on those individuals infected with subtype B HIV-1 for whom both sequence and ELISpot data were available, predominantly determined by specimen availability.pol, gag, nef and env genes were amplified separately using nested PCR reactions and primers described previously [Viral RNA was extracted from patient plasma  and the HIV-1 eviously , 37, 43.+ T-cell response was performed by interferon gamma ELISpot analysis in all subtype B patients, using methods described elsewhere [6 cells. ELISpot responses were measured at 0, 24 and 60 weeks post-recruitment.Quantification of CD8lsewhere . ResponsPatients’ HLA type was determined to the oligo-allelic level using Dynal RELITM Reverse Sequence-Specific Oligonucleotide kits for the HLA-A, -B and -C loci . To obtain four-digit typing, Dynal Biotech Sequence-Specific priming kits were used, in conjunction with the Sequence-Specific Oligonucleotide type.any amino acid variation in previously identified  escape sites within an ‘optimal’ defined epitope. Known escape sites were defined as within-epitope sites for which escape (E), calculated escape (CE), inferred escape (IE) or literature escape (LE) had been documented in the ‘CTL/CD8+ Epitope Variants and Escape Mutations’ HIV database table, as downloaded on 05/11/12 [A CTL escape mutation was defined as 05/11/12 . Any ami05/11/12  download+ T-cells, a comparison was made between the magnitudes of CD8+ T-cell ELISpot responses for the pre-defined optimal peptides with those for autologous epitope variants observed in the population at baseline. Variants for 7 Gag and 2 Pol epitopes were tested. In all cases except Gag EVD  the major variant(s) elicited a diminished response. In 6/9 cases responses to all ‘escape’ variants were reduced by at least 90%  of the Fisher’s exact test p-values for whether escape was independent of HLA-matching for that epitope. A permutation test was used to calculate the distribution of this mean under the null hypothesis; 2,000 random data sets were generated by permuting which patients the escape events occurred in for each epitope (i.e. there was no permutation between epitopes). The mean of the p-values for the observed data was then calculated and compared to the null distribution  across all epitopes in all patients and tp be the total observation time for all epitopes of a particular patient, p. Then, under the assumption that the average rate of incidence of escape in all patients is r = E/T, the probability of patient p driving i escape events is:tp = ×108.To account for missing data for , it was Note that this model does not impose the condition that only one escape is possible in each patient-epitope pair. We believe this to be a reasonable simplification given that the total number of escape events is small.2 goodness-of-fit test with 4 degrees of freedom  was used to test the null hypothesis that the observed data followed the expected distribution.A XSurvival analysis. For the survival analysis shown in Figs. S1 Text(PDF)Click here for additional data file.S1 FigHistograms show the distribution of the mean of the Fisher’s exact test p-values for independence of HLA-matching and escape in each epitope, under the null hypothesis that ‘escape’ is equally likely to be seen in HLA-matched and-mismatched hosts. (A) at baseline, (B) at 52 weeks. The red lines show the observed mean of the p-values. 2000 random simulations were used to obtain the null distributions. There is significant evidence to reject the null hypothesis at 52 weeks but not baseline, suggesting that little escape has already been driven before baseline.(EPS)Click here for additional data file.S2 FigEach patient-epitope pair was categorised according to if and when the patient reached the trial endpoint and how their time of clinical failure related to the last available sequence data point, as shown here diagrammatically. The three categories are (A) censoring may have resulted from clinical failure and there were no observations after failure, (B) censoring may have resulted from clinical failure but there was at least 1 further observation after failure, and (C) censoring is not related to clinical failure since long course ART was started after the last observed data point. Numbers on the right hand side represent the number of HLA-matched patient-epitope pairs and in brackets the number of distinct patients in each category. Note that three patients have epitopes in both A and B. One patient was removed as they were suspected to have been superinfected during the observation period.(EPS)Click here for additional data file.S3 FigAll HLA-matched host-epitope pairs are considered. Epitopes restricted by a protective HLA-allele as defined in (EPS)Click here for additional data file.S4 Figgag or nef, the two genes for which the most data was available, were not included so as not to skew the results due to lack of data. Time to first escape in an HLA-restricted epitope is plotted. These survival curves are very similar for the patients who have received no ART and who have received < 6 months of ART. Patients who have received > 6 mo ART were observed for less time off ART and hence the number of patients in this group with enough data to be included in the analysis is smaller (15 patients compared to 22 with no ART and 24 with < 6 mo ART). Therefore, while there appears to be a slower rate of escape in patients who have received > 6 months ART this difference is not significant (p = 0.24) and the effect of removing this group from the data is small Click here for additional data file.S5 Figgag or nef, the two genes for which the most data was available, were not included so as not to skew the results due to lack of data. (A) Time to first escape in an HLA-restricted epitope (solid line) is plotted along with 95% confidence intervals (dotted line). (B) Patients are split according to whether they have one of the more ‘protective’ HLA alleles or not. The set of beneficial alleles was taken to be B*58, B*27, B*57, A*26, B*51, A*11, B*14, B*18, B*08 . Having(EPS)Click here for additional data file.S6 Fig+ T-cell ELISpot response to them (light red) or not (dark red). As in -4, HR = 2.5) and then faster again if the host has a measurable CD8+ T-cell ELISpot response to that epitope . p-values and hazard ratios were calculated using a mixed effects model as in As with time to first escape in a patient, time to escape in epitopes was longer for epitopes in patients who have previously received > 6 mo ART, but there was no difference between epitopes in those who had received no ART and in those who had received < 6 mo ART. (A) Time to a first escape in epitopes within HLA-mismatched (dark blue) and-matched (light blue) hosts is shown. (B) Epitopes in HLA-matched hosts are split according to whether they have a measurable CD8(EPS)Click here for additional data file.S7 FigEpitopes are labelled where possible with their first three amino acids. Bars represent 95% binomial confidence limits (Agresti-Coull method). The dotted black line is y = x. Epitopes labelled in bold have a significantly higher proportion of escape in matched hosts than in mismatched hosts . Note that this plot is noisier that (EPS)Click here for additional data file.S8 Figgag or nef, the two genes for which the most data was available, were not included so as not to skew the results due to lack of data. (A) Time to first escape in an HLA-restricted epitope (solid line) is plotted along with 95% confidence intervals (dotted line). (B) Patients are split according to whether they have one of the more ‘protective’ HLA alleles or not. The set of beneficial alleles was taken to be all the HLA-A and-B alleles down to B*08 that are present in our data, taken from the ranking in [Patients who were either missing data for nking in . Having (EPS)Click here for additional data file.S9 Fig+ T-cell ELISpot response to them (light red) or not (dark red). As in -4, HR = 2.2) and then faster again if the host has a measurable CD8+ T-cell ELISpot response to that epitope . p-values and hazard ratios were calculated using a mixed effects model as in (A) Time to a first escape for epitopes in HLA-mismatched (dark blue) and-matched (light blue) hosts is shown. (B) Epitopes within HLA-matched hosts are split according to whether they have a measurable CD8(EPS)Click here for additional data file.S10 FigEpitopes are labelled where possible with their first three amino acids. Bars represent 95% binomial confidence limits (Agresti-Coull method). The dotted black line is y = x. Epitopes labelled in bold have a significantly higher proportion of escape in matched hosts than in mismatched hosts . (A) At baseline the majority of epitopes have escape at equal prevalence in HLA-matched and-mismatched hosts and the distribution of points is not significantly different from as would be expected if escape was equally likely to occur in HLA-matched and-mismatched hosts  (B) 52 weeks later within host evolution has resulted in a higher prevalence of escape in HLA-matched hosts for many epitopes, with the eight epitopes and the distribution as a whole lying significantly ‘below the line’ Click here for additional data file.S11 Figgag or nef, the two genes for which the most data was available, were not included so as not to skew the results due to lack of data. (A) Time to first escape in an HLA-restricted epitope (solid line) is plotted along with 95% confidence intervals (dotted line). (B) Patients are split according to whether they have one of the more ‘protective’ HLA alleles or not. The set of beneficial alleles was taken to be all the HLA-A and-B alleles down to B*08 that are present in our data, taken from the ranking in [Patients who were either missing data for nking in . Having (EPS)Click here for additional data file.S12 Fig-10, HR = 3.2). The effect of a response could not be analysed as CD8+ T-cell ELISpot responses were not measured for all of the extended list of epitopes. The p-value and hazard ratio was calculated using a mixed effects model as in Time to a first escape for epitopes in HLA-mismatched (dark blue) and-matched (light blue) hosts is shown. As in (EPS)Click here for additional data file.S1 TableNote that some sequences do not cover the whole gene and so the number given is an upper bound for the number of patients who have sequence data for any one epitope. For each patient ELISpot data was either available for all 181 optimal peptides across all four genes or not at all. Data was available in 115 patients at week 0, 109 patients at week 24 and 115 patients at week 60. The final column gives the number of patients with at least 2 time points of sequence data and ELISpot data at at least one of 0, 24 or 60 weeks.(PDF)Click here for additional data file.S2 Table+ T-cell responses to autologous variants were assessed and for which there was at least one HLA-matched patient with a positive response to the optimal epitope are shown. The top sequence for each epitope gives the pre-defined optimal ELISpot peptide, which is also the most prevalent variant in each case. The ‘Escape’ column records whether or not each variant is classified as escape according to our definition. The prevalence of each variant in the cohort at baseline and the proportion of patients with a particular variant who are HLA-matched for the epitope are also recorded. The final column gives the average ELISpot response to the variant (number of SFCs) in those that are HLA-matched and respond to the optimal, as a percentage of their response to the optimal.All epitopes for which CD8(PDF)Click here for additional data file.S3 TableAll p-values < 0.2 are given for a Fisher’s exact test with the alternative hypothesis that the proportion of escape in matched hosts is greater than that in mismatched hosts.(PDF)Click here for additional data file.S4 TableAll p-values < 0.2 are given for a Fisher’s exact test with the alternative hypothesis that the proportion of escape in matched hosts is greater than that in mismatched hosts.(PDF)Click here for additional data file.S5 TableSince a few patients did not have available sequence data at baseline, this table is similar but not identical to (PDF)Click here for additional data file.S6 TablePeptides that are also in the ‘A-list’ of optimal epitopes are shown in bold.(PDF)Click here for additional data file.


Advances in nanotechnology and nanomaterials have facilitated the development of silicon dioxide, or Silica, particles as a promising immunological adjuvant for the generation of novel prophylactic and therapeutic vaccines. In the present study, we have compared the adjuvanting potential of commercially available Silica nanoparticles  with that of aluminium hydroxide, or Alum, as well as that of complete and incomplete Freund's adjuvants for the immunisation of BALB/c mice with virus-like particles (VLPs) formed by recombinant full-length Hepatitis B virus core (HBc) protein. The induction of B-cell and T-cell responses was studied after immunisation. Silica nanoparticles were able to adsorb maximally 40% of the added HBc, whereas the adsorption capacity of Alum exceeded 90% at the same VLPs/adjuvant ratio. Both Silica and Alum formed large complexes with HBc VLPs that sedimented rapidly after formulation, as detected by dynamic light scattering, spectrophotometry, and electron microscopy. Both Silica and Alum augmented the humoral response against HBc VLPs to the high anti-HBc level in the case of intraperitoneal immunisation, whereas in subcutaneous immunisation, the Silica-adjuvanted anti-HBc level even exceeded the level adjuvanted by Alum. The adjuvanting of HBc VLPs by Silica resulted in the same typical IgG2a/IgG1 ratios as in the case of the adjuvanting by Alum. The combination of Silica with monophosphoryl lipid A (MPL) led to the same enhancement of the HBc-specific T-cell induction as in the case of the Alum and MPL combination. These findings demonstrate that Silica is not a weaker putative adjuvant than Alum for induction of B-cell and T-cell responses against recombinant HBc VLPs. This finding may have an essential impact on the development of the set of Silica-adjuvanted vaccines based on a long list of HBc-derived virus-like particles as the biological component. Although vaccines offer the most cost-effective solution to prevent infectious and possibly non-infectious diseases, the rate of success in the vaccine development is currently declining The existing vaccine adjuvants are comprised of different classes of substances including mineral salts, emulsions, and microorganisms-derived molecules However, application of Alum as the adjuvant is under controversial debate. Local reactions, swelling, indurations, cutaneous nodules and allergic reactions are found in the sites of Alum injections E.coli into mesoporous Silica particles induced antibody responses, which could be compared with the Alum- and incomplete Freund's adjuvant (IFA)-adjuvanted responses The rapid development of nanotechnology and nanomaterials raises new promises in the generation of novel adjuvant systems. Silicon dioxide (Silica) nanoparticles are one of the most favourable adjuvant candidates. The particles are solid with a diameter less than 100 nm. Adjuvanting capacities of different structures of Silica such as amorphous Silica E.coli are available at high levels of synthesis and quality standards Here, we evaluated the Silica nanoparticles as the possible adjuvants for the immunisation of mice with one of the most studied VLP models, recombinant hepatitis B virus (HBV) core (HBc) particles, which possess high proper immunogenicity on B-cell, T-cell, and cytotoxic T lymphocyte (CTL) level (for review see ayw2E.coli K802 cells producing HBc VLPs (wet weight 8 g) were resuspended in 4 volumes of the lysis buffer  and ultrasonicated at 22 kHz 8 x for 10 s. After clarification of lysate at 10,000×g for 30 min, soluble proteins were precipitated with ammonium sulphate at 10% saturation, at 4°C for 1 h, followed by centrifugation at 10,000×g for 30 min. VLPs in the supernatant were precipitated with ammonium sulphate at 35% saturation, at 4°C overnight, followed by centrifugation at 10,000×g for 30 min. The sediment was dissolved in 20 mL of PBS buffer with 0.5 M urea and 50 µM PMSF, and subjected to anion exchange chromatography on a Sepharose Q High Performance  XK26/20 column using AKTA Avant chromatography system . Elution was performed with buffers A  and B  until 100% of B in 300 mL at flow rate of 5 mL/min. Peak fractions were pooled and concentrated with 500 kDa MidGee Hollow Fiber Cartridge. Concentrated VLPs were subjected to size exclusion chromatography on a Sepharose 4 Fast Flow  120 mL column (16×600 mm) at a flow rate of 0.5 mL/min. The peak fractions were analysed using SDS-PAGE, native agarose gel electrophoresis, dynamic light scattering (DLS), and electron microscopy (EM). DLS analysis was performed on a Zetasizer Nano ZS instrument , in line with previously described VLP measurements HBc VLPs . Distilled water was used to prepare stock solutions of 20.0 mg/mL before immunisation. The stock solution was sterilised with UV irradiation for 2 h. An Alum adjuvant Alhydrogel was purchased from Brenntag Biosector (Denmark). Complete Freund's adjuvant (CFA) and Incomplete Freund's Adjuvant (IFA) were supplied by Statens Seruminstitut (Denmark).The Silica nanopowder, with the particle size of 10–20 nm, surface area  140–180 m230nm–75.8×OD260nm The percentage of the adsorbed VLPs was calculated against a control sample of HBc VLPs in PBS, without the added adjuvant.For adsorption experiments, 0.125 mg of the HBc VLPs in PBS were taken and combined with 0.5, 1.25, 2.5 or 5.0 mg of Silica or Alhydrogel in an Eppendorf tube and normalised with PBS to the final volume of 1.0 mL. The suspensions were gently mixed by overhead rotation at room temperature for 2, 24 or 48 h. For analysis of non-adsorbed HBc VLPs, the samples were centrifuged at 3,000×g, at room temperature for 20 min and protein concentration in the supernatants was measured by a NanoDrop analyser  at 230 and 260 nm. The HBc VLPs concentration was calculated by a formula: C (µg/mL)  = 183×ODFor DLS and EM analysis, 0.125 mg of the HBc VLPs and 1.25 mg of the appropriate adjuvant separately or in combination were used and normalised with PBS to the final volume of 1.0 mL and incubated at room temperature for 2 h. For DLS, the supernatants were analysed after centrifugation at 3,000×g, at room temperature for 20 min; for EM, samples were used without centrifugation.Pathogen-free, female BALB/c mice between 6-8 weeks of age were obtained from the Latvian Experimental Animal Laboratory of the Riga Stradins University and maintained under pathogen-free conditions in accordance with the principles and guidelines of the Latvian and European Community Laws. Five animals were used in each experimental group. The experimental protocol was approved by the local Animal Protection Ethical Committee of the Latvian Food and Veterinary Service (permission no. 55/15.03.2013).Mice were injected intraperitoneally (i.p.) or subcutaneously (s.c.) with 25 µg of HBc VLPs in PBS formulated with following adjuvants in a total volume of 0.2 mL : (a) 250 µg of Alhydrogel, (b) 250 µg of Silica, (c) with Complete Freund's adjuvant for the first injection followed by two injections with Incomplete Freund's Adjuvant (CFA/IFA) mixed with protein solution in 1∶1 ratio by volume, (d) with IFA for all three injections mixed with protein solution in 1∶1 ratio by volume and (e) with VLPs diluted in PBS. Animals were immunised on days 0, 14, 28 and bled on day 42. Mice were then sacrificed by cervical dislocation.For the T-cell proliferation assay and cytokine test, groups of BALB/c mice with five animals in each group were immunised s.c. with 25 µg HBc VLPs formulated as before (a) with 250 µg of Alhydrogel, (b) with 250 µg of Silica, or (c) with 250 µg of Alhydrogel supplemented with 10 µg of MPL , (d) with 250 µg of Silica supplemented with 10 µg of MPL, and (e) with 10 µg of MPL only. On day 10, mice were bled for an ELISA test; they were then sacrificed, and lymph node cells were obtained.For detection of anti-HBc antibodies, ninety-six-well plates  were coated with HBc VLPs  at 4°C overnight. After blocking with 1% BSA in PBS at 37°C for 1 h, serial dilutions of mouse sera were added to the plates and incubated at 37°C for 1 h. After washing 3 times with PBS containing 0.05% Tween-20, 100 µL horseradish peroxidase conjugated anti-mouse antibody  at a 1∶10,000 dilution was added per well. Following incubation at 37°C for 1 h, plates were washed and substrate OPD  was added for colour development. Optical absorbance measurements were performed with Multiscan (Sweden) at 492 nm. The end-point titers were calculated as the highest serum dilution that resulted in an absorbance value exceeding three times that of the negative control (serum obtained from non-immunised mice).An isotype-specific ELISA was performed for the detection of IgG1 and IgG2a subclasses of anti-HBc antibodies, using mouse monoclonal antibody isotyping reagent ISO2 , as secondary antibodies using peroxidase conjugate of monoclonal anti-goat/sheep IgG antibodies . The end-point titers were calculated as stated above.6 cells/mL) and co-cultured with HBc VLPs (10 µg/mL) in RPMI 1640 medium  at 37°C for 96 h in a humidified 5% CO2 atmosphere. 1 µCi of [3H]-thymidine  was added to each well for a final 18 h period of incubation, and [3H]-thymidine incorporation was measured by Liquid scintillation β-counter . The proliferative response was defined as a stimulation index (SI) calculated as the mean cpm corrected for background [3H]-thymidine incorporation in the absence of the HBc VLPs.Murine splenocytes were harvested using Red blood cell lysis buffer , lymphocyte suspensions were prepared , according to the manufacturer's instructions.The DLS analysis was performed to characterise the supernatants after low-speed centrifugation of suspensions of HBc VLPs before and after formulation with Silica and Alhydrogel . AnalysiTo compare the adsorption capacity of Silica and Alhydrogel for HBc VLPs, the level of non-adsorbed HBc VLPs was measured spectrophotometrically in the supernatants of appropriate HBc/adjuvant-formulated probes for several doses of adjuvants . Thus, fTo characterise components before and after the formulation, the EM analysis was performed . The EM Intraperitoneal and subcutaneous immunisations of BALB/c mice were performed with HBc VLPs adjuvanted in parallel by Silica and traditional adjuvants Alhydrogel, CFA and IFA alone . Immune Isotyping of the induced anti-HBc antibodies revealed a regular excess of IgG2a over IgG1 in all cases , which hThe lowest IgG2a/IgG1 ratio was found for HBc VLPs formulated in IFA or diluted in PBS; however, dependence on the immunisation route was also observed in both of these cases .To increase the induction of the cell-mediated immunity, HBc VLPs formulated with Silica or Alhydrogel were supplemented with MPL and both humoral  and cellThe IgG2a/IgG1 isotype ratios  were higin vitro, both Silica- and Alhydrogel-adjuvanted injections provided a definite increase in the T-cell proliferation as a result of the MPL contribution: 2.0 and 1.4 times, namely, SI = 11.0 and 9.5, respectively.The HBc-specific T-cell proliferation response  of mice A slight increase of the stimulation index was observed in immunisation of mice with HBc VLPs in PBS (SI = 3.6) compared to immunisation with MPL alone (SI = 2.7) and splenocytes obtained from naive mice (SI = 1.9).in vitro. While the level of IFN-γ was undoubtedly enhanced, the level of IL-2 synthesis did not demonstrate any increase as the result of the MPL addition to Silica- and Alhydrogel-adjuvanted HBc VLPs.Because the Th1 and Th2 subsets of T-helper cells can be characterised by a specific pattern of cytokines produced, the levels of the Th1-characteristic IFN-γ  and IL-2The main goal of the present study was the evaluation of the adjuvant properties of the commercially available Silica nanopowder, with an original particle size of 10–20 nm, to provide a putative vaccine with high humoral and cell-mediated immune response in comparison to the traditional Alum, CFA and IFA adjuvants.The role of the biological component of the vaccine is assigned here to HBc VLPs, which belong to the most studied VLP carriers and appear as a basis for a long list of promising vaccines against hepatitis A and B, anthrax, influenza, malaria, borreliosis, cattle theileriosis, infectious bursal and many other diseases  as a shell, with a diameter of approximately 210 nm, was proposed recently as a promising adjuvant for the delivery of a DNA vaccine Noteworthy, SiO


In vitro study of the PDAPEI/pDNA complex revealed a lower cytotoxicity and higher transfection efficiency when transfecting TNF-α shRNA to macrophages by significantly down-regulating the expression of TNF-α. Moreover, the complex was extremely efficient in decreasing the severity of arthritis in mice with collagen-induced arthritis. PDAPEI delivered TNF-α shRNA has great potential in the treatment of RA.Small interfering RNA (siRNA) is an effective and specific method for silencing genes. However, an efficient and non-toxic carrier is needed to deliver the siRNA into the target cells. Tumor necrosis factor α (TNF-α) plays a central role in the occurrence and progression of rheumatoid arthritis (RA). In this study, we pre-synthetized a degradable cationic polymer (PDAPEI) from 2,6-pyridinedicarboxaldehyde and low-molecular-weight polyethyleneimine  as a gene vector for the delivery of TNF-α shRNA. The PDAPEI/pDNA complex showed a suitable particle size and stable zeta potential for transfection.  Rheumatoid arthritis (RA) is a chronic systemic autoimmune disorder with complex causes, which leads to cartilage loss and synovial inflammation , 2. ManyAnti-TNF-α antibodies has shown its efficacy in RA and is widely used clinically , 10. HowHerein, we investigated TNF-α shRNA delivered by PDAPEI for the treatment of RA. We examined its characteristics, cytotoxicity, transfection efficiency, and gene expression on macrophages. PDAPEI/pDNA was also used to treat mice with collagen-induced arthritis (CIA), and the therapeutic efficacy was evaluated using histological examination.Polyethylenimine 1.8 kDa and 25 kDa and anhydrous ethylene dichloride were purchased from Sigma-Aldrich. 2,6-PDA was purchased from TCI (Shanghai) Development Co., Ltd. Cellulose membranes  were purchased from Thermo Scientific. Poly (ethylene glycol) (PEG) standards kit  was purchased from Polymer Standards Service GmbH. Water was purified using a milli-Q instrument (Millipore). All the reagents were used without further purification. For TNF-α gene silencing, Plasmid DNA encoding green fluorescence protein (GFP) and mouse TNF-α shRNA were constructed by Bioroot Biology  according to a previous report that used the following sequence: 5′-GACAACCAACUAGUGGUGCdTdT-3′. The RAW 264.7 macrophage cell line was purchased from the cell bank of the Chinese Academy of Sciences .First, 1 mmol PEI 1.8 kDa and 20 ml anhydrous ethylene dichloride solution were mixed until completely dissolved. Then, 2 mmol PDA in 20 ml anhydrous ethylene dichloride solution was added and stirred for 48 h at room temperature. After removing the solvent by evaporation, the residue was dissolved in deionized water and dialyzed through the cellulose membrane (<10 k). Then, following lyophilization, we were left with the yellow polymers, referred to as PDAPEI.PDAPEI/pDNA complexes were prepared at different weight/weight (w/w) ratios of 1–50. First, PDAPEI was dissolved with deionized water (2 mg/ml) and pDNA (2 mg/ml). Then, the PDAPEI solution was diluted to different concentrations and added rapidly into the pDNA solution. Next, the solutions were incubated at room temperature for 30 min, and PEI/pDNA complexes and naked pDNA were simultaneously prepared as controls.Agarose gel electrophoresis assay was used to evaluate the condensation ability of PDAPEI/pDNA complexes at different w/w. The particle size and zeta potential of PDAPEI/pDNA complexes were measured using a particle size analyzer (Brookhaven Instruments) and a zeta potential analyzer  at different w/w ratios. Zeta potential across a range of pH  was measured to analysis its stability and degradation. The morphology of the PDAPEI/pDNA complexes was observed by Transmission electron microscopy (JEOL JEM 2010 system).The RAW 264.7 macrophage cell line was cultured in Roswell Park Memorial Institute-1640 (RPMI-1640) containing 10% fetal bovine serum, 100 IU/ml penicillin, and 10 μg/ml streptomycin at 37°C inside a 5% CO2 atmosphere.4/well) were seeded into 96-well plates and incubated for 24 h. Then, a 10-μl PDAPEI/pDNA solution (w/w from 1 to 50) was added into each well for an additional 4-h incubation. A cell counting Kit-8 (CCK-8) reagent was used to evaluate the cytotoxicity of the complexes. Briefly, 10 μl of the CCK8 reagent was added into each well and cultured for another 2 h and measured at 450 and 630 nm using multifunctional microplate reader (SpectraMax M3 Multi-Mode Microplate Reader). Similarly, PEI/pDNA complexes and PEI 1.8 kDa/pDNA were examined as controls.For the cytotoxicity of the PDAPEI/pDNA complex in RAW 264.7, cells (1.0 × 104/ml) were seeded into 12-well plates and cultured for 24 h. Next, the medium was replaced by solutions containing 200 μl of PDAPEI/pDNA complexes of different w/w ratios and 800 μl RPMI-1640 for an additional 4 h of incubation. Then, the transfection medium was removed, and 1 ml of RPMI-1640 (10% FBS and 1% antibiotics) was added for 48-h incubation. We also prepared PEI 1.8 kDa/pDNA as the negative control and PEI 25 kDa/pDNA (w/w ratio of 2) as the positive control. A fluorescent microscope  and flow cytometer  were used to quantify the GFP-positive cells.The constructed shTNF-α was encoded with a GFP for determining the transfection efficiency. RAW 264.7  for 4 h, PEI 1.8 kDa/pDNA and PBS was used as a negative control and PEI 25 kDa/pDNA (w/w ratio of 2) was used as a positive control. Then, the transfection medium was replaced with fresh RPMI-1640 (10% FBS and 1% antibiotics) and incubated for 24/48 h. ELISA measurement was applied to detect TNF-α expression in the supernatant according to the manufacturer’s instructions . Total RNA was extracted from the cells using RNeasy®Mini Kit  and reverse transcribed into cDNA using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). The cDNAs for TNF-α were amplified by RT-PCR under the following conditions: denaturation at 95°C for 10 s; 40 cycles of amplification at 95°C for 15 s, and 60°C for 30 s. The specific primers used to detect mouse TNF-α  were synthesized and purified by TAKARA, Inc. Then, the qPCR products were subjected to electrophoresis on 2% agarose gels and visualized with UV light.To evaluate the gene silencing efficiency of PDAPEI/pDNA in vitro study results 1 week after booster immunization. The treat injection was repeated once a week until 5 weeks when the therapeutic efficacy was analyzed.Healthy male DBA/1 mice were purchased from the Institute of Zoology, Chinese Academy of Sciences . All animals survived under specific pathogen-free (SPF) environment in a laboratory within the animal facility located at the school of pharmacy, Shanghai Jiao Tong University. All mice were housed under standardized laboratory conditions and monitored to observe changes in ordinary conditions and activities. Animal care and use were in accordance with the guidelines established by the Administration of Affair Concerning Laboratory Animals for Shanghai JiaoTong University, the National Institutes of Health Guide for care and Use of Laboratory Animals (GB14925-2010) and the Regulations for the Administration of Affairs Concerning Experimental Animals . The murine CIA model was prepared according to a previous study . Type IIArthritic severity was recorded based upon the clinical scores of all four paws. Clinical severity of arthritis for each paw was classified as follows: 0 , 1 (slight redness and/or swelling), 2 (pronounced edematous swelling), and 3 (joint deformity and rigidity). The total clinical score was calculated by averaging all four paws scores. Hind paw edema was also measured by digital Vernier caliper to determine the severity.Before the mice were sacrificed for histological examination, synovial fluid was collected from the knees to measure TNF-α by ELISA and western blot, as per the instructions of the manufacturer and our previous study.At 6 weeks after the first immunization, the knee joint tissues of each group were collected and fixed in fixative/decalcifier for safranin O/fast green staining. Anti-TNF-α immunohistochemical analysis was applied to each knee joint. Briefly, 5-μm-thick sections of frozen tissue blocks were stained with mouse monoclonal anti-TNF-α antibody  and peroxidase-conjugated goat anti-mouse immunoglobulin G (H + L) secondary antibody  for further detection.P < 0.05 being considered statistically significant.The statistical analyses were performed as the mean ± SD by one-way ANOVA with a value of *1H NMR) and Fourier transform infrared spectroscopy (FT-IR) were used to confirm the structure of PDAPEI. The chemical shifts of 1H NMR and transmittance attribution in FT-IR were in agreement and as had been desired. The average molecular weight of PDAPEI was 21,000 Da, as measured by gel permeation chromatography , the PDAPEI/pDNA showed a similar percentage of GFP-positive cells (w/w ratio from 1 to 10) and a significantly higher percentage of GFP-positive cells (w/w ratio of 20–50) Figure B.in vitro was measured by ELISA. As shown in Figure p < 0.05) and PDAPEI/pDNA group  48 h after transfection. To further determine the in vivo silencing effect of TNF-α shRNA transfected by PDAPEI, real-time RT-PCR was utilized to measure the TNF-α microRNA expression. The mean levels of TNF-α microRNA expression in the PDAPEI/pDNA group were 23% of those in the PBS control group 48 h after transfection . The levels of 11 in both the PEI 25 kDA/pDNA (103.56 ± 29.82 ng/L) and PEI 1.8 kDa/pDNA (93.78 ± 30.42 ng/L) groups were significantly higher than that in the PDAPEI/pDNA group, but lower than that in the saline control group (p < 0.05)  was used for ELISA and western-blot analysis. The level of TNF-α expression in synovial fluid of the knees was 212.45 ± 62.70 ng/L in the saline control group, and a substantial decrease in the production of TNF-α was found in the PDAPEI/pDNA group  molecules to inhibit the expression of specific gene homologs , 28. It in vitro study, TNF-α shRNA was successfully transfected into macrophages and its expression level was significantly decreased. PDAPEI/pDNA had significantly better TNF-α inhibition results than the other three groups because of its high transfection efficiency and low cytotoxicity. TNF-α participates in cartilage/bone degradation and promotes other the secretion of other cytokines (TNF-α plays a central role in the development and progression of RA and the main gene target for treatment . In our ytokines . Upon hiRheumatoid arthritis is a systemic autoimmune disease, characterized by non-organ-specific autoantibody production and chronic inflammation of the synovial tissues, leading to cartilage and bone destruction . Treatmein vitro and proved to have an excellent efficacy in decreasing the severity of arthritis in mice with CIA. PDAPEI delivery of TNF-α shRNA has great potential in the treatment of RA.In conclusion, our results have shown that PDAPEI is an efficient delivery vector for TNF-α shRNA into macrophages. PDAPEI/pDNA can significantly inhibit TNF-α expression JS and YC participated in its design, did tests, searched databases, extracted and assessed studies, and helped to draft the manuscript. SJ, KY, XL, and XZ participated in doing some tests. YO, CF, and WY participated in the conceptualization and design of data extraction and analysis, and wrote and revised the manuscript. All authors read and approved the final 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. The reviewer SH and handling Editor declared their shared affiliation, and the handling Editor states that the process nevertheless met the standards of a fair and objective review.


Vascular anatomy of the liver is subjected to many variations. The most common hepatic artery (HA) replacement is the right hepatic artery (RRHA). Variations of the HA are particularly important consideration when choosing the best surgical procedure or if radiological abdominal intervention is required. In this study, we evaluated the anatomical details of the RRHA origin.Retrospective investigation of clinical data from 1569 patients who underwent an abdominal MDCT was performed. The anatomy of RRHA origin was described based on four parameters measured: D—the distance between SMA origin and the RRHA origin, L—the lumen at the place of origin, AH—the origin angle from the SMA in horizontal plane, and AV—the origin angle from the SMA in vertical plane.RRHA arising from SMA was detected in 10.13 % of cases (159/1569) and its anatomy was subjected to variations. Mean (±SD) of parameters D, L, AH and AV was 27.34 mm ± 6.83, 3.29 mm ± 1.17, 97.27º ± 26.69 and 89.73º ± 20.81, respectively. Values of parameters D and L were significantly higher in males compared to females.Although radiologists are not always aware of the clinical significance of the RRHA origin, the evaluation of its anatomy is thought to help reduce the risk of inadvertent vascular injury, especially in pancreatoduodenectomy. Detection and evaluation of the RRHA does not necessarily require angio-CT examination. Our study demonstrated that the MDCT, the standard imaging modality for diagnosing the abdominal symptoms, is sufficient to provide the knowledge of the HA abnormalities. Vascular anomalies in the peripancreatic region include variations of the superior mesenteric artery (SMA), the celiac trunk (CT) or the hepatic artery (HA) . In normThe resection of the tumour is the treatment of choice for liver cancer ; howeverPrior to surgical intervention, the radiological evaluation of the operated area is initially performed using multi-detector computed tomography angiography (MDCT angiography) rather than conventional angiography , 22. TheThe study outlines the anatomical details of the right hepatic artery that takes its origin from the superior mesenteric artery. To the best of our knowledge, this is the first description to emphasize the variations existing in the place where RRHA originates.Retrospective investigation of clinical data concerning patients after abdominal MDCT in the Department of Radiology; Clinical Hospital No. 4 in Lublin, Poland, that were performed in 2012. The study consisted of 1569 patients (F:M = 829:740). The mean age of patients was 58.0 years (age range 16–96 years).Abdominal multi-detector CT exams were performed using a 64-row scanner , with 64 × 1.25 collimation, slice thickness of 1.25 mm, pitch 1.375:1, at 120 kV current and automatic mA adjustment. During examination 80 ml of non-ionic iodinated contrast was injected into the patients’ antecubital vein.n = 159) were selected from the examined population. Afterwards, they were analysed using the volume rendering technique. For the more detailed anatomical description of the RRHA origin, four precisely defined parameters were measured:Parameter D: the distance between SMA origin from the abdominal aorta and the place of RRHA origin Fig. ;Fig. 1ViParameter L: the lumen of the RRHA at the place of origin Fig. ;Fig. 2ViParameter AH: the origin angle of RRHA from the SMA in horizontal plane Fig. ;Fig. 3ViParameter AV: the origin angle of RRHA from the SMA in vertical plane Fig. .Fig. 4ViNext, the RRHA cases —transversing pancreatic head Fig. bType III: RRHA located within the superior mesenteric vein (SMV) groove Fig. cThe course of the RRHA in relation to the pancreas was analysed and three types of RRHA were distinguished as described by Jah et al. :Type I: U test. Differences of p less than 0.05 were considered to be significant. Statistical analysis was performed using SPSS Version 16.0 .Data were presented as mean ± standard deviation (SD) together with minimum and maximum values. The coefficient of variation (CV %) indicating the extent of variability in relation to mean of the population was calculated for the measured parameters. Differences between males and females were analysed using the Mann–Whitney Classical liver vascularization pattern was found in 1410 of 1569 patients (89.87 %). Replaced right hepatic artery (RRHA) arising from the superior mesenteric artery (SMA) was detected in 159 cases (10.13 %).Each anatomical parameter of RRHA was subject to variation in the studied population. However, the analysis of coefficients of variation suggested that the parameters D and L were more variable than the parameters AH and AL Table .Table 1MThe RRHA arises from the SMA approximately 27.34 mm after its origin from the AA. The distance between SMA origin from the abdominal aorta and the place of RRHA origin (parameter D) was significantly longer in males in comparison to that of females (29.38 mm vs. 25.62 mm). The mean lumen of RRHA in the place of origin (parameter L) was 3.29 mm and was significantly wider in males than in females (3.56 mm vs. 3.07 mm). The RRHA origin angle from the SMA in horizontal (parameter AH) and vertical plane (parameter AV) was 97.27º and 89.73º, respectively.Anatomical parameters of the RRHA origin compared males and females Table . The staThe distribution of the values of the anatomical parameters of the RRHA origin indicate the most frequent values of the parameter D is between 20 and 30 mm , and for the parameter L is <3 mm for females and between 3 and 5 mm for males Fig. .Fig. 6ThAnatomical parameters of the RRHA were evaluated according to the RRHA course in relation to the pancreas Table . Type I Vascular anatomy of the liver is subject to many variations . The preRRHA usually passes lateral and behind the portal vein and joins the structures of the portal triad when entering the hepatoduodenal ligament posterolateral to the common bile duct. At this point, palpation can be used in the epiploic (Winslow) foramen .The vascular anatomy of the operated region is highly required during pancreatoduodenectomy (PD). Typically, the kocherization of the duodenum and opening of the pars flaccida of the lesser omentum is followed by the palpation of the proper hepatic artery . The addThe evaluation of the RRHA anatomy is also important during liver transplantation, the therapy has progressively increased in numbers over the last decade . FrequenMoreover, vascular anomalies are reported to increase the rate of morbidity and mortality during PD, as they adversely affect the outcomes of patients . AdditioIt is crucial to define the model of hepatic arteries for surgeons, as well as for diagnostic and interventional radiologists. The pattern of hepatic arteries is significant in the treatment of hepatocellular cancers identified at the initial stage as the cancer does not yet qualify for the transplant waiting list . If the In this study, the origin angle of RRHA from the SMA in vertical plane lower than 90° was observed with frequency of 36.5 %. In this group of patients, the acute angle makes the accessibility to the RRHA very difficult. Therefore, additional evaluation of the distance between SMA origin and RRHA origin may help to recognize where the RRHA origin from the SMA can be expected.Preoperative recognition of abdominal vascular architecture in the peripancreatic region provides valuable information that can help to choose the most appropriate strategy during surgery. Commonly performed radiological examinations include CT angiography or MDCT and MRCA, as a routine preoperative examination in case of potential radical resection for pancreatic cancers . AdditioThe report revealed that the anatomy of origin of RRHA, the most common anomaly of the hepatic arteries, is subject to considerable variations. The findings are relevant for radiologists responsible for providing accurate anatomical information and for surgeons involved in the treatment of pathologies in the upper abdomen. It was shown that preoperative MDCT routinely performed could successfully be used to detect and evaluate the RRHA.